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CATEGORY A: ANALYTICAL CHEMISTRY: METHODS DEVELOPMENT AND APPLICATIONS
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W. C. Andersen1 , S. B. Turnipseed1 , C. M. Karbiwnyk1 , L. E. Carr2 , R. H. Lee2 , M. R. Madson2 , K. S. Kreuzer2 , 1Animal Drug Research Center, FDA, Denver, CO 80225, 2Denver District Laboratory, FDA, Denver, CO 80225
Background: The FDA analyzes hundreds of shellfish samples annually to screen for illegal residues of chloramphenicol (CAP). Current methodology is based on mass spectrometric determination of sample extracts using a triple quadrupole LC-MS-MS. While the method provides excellent sensitivity and selectivity, shellfish requires a lengthy extraction and cleanup to render samples suitable for analysis. Surface plasmon resonance (SPR) biosensor technology based on an analyte-specific binding reaction has become standard methodology for the analysis of drug residues in food in Europe, but it has not yet been adopted for this use by the FDA.
Methods: Shrimp or crab was mixed with ethyl acetate then centrifuged. The organic phase was collected, evaporated, and redissolved in HBS-EP buffer. The sample was defatted with cyclohexane, and the aqueous phase collected for direct analysis. A Biacore Q Biosensor instrument and commercially available CAP test kit was used to analyze samples.
Results: The Biacore Q was evaluated by analyzing 400 shrimp and crab samples for CAP residues. Samples had previously been analyzed using the current FDA LC-MS-MS testing method. The method provides a limit of detection of 0.073 ppb for shellfish, exceeding the FDA requirements for shellfish testing. Biosensor and LC-MS-MS methods were compared with respect to residue detection, instrument performance, extraction efficiency, analysis time, and cost for sample preparation and analysis.
Conclusions: The biosensor method was found to be well-suited for routine shellfish testing providing lower CAP detection limits, higher sample throughput, and lower instrumentation cost than the current LC-MS-MS method.
D. L. Anderson, W. C. Cunningham, CFSAN, FDA, College Park
Background: Instrumental neutron activation analysis (INAA) is a non-destructive technique used by CFSAN for multi-element analysis of foods. High levels of NaCl in foods lead to high limits of detection (LODs) for other elements because of spectral background interferences. To improve INAA's capabilities, a procedure involving background suppression was developed.
Methods: Compton suppression INAA electronics were implemented and procedures developed and used to analyze foods for 16 elements with short half-life irradiation products. Irradiation and gamma-ray counting conditions were optimized for iodine detection to provide quality assurance analyses for FDA's Total Diet Study.
Results: Iodine mass fractions (0.075 to 2.03 mg/kg) were measured in 19 of 42 foods analyzed. LODs ranged from 0.03 to 1.4 mg iodine/kg for foods containing 0.007 to 3.6% NaCl. Al, Br, Ca, Cl, Cu, K, Mg, Mn, Na, Sr, Ti, V, and Zn mass fractions were determined for some or all foods. Control analyses of certified reference materials yielded very good agreement with known values.
Conclusions: Compton suppression spectrometry significantly improved INAA results. Compared with previous procedures, iodine LODs were lowered by about a factor of 4. Similar improvements were found for Al, As, Ba, Br, Ca, Cl, Cu, K, Mg, Mn, Na, Sr, Ti, V, and Zn. Improvements are due to the combined factors of Compton suppression, greater detection efficiency, count timing and times, and choice of irradiation location.
T. H. Begley, W. Hsu, G. W. Diachenko, CFSAN, FDA, College Park, MD
Perfluorochemicals because of their stability and chemical resistance are widely used in the manufacturing and processing of a vast array of consumer goods, including electrical wiring, medical devices, clothing, household, and automotive products. Furthermore, relatively small quantities of perfluorochemicals are also used in the manufacturing of food contact substances (FCS) which represent potential sources of oral exposure to these chemicals. The most recognizable products to consumers are the uses of perfluorochemicals in non-stick coatings (polytetrafluoroethylene (PTFE)) for cookware and also their use in paper coatings for oil and moisture resistance in microwave popcorn bags. Recent epidemiology studies have demonstrated the presence of two particular perfluorochemicals, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in human serum at very low part per billion (ppb) levels. These perfluorochemicals are biopersistent and are the subject of numerous studies investigating the many possible sources of human exposure. Because of this potential for biopersistence, FDA is evaluating the migration characteristics of perfluorochemicals from food contact paper.
In this paper, the types of perfluoro chemicals used in food contact paper will be illustrated, along with methods for their determination. Additionally, research will be presented on the migration of these chemicals into foods or food simulating liquids. Results from migration tests show that fluorotelomers from the paper additives/coatings do transfer to food.
H. W. Yang1 , H. T. Mai1 , A. Weisz2 , 1Office of Cosmetics and Colors, Color Certification Branch, FDA College Park, MD, 20740, 2Office of Cosmetics and Colors, Color Technology Branch, FDA College Park, MD 20740
Background: D&C Red No. 21 (R21, Colour Index No. 45380:2, mainly 2',4',5',7'-tetrabromofluorescein), its disodium salt, D&C Red No. 22 (R22, Colour Index No. 45380, Eosin Y), and their lakes are color additives listed in the U.S. Code of Federal Regulations (CFR) for use in drugs and cosmetics (Only material which has been batch certified by the FDA in accordance with CFR specifications is appropriately identified with the D&C designation.) R21 and R22 are manufactured by condensing phthalic anhydride (or acid) with two equivalents of resorcinol, partially purifying, and brominating the resulting fluorescein to yield the main component of R21. R21 is hydrolyzed with sodium hydroxide to produce the main component of R22. During manufacture, various impurities may occur. CFR specifications include certain side-reaction products: brominated resorcinol, not more than 0.4%, and 2-(3,5-dibromo-2,4-dihydroxybenzoyl)benzoic acid, not more than 0.5%; and the intermediate phthalic acid, not more than 1%. Currently, the Color Certification Program determines these impurities using gravity-column chromatography followed by UV-Vis spectrophotometry. A more efficient, automated method was needed for their determination.
Methods: Brominated resorcinol was purified for use as a reference standard. Appropriate standards were obtained for the other two analytes. A high-performance liquid chromatographic (HPLC) method was developed to determine the three specified impurities in R21 and R22.
Results: Five-point calibration curves were prepared by analyzing samples of R21 spiked with varying amounts of the three analytes. Twenty previously certified samples of R21, R21 lakes, and R22, representing eight manufacturers, were analyzed by the new method. The results compared well with those for the gravity-column chromatography method.
Conclusions: The present study reports the development of an automated, efficient, and reliable high-performance liquid chromatographic (HPLC) method for the determination of two side-reaction products and an intermediate that often contaminate batches of R21 and R22.
I. Vertsman, Y. T. Cain, Wyeth Research, Wyeth
Background: Sample preparation is the most time consuming part of the analytical testing. To be a front runner in today's competitive market place, dramatically increase in efficiency and productivity is crucial. Wyeth has recognized that utilizing new technology and automation is the answer. One of the techniques to automate sample preparation is using Tablet Processing Workstation (TPW).
Methods: Automated sample preparation methods using TPW were developed for the content uniformity testing in supporting of a several late stage development projects. TPW system weighs each tablet, adds assigned amount of solvents, performs extraction, filters sample, dilutes and transfers in the vials for the further analysis. Analyst time required for the set up of the instrument is about 2 hrs per 50 samples.
Results: Excellent equivalency was obtained between manual and automated sample preparation methods. Difference between manual and automated results was in the range of 0% to 1.8%. Reproducibility (Precision and Intermediate Precision) of the automated results was less then 1%. Recovery of the known standard concentration, processed through the TPWII system the same way as samples ranged between 97.9 and 102.1%.
Conclusions: The biggest advantage of automated sample preparation technique was observed for high volume projects, especially process validation testing, when robotic systems can prepare samples 24 hrs a day 7 days a week. Time consuming manual preparation steps can be fully automated. Minimum analyst time is required for the set up of the instruments, which increases efficiency about 4 times.
Y. T. Cain, M. Meyers, Wyeth Research, Wyeth
Background: Ion Mobility Spectrometry (IMS) has been successfully implemented as a rapid analysis technique for cleaning verification/validation.
Methods: IMS is a sensitive and fast analytical technique that has proven advantageous when used in cleaning validation/verification applications. The instrument is easy to operate and has a lower cost of operation in comparison with HPLC. Two examples of technical challenges that have been overcome are discussed. IMS technology has been enhanced using the new High Performance Injection (HPI) system. Improved precision and method simplification via elimination of dilutions have been demonstrated with HPI. Future applications using IMS with HPI are discussed.
Results: Several cleaning methods have been developed and validated using the limit test approach. The technology has been deployed to several commercial manufacturing sites and successful technical transfers have been completed. Sensitivity is a function of the attributes of the analyte, but detection limits at the low pictogram level have been achieved with data acquisition times as short as one minute per injection. HPI technology significantly improves reproducibility both short-term (precision) and long-term (signal drift).
Conclusion: IMS provides rapid, low cost results which dramatically increases cleaning sample throughput and reduces manufacturing equipment down time.
Y. T. Cain, M. Meyers, Wyeth Research, Wyeth
Background: The development of a fiber optic probe coupled to a fluorescence spectrophotometer has made possible the rapid identification of pharmaceutical tablet dosage forms with minimal sample preparation.
Methods: UV light at the excitation wavelength is transmitted from the spectrophotometer source directly to the solid tablet surface, in which the coating has been removed with light sanding, by way of the fiber optic bundle and probe. Fluorescence from the tablet is transmitted back though the bundle to the detector. The active pharmaceutical ingredient (API) is positively identified by its fluorescence spectrum.
Results: Label claims with similar API to excipient ratios are differentiated though volume adjusted concentration differences of the API in the bulk tablet compared with a standard in powder form.
Conclusion: A tablet can be identified in a matter of seconds with this technique.
Y. T. Cain, J. L. Edgar, Wyeth Research, Wyeth
Background: The future of dissolution is moving toward increasing productivity in laboratories. This can be accomplished is by utilizing dissolution with online fiber optic UV analysis.
Methods: Fiber optics changes the traditional way of sampling for dissolution by bringing the UV spectrometer to the sample solution in-situ.
Results: The use for fiber optics allows real-time determination of drug release levels in-situ and effectively eliminates many common issues with traditional dissolution testing and analysis including carryover, sipper, line or filter malfunction and cost of consumables such as filters, test tubes and HPLC solvents. Since no sample is removed from the dissolution vessels, readings can be taken as often as every few seconds if necessary; thereby setting a new precedent for dissolution profiles. Direct comparison of fiber optic analysis with conventional quartz cell UV analysis shows agreement well within 2%.
Conclusion: The use of fiber optic analysis for dissolution testing saves analyst time, which reduces laboratory operating costs. Product development data is more meaningful since the number of sampling time points is not limited by volume removal. This technology has been successfully implemented at Wyeth research and commercial manufacturing sites.
S. Smith, C. S. Cheely, C. Gieseker, R. Reimschuessel, M. C. Carson, CVM, FDA, Laurel, MD
Background: Prior to approving a drug for food animals, a method for determining residues is needed and a depletion study is conducted using this method. A microbiological (micro) method for erythromycin was used in the salmon depletion study, however a liquid chromatography (LC) assay developed by NCTR gave much lower concentrations in incurred tissue than the micro assay. Using LC-ion trap mass spectrometry, we identified at least two additional compounds in dosed salmon, N-demethylery A and anhydroery A. These may contribute to the difference between the NCTR LC method and the micro method.
Method: Ground salmon is extracted with acetonitrile, the extract defatted with hexane, evaporated, and reconstituted in a solution of Ery C, the internal standard (IS). Extracts are chromatographed on a phenyl column. Detection is by + ESI, with monitoring of ion trap transition products of m/z 734.5 (Ery A), 720.5 (Ery C and N-demethylery A), 718.5 (Ery B), and 716.5 (anhydroery A).
Results: Accuracy ranged from 65% at 50 µg/kg to about 80% at concentrations of 500 µg/kg and above. CVs were 7-20%, reflecting the variable nature of the ion trap. Limits of detection were estimated to be <10 µg/kg. Salmon were dosed with 100 mg/kg Ery A and sacrificed following 1 to 28 days. Ery A and both metabolites were detected at all time points. By 3 days withdrawal, metabolite exceeded parent concentration. Parent Ery A depleted to <100 µg/kg by ~3 weeks, but total residue remained above 1 mg/kg.
Conclusion: Ery A is metabolized in salmon to compounds which deplete much slower than the parent drug.
P. Chu, M. Lopez, CVM, FDA, Laurel, MD
Background: Nitrofurans are antibacterials commonly used in the treatment of aquatic and terrestrial animals to control bacterial and protozoan infections. Although widely used since the 1940s, nitrofurans have been banned for use in food-producing animals in most countries because of their mutagenicity and potential carcinogenicity. Recently, concerns about the illegal use of this class of compounds in food-producing animals have developed. Therefore, methods are needed to monitor nitrofuran residues in food and for research purposes. We describe a method capable of detecting and quantitating total bound and free residues of nitrofurans in milk.
Method: To 2 mL of milk is added 5 mL of 0.125 M HCl followed by 400 µL of 50 mM 2‑nitrobenzaldehyde. The mixture is placed in an incubator at 37°C overnight with gentle shaking. During this step, the side-chains of the bound residues of nitrofurans are released through hydrolysis and simultaneously derivatized with 2-nitrobenzaldehyde. After adjustment of pH to ~7, the sample is cleaned up on a solid-phase extraction column. The derivatives are detected and quantitated using LC-MS/MS in the positive ion mode.
Results and Conclusions: The method has been validated at 1, 2, and 4 ppb using fortified milk and milk obtained from a treated cow. The method accuracy is >60% with coefficients of variation <20%. Among the four nitrofurans studied in milk of the treated animal, nitrofurantoin exhibits the lowest level of residues.
D. Dai, S. Wang, NRL, FDA, Jamaica, NY
The cephalosporins are a group of antibiotics closely related to the penicillins. The presence of penicillin residues in cephalosporins has the potential to cause health effects to patients allergic to penicillin.
The presence of trace levels of spiked penicillin G in cephalosporins was determined by HPLC with a PDA detector using a Waters Symmetry C18 5µm, 4.6 x 250 mm column; a gradient elution of 0.01M orthophosphoric acid/acetonitrile from 80/20 to 40/60 in 20 min. The UV spectra were collected from 190 to 400 nm and extracted at 220 nm. Identification was confirmed by LC/MS/MS method.
This HPLC method provided excellent separation between penicillin G and 12 cephalosporins and PDA detection provides spectra identity of the peaks. Excellent linear response of penicillin G was found in the range of 0.5 to 100 µg/mL and the estimated MDL was 0.04 µg/mL. A peak very close to the retention time of penicillin G was detected in the screening of selected cephalosporins APIs and finished products; however, the UV and mass spectra data indicated it was not penicillin G. The stability of penicillin G and Cephalosporins in various solvents has been examined. The optimum solvent for dissolving and diluting penicillin G and Cephalosporins was found to be the mixture of acetonitrile and water at a neutral pH. Penicillin G rapidly changed to its isomer in an acidic environment. In the presence of methanol, penicillin G rapidly converted to its methanol adduct.
L. S. de Jager, G. A. Perfetti, G. W. Diachenko, CFSAN, FDA, College Park MD
Vanilla extract is widely used as a flavoring in food. Because authentic vanilla extract is expensive, artificial vanilla flavorings containing synthetic vanillin and ethyl vanillin are often used. Some foreign manufacturers add coumarin to vanilla products to increase the vanilla flavor perception. Coumarin is a phytochemical found in many plant species, with the main source being the tonka bean. It has a sweet herbaceous odor and has been used in food, tobacco, and cosmetics as a flavoring and fragrance material. Coumarin has been shown to be hepatotoxic and has been prohibited from being added to food in the U.S. since 1940 (CFR part 189.30).
Two official AOAC methods have been published for the detection of coumarin in vanilla extract. One involves separation using column chromatography followed by spectophotometric detection and quantification (AOAC Official Method 955.3) and the other is a purely qualitative TLC method (AOAC Official Method 964.11). These methods have limited selectivity and sensitivity. A solid phase microextraction (SPME) GC-MS method for determining coumarin and vanilla extract components has been developed. This method has greater sensitivity than the AOAC methods and provides MS confirmation. Method development, optimization and validation and the results of a survey of vanilla extract products will be presented.
Q. Ge, R. V. Bahulekar, P. M. Amin, M. W. Diamond, F. M. Buevich, F. T. Do, R. A. Ebelle, S. K. Pulapura, A. C. Moses, W. C. McJames, TyRx Pharma, Inc.
Background: Gravimetric analysis is accepted and the standard method for measuring the mass loss of bioresorbable polymers (ASTM Standard F1635-04a). The current method involves drying the sample to a constant weight, prior to weighing. Since resorbable polymers degrade via hydrolysis, there is often an initial increase in mass at earlier assessment time points, which one can assume is the result of bound water molecules. The introduction of the use of polyarylate polymers on implantable medical devices has permitted the development and use of an HPLC method that calculates mass loss by measuring the polymer degradants as they become solubilized in the buffer over time.
Methods: Polyarylate polymer-coated polypropylene mesh was incubated in PBS at 37°C with no agitation. PBS buffer was changed twice each week and the buffer analyzed via HPLC for the presence of tyrosine-related compounds (i.e., monomers and end stage degradants). The mass loss at each time point was calculated by adding the mass of the measured compounds and the mass of equivalent molar amounts of the non-measurable degradants to the mass equation. The experiment was terminated when no degradants were detected at three consecutive time points.
Results: This experiment characterized the in vitro mass loss profile and degradation rate of the polyarylate polymer coating of the polypropylene mesh.
Conclusions: HPLC is a reliable and accurate method to measure the mass loss of resorbable polymers containing optically active compounds. When it is possible to use it, it may be more accurate than gravimetric analysis.
S. M. Etheridge1 , J. R. Deeds1 , S. M. Conrad1 , S. Hall1 , P. DiStefano2 , M. Ellwanger2 , K. Chu3 , F. Pettengill4 , M. Hickey5 , D. Couture6 , 1OS, CFSAN, FDA, Laurel, MD, 2OS, CFSAN, FDA, College Park, MD, 3NOAA Fisheries Service, Gloucester, MA, 4Div. of Marine Fisheries, Gloucester, MA, 5MA Marine Fisheries, Pocasset, MA, 6Dept. of Marine Resources, W. Boothbay Harbor, ME
An extensive Alexandrium bloom occurred off the New England coast from May to July 2005 creating an unprecedented paralytic shellfish poisoning (PSP) event severely impacting the shellfish industry with toxicity exceeding the action level (80 micrograms saxitoxin equivalents per 100 grams tissue). High-resolution coastal sampling by management programs allowed safe shellfish to be marketed, whereas shellfish beds threatened by PSP were closed to protect public health. At the request of FDA, NMFS closed approximately 15,000 square miles of federal waters in the northwestern Atlantic Ocean on 14 June. A time-series of offshore shellfish toxicity was monitored since the beginning of the closure. The receptor binding assay was used as the primary detection method, with the AOAC mouse bioassay providing confirmation for regulatory decisions. Shellfish toxicities varied with species, with the highest toxicity (2045 micrograms saxitoxin equivalents per 100 grams) found in whole scallops sampled in July. Toxicity decreased over time with depuration rates differing between species. Analytical data supported reopening a portion of the closure on 9 September (except for whole and roe-on scallops); although the remaining northern area still remains closed. Due to an effective, long-standing cooperative shellfish program managed by FDA and implemented by states, there were no human illnesses despite remarkably high toxicity in the unmarketed product. Current/future efforts to improve seafood biotoxin risk management include: 1) implementing a dockside testing protocol and 2) conducting a collaborative investigation with academia, government, and industry to establish a comprehensive regional-scale understanding of Alexandrium blooms and associated shellfish toxicity.
R. B. Shah, Y. Yang, M. A. Khan, P. J. Faustino, Division of Product Quality Research, Office of Testing and Research, Office of Pharmaceutical Science, FDA, Silver Spring, MD
Purpose. Development of a validated SEC method to efficiently assess the MW of polysaccharides. Heparin, a mucopolysaccharide whose antithrombic activity depends on MW was investigated.
Methods. Separation was achieved on two gel filtration columns (TSK-GEL-G4000SWXL) with a mobile phase of sodium azide (0.02%, pH, 6.2) delivered isocratically at 0.6 ml/min with refractive index detection. Standard curves were constructed using dextran MW standards (1 to 670 KDa). The method was validated according to ICH. Heparin sodium from porcine intestinal mucosa (Product A) and bovine lung tissue (Product B) in different pH media were analyzed.
Results. Log-linear function was used for MW standard curve (12 to 670 KDa). A second order polynomial fit was applied for the MWs below 5 KDa. Accuracy ranged from 90-103% and precision was <2.7%. Product A showed three MW fractions of 580, 30, and 1.5 KDa at pH 3, 4, and 5. However at pH 6.2, only two fractions were obtained (590 and 2 KDa). At pH 7.4, the two significant fractions were 23 and 1 KDa. Product B showed two fractions of different MW from pH 3-7.4. The major fraction at pH 3, 4, and 5 was 30 KDa. However at pH 7.4, a fraction of 12 KDa was achieved.
Conclusions. A highly specific, selective, and efficient SEC method was developed for the accurate MW determination of polysaccharides. Two heparin products exhibited different MW fractions depending upon isolation source and media pH. The method can be used to assess the quality of therapeutic polysaccharides.
S. R. Gratz, R. A. Flurer, M. R. Witkowski, C. L. Flurer, Forensic Chemistry Center, FDA, Cincinnati, OH
For several years, synthetic phosphodiesterase type 5 (PDE-5) inhibitors have been identified routinely in "all natural" herbal remedies and dietary supplements, as well as in counterfeit and unapproved pharmaceutical products. A liquid chromatography-electrospray mass spectrometry (LC-MS) method was developed and implemented at the Forensic Chemistry Center to screen for sildenafil (Viagra®), tadalafil (Cialis®) and vardenafil (Levitra®) in both herbal and pharmaceutical matrices. More recently, there has been a trend toward the development of designer drugs, or analogs, based on sildenafil, tadalafil and vardenafil. As a result, methods that only screen for the FDA-approved compounds are inadequate. Among the growing list of such compounds are homosildenafil, hydroxyhomosildenafil, acetildenafil, hydroxyacetildenafil, aminotadalafil and piperadino-vardenafil. Based on the structural similarities of these drugs to their FDA-approved counterparts, it is reasonable to expect that they would exhibit similar biological activity.
The general approach used for identification of these analogs will be shown. The method of choice for initial identification of this class of compounds is LC-MS, while Fourier-Transform infrared spectrometry also provides valuable information. Elucidation of analog structures is typically accomplished through MSn experiments, and by comparing spectra to those of sildenafil, tadalafil and vardenafil. Fourier-Transform ion cyclotron resonance mass spectrometry is used for accurate mass determinations, to further characterize analog structures.
Z. Gao, T. Moore, A. Smith, W. H. Doub, B. J. Westenberger, L. F. Buhse, FDA
Background: In-vitro dissolution tests on finished dosage forms have been performed for many years. Currently, the repeatability and reproducibility of dissolution tests are of concern to the pharmaceutical industry. The large variability observed in dissolution test results may be a result of uneven distribution of hydrodynamic forces within the dissolution vessel. Random positioning of tablets/capsules, even in well calibrated USP apparatus 2 dissolution vessels, can lead to high variability in dissolution results. Method: The gauge repeatability and reproducibility (Gauge R&R) method was used to analyze variability in a dissolution testing system. Additionally, perturbation studies were performed to study the variability caused by changes in the sample position. Results: Evaluation of dissolution testing results at 30 minutes using an internal DPA NCDA #2 tablet indicate that the main contribution to total variance (~70% of total variance) is due to the sample tablets, ~25% of the total variance arises from the vessels (apparatus) and ~5% of the total variance is due to the operators. In addition, the sample position (centered and off-centered positions) can have a serious effect on the dissolution results. Conclusions: Gauge R&R analysis is a useful tool for determining the sources of variability in a dissolution measurement system. Although there are sample position effects, the impact of variability from sample position depends on the drug product.
P. E. Nwakama, S. H. Haidar, Y. S. Yang, B. M. Davit, D. P. Conner, L. X. Yu, CDER, FDA, Rockville, MD
Background: Bioequivalence (BE) studies are conducted "...to ensure therapeutic equivalence between a pharmaceutically equivalent test (T) drug product and a reference (R) listed drug." The current confidence interval limits of 80 -125% for Cmax and AUC ratios were established based on physicians' thinking that as much as a 20% difference in dose would not be clinically significant for most drugs. This study was to evaluate performance of the BE criteria, by examining differences in extent (AUC) and rate (Cmax) of bioavailability between reference listed drugs (RLD) and their generic equivalents.
Methods: With few exceptions, data used in this study were from single-dose, fasting BE studies of approved oral drug products submitted to the Office of Generic Drugs from 1996 to 2005. Statistical analysis was performed on differences in extent and rate of absorption between the RLD and its generic equivalent using point estimates for AUC and Cmax, respectively.
Results: A total of 1636 fasting BE studies were examined; the sample size of each study ranged from 12 to 127 subjects. The mean (±S.D.) of the absolute value of the difference in point estimates |T - R| for AUC0-T was 3.19% (±2.72) and 3.12% (±2.66) for AUC0-inf. Mean difference for Cmax was 4.50% (±3.57). All were well within the 90% CI criteria of 80 - 125%.
Conclusion: The results show that generic products have averaged less than 4% difference in extent of absorption relative to the RLD. This illustrates the effectiveness of the BE acceptance method and criteria used in the approval of generic drugs in the U.S.
M. K. Halbert, J. C. Archer, ARL, FDA
Background: According to the guidelines of the International Olive Oil Council, olive oil may by labeled as "extra virgin" if it has no more than 0.8 percent acidity and has not undergone any treatment which would lead to alterations in the oil. Oils not labeled as "virgin" have undergone some refinement to alter the acidity, taste and appearance, and may be a mixture of refined and virgin oils. Since virgin olive oils have undergone no refinement, they may contain higher concentrations of contaminants, such as dioxins and furans.
Methods: Thirty-eight samples of olive oil were collected from store shelves during the period 2003-2005, and were analyzed for dioxins and furans using EPA Method 1613, including HRMS analysis. These oils were described variously as "extra light tasting," "100% pure," "extra virgin," or simply "olive oil."
Results: The oils were divided into two categories for the sake of comparison —"extra virgin olive oil" and "non-virgin olive oil." The extra-virgin oils exhibited an average toxic equivalence (TEQ) overall of 0.035 pg/g, with a range of 0.0014-0.10 pg/g. The average TEQ for the non-virgin olive oils was lower, at 0.010 pg/g, with a range of (non-detect)-0.041 pg/g. The greatest contributors to TEQ for both groups were 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF, with these congeners reduced by 73% and 77%, respectively, in the non-virgin olive oils. 1,2,3,7,8-PeCDD was reduced to a non-detect level in the non-virgin olive oils from an average of 0.0058 pg/g in the extra-virgin oils.
Conclusions: As expected, refinement of the olive oils apparently reduces the levels of dioxins and furans, although the levels in all the samples were acceptably low.
S. Hall, S. M. Etheridge, J. R. Deeds, S. M. Conrad, WSL, OS, CFSAN, FDA, Laurel, MD
Marine biotoxins, produced by unicellular algae and accumulated in seafood, can cause illness and death in human consumers. Purified toxins are needed for determination of structure, for chemical and toxicological characterization, for the development and validation of detection methods, and for the field deployment of those methods. In most laboratories, work with these toxins is constrained by their availability since they tend not to be commercially available and are difficult to obtain or produce. Due to the facilities established and maintained by the Washington Seafood Laboratory for the production and purification of marine biotoxins, the FDA needs for the saxitoxins and domoic acid can be well supported and many other needs can be met as they become priorities. Current priorities include the purification and certification of reference standard saxitoxin to replace the historical standard now in use, purification and characterization of decarbamoylsaxitoxin as an alternative to saxitoxin and as a starting material for producing other needed derivatives, and purification of other saxitoxin congeners for the evaluation of new detection methods.
D. G. Hayward1 , T. S. Pisano2 , 1CFSAN, FDA, College Park, MD, 2JIFSAN, University of Maryland, College Park, MD
Background: Polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBDEs) are known to persistent in the environment. Foods are the major route of exposure to nonoccupationally exposed persons. FDA routinely monitors PCDD/Fs in 1000s of foods of many types annually. PCBs are monitored through the total study program, but no isomer specific data is generated. PBDEs have never been measured routinely by FDA in foods.
Methods: These three compound classes are often determined by separate methods (e.g. EPA 1613b/8290, 1668 and 1614). A study was conducted to evaluated four separate approaches to determining all target analytes by a single method that might be easily automated using four criteria to include or exclude elements in each approach. The method selected must be 1) automated, 2) produce reliable recovery and chromatography, 3) be applicable to any food or feed, 4) use a sample size suitable for detecting congeners with toxic equivalency factors (TEFs).
Results: An automated procedure using accelerated solvent extraction with integrated clean up followed by "express" gel permeation chromatography with on-line SPE clean up made the final selection. Eight fish oils, a corn oil, 3 fish fillets, carrots and butter were successfully measured isomer specifically for 50 target analytes from the three compound classes. Recoveries were assessed using fortified corn oil and native incurred levels in fish fillets measured by a LIB 4084 and the new method. Recoveries fell within the acceptable range (50-100%).
Conclusions: A single automated approach was identified that met the four criteria.
T. S. Pisano1 , D. G. Hayward2 , 1JIFSAN, University of Maryland, College Park, MD, 2CFSAN, FDA, College Park, MD
Background: Persistent organic pollutants (POPs) such as polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs), polychlorinated biphenyls (PCBs), and polybrominated biphenyls (PBDEs) are associated with lipid compartment in foods. FDA plans to report milk POPs levels on a lipid weight basis and will need to determine the lipid content of all milk samples as part the PCDD/F analysis. Traditional liquid/liquid extraction (LLE) methods such as AOAC 970.52H, AOAC methods vol.2, p278, require large amounts of solvent and tedious manual manipulations. Pressurized liquid extraction (PLE) uses less solvent and is automated.
Methods: A study was performed to find the optimal conditions for extracting fat from freeze dried cow's milk using PLE then following with purification of the fat for POPs analysis1. Freeze-dried milk was extracted with several solvent systems by PLE. PLE was performed by an ASE300™ (Dionex, Sunnyvale, CA). Using butter as a test matrix, automated fat removal with sulfuric acid silica gel as fat retainer was tested with PLE.
Results: The optimal solvent system was determined to be methanol/ dichloromethane/ hexane (1/2/2). The PLE gravimetric fat results were not significantly different from those by LLE at 95 % confidence level. A 99% fat removal from 4g of butter was achieved with 60g of 44% sulfuric acid on silica gel fat retainer in the ASE with the following conditions: 100% petroleum ether in three 5min extractions at 1500psi, 100ºC and a 5% flush.
Conclusions: PLE based extraction method produces comparable results using less solvent and man-hours for the milk fat determination.
1 Hayward, D.G., Pisano, T.S., 2006 FDA science form, Automation for isomer specific determination of polychlorinated dibenzo-p-dioxins/furans, polychlorinated biphenyls and polybrominated diphenyl ethers in foods and feeds.
N.M. Hepp, Office of Cosmetics and Colors, CFSAN, FDA, College Park, MD
Background: Bone Black is proposed to be listed in the Code of Federal Regulations as the color additive D&C Black No. 3, subject to FDA batch certification. Methods are needed to enforce specifications of <10 ppm for lead (Pb) and <3 ppm for arsenic (As). X-ray fluorescence spectrometry (XRF) standards were prepared and validated by atomic absorption spectrometry. The calibration curves generated from the standards were evaluated for suitability in the routine analysis of Bone Black samples for Pb and As.
Methods: Standards were prepared from Bone Black fortified with varying amounts of Pb and As. The fortified standards were used to develop appropriate grinding and pellet-pressing procedures and to obtain XRF calibration curves. Portions of each standard were analyzed by atomic absorption spectrometry to verify fortification levels. Electrothermal atomization atomic absorption spectrometry was used to determine Pb, and flow injection/hydride atomic absorption spectrometry was used to determine As.
Results: Very good XRF calibration data were obtained for both Pb and As in fortified Bone Black samples using secondary XRF lines to deconvolute overlap of the primary lines for the two analytes. Detection limits for Pb (1.0 ppm) and As (0.3 ppm) indicate that XRF is appropriate for routine batch certification analysis of these elements in Bone Black.
Conclusion: XRF can be used for FDA batch certification to efficiently analyze multiple samples of Bone Black (D&C Black No. 3) at the proposed specification levels for Pb and As.S. C. Hight, J. Cheng, FDA
A method was developed for determination of methylmercury and estimation of total mercury (Hg) in seafood. Hg compounds were extracted from 0.5 g edible seafood or 0.2 g lyophilized reference material by adding 50 ml aqueous 1% w/v L‑cysteine•HCl•H2O and heating 120 min at 60°C in glass vials. Hg compounds in 50 µl of filtered extract were separated by reversed‑phase HPLC using a C‑18 column and aqueous 0.1% w/v L‑cysteine•HCl•H2O+0.1% w/v L‑cysteine mobile phase at room temperature and were detected by ICP-MS at mass‑to‑charge ratio 202. Total Hg was calculated as the mathematical sum of methyl‑ and inorganic Hg determined in extracts. Precision of analyses was ≤5% relative standard deviation (RSD) for methylmercury and ≤9% RSD for inorganic Hg. Recovery of added analyte was 94% for methylmercury and 98% for inorganic Hg. Methyl‑ and total Hg results for reference materials agreed with certified values. Limits of quantitation were 0.007 mg/kg methylmercury and 0.005 mg/kg inorganic Hg in edible seafood and 0.017 mg/kg methylmercury and 0.012 mg/kg inorganic Hg in lyophilized reference materials. Evaluation of analyte stability demonstrated that L‑cysteine both stabilized and de‑alkylated methylmercury, depending on holding time and cysteine concentration. Polypropylene adversely affected methylmercury stability. Total Hg results determined by this method were equivalent to results determined independently by cold vapor‑atomic absorption spectrometry. Methylmercury was the predominant form of Hg in finfish. Ratios of methylmercury/total Hg determined by this method were 93‑98% for finfish and 38‑48% for mollusks.
J. C. Hubinger, D. C. Havery, Office of Cosmetics and Colors, CFSAN, FDA, College Park, MD
Background: Retinol and retinyl palmitate are frequently used in cosmetic skin care products but may be irritating to the skin at higher concentrations.
Methods: A simple, rapid and sensitive reversed‑phase HPLC method with UV detection was developed for the quantitation of retinol, retinyl palmitate and retinoic acid in cosmetic preparations. The analytes were extracted from a cosmetic/Celite mixture using a solvent system composed of equal amounts of hexane, isopropanol, and ethyl acetate, and the extract was injected directly into an HPLC with a C18 column and UV detector set at 330 nm. Chromatographic separation was achieved by gradient elution with a mobile phase starting with aqueous ammonium acetate buffer/methanol that was gradually changed to methanol/dichloromethane.
Results: The method was validated, and average recoveries of retinol, retinyl palmitate and retinoic acid were 95% or higher. In a survey of 29 consumer cosmetic skin care products, most products were found to contain either retinol or retinyl palmitate at concentrations up to 2.0%, while a few products contained both ingredients. Several cis isomers of retinol and retinoic acid were also isolated from cosmetic products with the method and could be quantitatively distinguished from the all-trans compounds.
Conclusions: The method can be used to accurately quantitate levels of several retinoids and their isomers in cosmetic products.
J. N. Jean-Giles, G. L. George, J. E. LeClerc, T. A. Cebula, Mod-1
Background: In order to evaluate tools for the differentiation of strains of foodborne pathogens, we have applied the multilocus variable-number tandem repeat analysis (MLVA) to a reference collection of diverse isolates of Escherichia coli O157:H7. The method differentiates strains by analysis of the expansion and contraction of short tandem repeats (TRs) in the genome. Since these elements vary in number at several loci, the method discriminates among strains of the same species.
Methods: MLVA analysis using a multiplex of seven targeted loci of TRs (TR1-TR7) was used to compare isolates in a collection of 120 strains of E. coli O157:H7. The MLVA pattern of each strain was determined based on the size (bp) of TRs of each locus to determine the number of repeats. The patterns were categorized using a letter coding system.
Results: From 120 strains of E. coli O157:H7 examined, 89 different TR patterns were determined. The most variable locus analyzed, a tandem repeat of six bp (TR2), showed 30 alleles and as many as 69 repeat elements.
Conclusion: Application of the MLVA analysis to a large set of diverse isolates of E. coli O157:H7 showed the duplication of MLVA patterns in independent strains. While the variability in MLVA patterns showed the genetic diversity present among many of the strains examined, the results demonstrated that additional molecular markers would be required for conclusive strain differentiation. Further evaluation of rapidly evolving loci, such as TR2, is also needed for proper interpretation of MLVA results.
A. N. Joshi, J. Nickelsen, G. Gavini, FDA
Abstract: There is a continuous demand on ORA labs to develop improved qualitative analytical methods for the enforcement of FDA regulations. The method described is for identification of color additives in imported and domestic food and cosmetic products, using FT-IR Spectroscopy. Two parallel procedures are run. One the standard separation and identification procedure and the other using the FTIR identification. The results so obtained are compared for cost, quality, time and reproducibility. If a color is observed and FTIR confirms absence of a certifiable color then it is an indication of non permitted color additives. The results of samples analyzed and spectra generated are reported.
P. A. Jupp, D. Trew, C. H. Turner, Wyeth Research
The measurement of pKa by NMR gives a specific approach to both the determination and the assignment of pKa values to ionisable centres in a molecule. The traditional approach for the measurement of pKa by potentiometric titrations can give the value for the ionisable centre but cannot show where the protons actually resides. Also at low concentrations the influence of the water as a media can distort the values as well as causing determination difficulties if the compound has low solubility. NMR, however can precisely determine the pKa in aqueous as well as mixed solvent systems. Work has been carried out to assess the impact on measuring pKa in organic/aqueous mixtures and clearly demonstrates that protonation is dependant of the nature of the media in which the measurement is made.C. M. Karbiwnyk1 , K. C. Faul2 , S. B. Turnipseed1 , W. C. Andersen1 , K. E. Miller3 , 1ORA, Animal Drugs Research Center, FDA, Denver, CO, 2ORA, FDA, Denver, CO, 3Univ. of Denver, Denver, CO
Background: The drug most commonly used to induce labor in the U.S., oxytocin, is a peptide hormone composed of nine amino acids. Oxytocin is often reconstituted in IV saline solutions at less than 0.05 Units/mL to be delivered at 1 - 4 drops/minute. Existing LC-UV methods for oxytocin do not have sufficient detection limits to quantitate and/or confirm oxytocin in IV solutions. LC-MSn was utilized as a rapid and sensitive method for the determination of oxytocin in a saline solution at less than 0.02 Units/mL.
Method: A determinative and confirmatory method for oxytocin was developed using an LC-MSn ion trap instrument with an electrospray ionization (ESI) interface in positive ion mode. Separation was achieved on a C-18 column with 250 µL/min isocratic flow of 36% acetonitrile:water (1:1) and 64% dilute acetic acid (0.05%) mobile phase.
Results: Calibration standards, prepared in de-ionized water from 0.006 - 0.046 Units/mL, were linear with an R2 value of 0.9983 using results from a SIM scan. A method detection limit (MDL) was tested at 0.003 Units/mL (7.5 ng/mL) and met all criteria for quantitation and confirmation. This LC-MSn method was used to determine the amount of oxytocin in a 0.04 Unit/mL clinical sample that was prepared in 0.9% sodium chloride IV solution. The sensitivity of the method allowed the sample to be diluted 1:1 with de-ionized water to reduce the salt content.
Conclusions: This study demonstrates the application of LC-MSn for low concentration (ng/mL) determination and confirmation of oxytocin in saline solution.
E. Karnaukhova, B. Golding, Y. Ophir, Division of Hematology, CBER, FDA, Bethesda, MD
Human alpha-1-proteinase inhibitor (a1-PI) is the most abundant serine protease inhibitor in plasma. A major function of α1-PI is the inhibition of neutrophil elastase in lungs. Three plasma-derived (pd-) a1-PI products are licensed in the US for augmentation therapy of deficient patients. The recombinant versions (r-α1-PI) provide as an alternative to pd-a1-PI products and have been under intensive investigation. For accurate determination of α1-PI from different sources and in various forms, there is clearly a need for reliable standardized assays. As a part of our multi-step research focused on α1-PI structure-function investigation, we have established an ELISA with high specificity and a low limits of detection (1.10 ng/mL) and quantification (3.34 ng/mL), which allows determination of pd-α1-PI as well as r-α1-PI in complex matrices. A validation of ELISA was performed with the working range of the assay from 3.1 to 50 ng/mL with an average correlation coefficient of 0.995 established by analyses of 108 calibration standard curves.
The analytical performance of the a1-PI ELISA has been demonstrated for: (a) quantification of r-α1-PI in various fermentation mixtures (E. coli and A. niger), (b) investigation of α1-PI enzymatically digested in the conditions of harsh fungal proteolysis, (c) evaluation of α1-PI thermally polymerized, (d) quantification of α1-PI in human serum, as well as (e) for comparative quantification of α1-PI in commercially available products.
E. Karnaukhova, Division of Hematology, CBER, FDA, Bethesda, MD 20895
Human α1-PI derived from plasma is an US FDA licensed product recommended for intravenous treatment of patients with hereditary α1-PI deficiency in order to slow down the progression of emphysema. Retinoic acid (RA) is another FDA-approved (orphan) drug recognized for its activity against acute promyelocytic leukemia. In regards to lung diseases, RA plays a key role in activation of genes involved in lung development and alveolar regeneration.
This study is focused on obtaining complexes of α1-PI with RA, which may have a dual function and thus may possess an enhanced efficacy in regards to current treatment.
The results of our UV/Vis, circular dichroism and fluorescence study show the following: (1) RA does bind to α1-PI in a non-covalent fashion, (2) There are 2 hydrophobic sites in the α1-PI interior available for RA, (3) Due to a complex formation RA, which solubility in water is extremely low (0.2 µM), behaves as water-soluble with concentration range consistent with α1-PI content and stoichiometry of binding (up to 90 µM); (4) Stability of RA in complex with α1-PI is significantly increased.
To the best of our knowledge, this is the first report of a binding of lipophylic ligand to α1-PI. Our study reveals a new structural feature, which may have a significant impact on α1-PI research and development, including clinical application.
E. Karnaukhova, Division of Hematology, CBER, FDA, Bethesda, MD 20895
Human serum albumin (HSA) is an US FDA licensed drug recommended at high doses for various therapeutic indications, including surgery or trauma, cardiopulmonary bypass, hypovolemia, shock, hemodialysis, burns, and acute respiratory distress syndrome.
HSA is the most abundant protein in plasma. As a major carrier protein, HSA is capable of binding a great variety of metabolites and drugs. Binding of new chemical entities to plasma proteins is an important concern confronting the development of new therapeutics.
The scope of this work includes investigation of the interactions between the major plasma proteins and small drugs and metabolites. Retinoic acid and retinaldehyde have been selected as sensitive probes to study these interactions. Titration of HSA with RA over the range of ligand-to-protein ratio from 0.05 to 6 has been monitored by UV/Vis, circular dichroism and fluorescence. The results indicate that retinoic acid specifically binds HSA at least at 2 binding sites of different affinity. Non-covalent binding of retinaldehyde is not specific, but proceeds at higher extent (6 and above). Not only does this research allow characterization of a type of interactions between HSA and different retinoids, but it provides a valuable methodological approach for investigation of protein complexes with small exogenous ligands by exploring phenomena of protein-induced chirality and fluorescence quenching.
We suggest that this approach can be more extensively used for the investigation of the interactions between native plasma proteins, protein therapeutics, and small chemical drugs and/or native metabolites of plasma.
I. P. Leader, P. A. Jupp, P. Weatherhead, V. Huynh, D. Trew, C. H. Turner, T. Day, D. Wheatley, WYETH RESEARCH
Background - Finding an unexpected degradant in a developmental drug formulation is a critical occurrence that requires swift action to either identify or eliminate. Tocopherol succinate polyethylene glycol (TPGS) was used as a wetting surfactant in a developmental drug formulation to aid bioavailability of the active. Analysis of an aged product by HPLC found that at least one unexpected degradant was being formed.
Methods - A combination of reverse-phase HPLC, LC-MS and NMR were utilised to identify the unknown entity. Preparative HPLC was used to collect fractions of the entity; mass spectrometry and NMR were used to elucidate the structure of the entity. Once identified, an HPLC method was developed to quantify levels of both the precursor and degradant species. Further testing and literature searches as to the stability of TPGS showed that the degradation process was actually catalysed by the presence of the drug substance.
Results - The unknown degradant was conclusively identified as tocopherol succinate (TS). With the use of a reference material and a stability indicating method the levels of TPGS and TS were tracked over the stability programme. The most likely pathway of formation seems to be related to a carboxylate functionality present in the drug substance.
Conclusions - A combination of HPLC, MS and NMR have been used to identify and quantify an unknown degradant in a developmental drug formulation. The work also enabled an understanding of how to prevent the formation of the degradant via storage of the product at <5oC.
H. Li, P. J. Kijak, J. von Bredow, A. Chiesa, M. L. Smith, CVM, FDA, Laurel, MD
Background: The misuse of sulfadimethoxine (SDM) in cattle is considered to be one of the major causes of illegal drug residues in slaughtered animals. FDA's official tolerance of SDM in edible cattle tissues is 100 ppb.
Methods: We developed a quantitative method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assay sulfadimethoxine residue and its major metabolite, 4N-acetyl-sulfamethoxine (N-AcSDM), in bovine kidney, liver, plasma, and urine. It includes simple lab procedures to extract the target drug and metabolite residue from these matrices. An additional clean-up step is only necessary for kidney and liver samples. For these tissues, analysis can be performed on a very small sample size (e.g., about 100 mg kidney homogenate or cortex), which makes it possible to analyze laparoscopic samples from live animals.
Results: With appropriate choice of internal standard, the overall method is able to analyze SDM and N-AcSDM in a range of 4,000 to 10,000 fold with lower limit of quantitation (LLOQ) down to low ppb level. Validation of the method is being conducted.
Conclusions: This analytical method will provide quantitative data of SDM and N-AcSDM residue in bovine tissues with high sensitivity, specificity, and wide dynamic range. It will contribute to CVM's tissue-fluid drug residue correlation program. In addition, it will be used to evaluate the FAST test kit (Fast Antibiotic Screen Test) for detecting SDM in bovine kidney.B. S. Liao, T. T. Cain, PRL/SW, FDA, Irvine, CA
Background:
A method has been developed to separate and quantify putrescine, cadaverine, and histamine simultaneously for scombroid fish samples and fishery products by cation ion chromatography. This method is an alternative to the two official methods currently used to analyze for these three biogenic amines. Putrescine and cadaverine are analyzed by Gas Chromatography (AOAC Method 996.07) and histamine is analyzed by Fluorescence Spectroscopy (AOAC Method 977.13). This method eliminates the need for chemical derivatization and the sample preparation is quick and simple.
Methods:
Aqueous 5mM sulfuric acid mobile phase is used with a cation exchange column under isocratic condition at a flow rate 0.8 mL/min and a run time of 30 minutes. Fish samples are homogenized with 75% methanol/water, incubated at 60ºC for 15 minutes and filtered before injection.
Results:
The three biogenic amines are baseline separated regardless of scombroid fish matrix in the order of putrescine, cadaverine, and histamine, without the interferences traditionally seen for tuna with cation ion chromatography. Five different scombroid fish samples, tuna, mackerel, anchovy, mahi mahi and sardines, were spiked in duplicate with standard solutions of the three amines with recoveries within 80% - 120%. The correlation coefficients (r2) of the standard calibration curves for putrescine, cadaverine and histamine are all higher than 0.999. The lowest standard concentrations which were spiked into tuna fish matrix are 0.055 ppm for putrescine, 0.05 ppm for cadaverine, and 1.0 ppm for histamine with RSDs of 1.5%, 5.4%, and 4.7%, respectively.
Conclusion:
This method is simple, direct, effective, and is also environmentally friendly because no chemical derivatization or time-consuming sample preparation is necessary.
M. Lopez1 , M. Feldlaufer2 , P. Chu1 , 1CVM, FDA, 2ARS, USDA
Background: Many countries, including the United States, have banned the use of nitrofurans in food-producing animals due to the carcinogenicity and mutagenicity of these drugs and their metabolites. Furazolidone, furaltadone, nitrofurantoin, and nitrofurazone are among the most commonly misused nitrofurans. We present a quantitative and confirmatory LC/MS/MS method for the determination of these four nitrofurans as their metabolites in honey at a target level of 1.0 ppb.
Method: The assay begins with a solid-phase-extraction cleanup of the honey sample, followed by overnight acid hydrolysis and derivatization of the nitrofuran metabolites with 2‑nitrobenzaldehyde. After pH adjustment and liquid-liquid extraction, the extract is assayed by LC/MS/MS using electrospray ionization in the positive mode.
Results: The method has been validated using incurred honey and fortified honey at 0.5, 1.0, and 2.0 ppb. For all four metabolites, the reproducibility errors are < 8% and the accuracies > 95%. The details of the quantitative and confirmatory performance of the method for each of the individual nitrofuran metabolites are presented.
Conclusion: The method complies with CVM performance criteria for the analysis of veterinary drug residues in animal products and can, therefore, be used to monitor the presence of nitrofuran metabolites in honey which may result from the misuse of nitrofurans in bee colonies.
J. C. May1 , N. M. Etz1 , H. Wang1 , L. Rey2 , 1CBER, FDA, Rockville, MD, 2Conseiller Scientifique, Lausanne, Switzerland
Residual moisture specifications which are set by the US Food and Drug Administration must be met to ensure potency and stability of freeze-dried biological product throughout the product's licensed shelf life. Thermogravimetry, thermogravimetry/mass spectrometry, and Karl Fischer methodology and vapor pressure moisture measurements are used to provide moisture information for freeze-dried biological products. Residual moisture measurements are compared for a Pertussis Vaccine and other freeze-dried biological products. Residual moisture data for the freeze-dried cake and vapor pressure moisture determinations in the space above the freeze-dried cake in the final container are discussed in regard to stability. Residual moisture stability data are presented and results for biological products in glass vials with elastomeric container closures are studied and compared to freeze-dried products in flame-sealed glass ampoules. Near-infrared spectroscopy measurements in the OH combination bands region (near 1920 nm) for Pertussis Vaccine incubated at 22, 37 and 50o C indicate moisture migration within the flame-sealed glass ampoule.
S. E. McMullen, V. A. Vega, F. J. Schenck, SRL, Atlanta, GA
Aquaculture is rapidly becoming a prominent source of seafood especially for finfish such as salmon, catfish, basa, and tilapia. Currently, there are only 5 veterinary drugs approved in the USA for use in aquaculture. Unfortunately, these drugs do not address all the pests/diseases encountered in aquaculture. Unapproved drug use is widespread especially in third world countries where misinformation on US tolerances is common. Recently, many import fish samples were found to have residues of unapproved fluoroquinolones, specifically enrofloxacin and ciprofloxacin. This poster presents an improved extraction method that facilitates rapid determination (Liquid Chromatography/Fluorescence Detection) and confirmation (Liquid Chromatography/Tandem Mass Spectrometry) of four fluoroquinolone residues including enrofloxacin and ciprofloxacin. This methodology presents several improvements over existing methodology including simplified extraction, less solvent consumption, and a reduction in analysis time.J. C. Moore1 , J. J. Yin2 , L. Yu1 , 1Dept. of Nutrition and Food Science, University of Maryland, College Park, MD, 2FDA, CFSAN, College Park, MD
Growing evidence suggests the role of antioxidants in preventing chronic diseases by scavenging reactive oxygen species such as the highly reactive hydroxyl radical. This has created a need for simple, reliable, high-throughput, in-vitro methods to investigate the free radical scavenging capacities of dietary antioxidants. The objective of this study was to develop an optimized hydroxyl radical scavenging capacity (HOSC) assay to meet the needs of antioxidant research and development. A novel method was developed for micro-plate reader high-throughput analysis using fluorescein as a fluorescent probe and the Fe(III)/H2O2 system to create a steady flux of pure hydroxyl radicals at pH 7.4 verified by electron spin resonance (ESR) spin trapping studies. The method was tested for solvent system compatibility and evaluated for performance characteristics and found to have compatibility with aqueous and 50% acetone solvents, a linear range with trolox of 20-100 µM with a linear correlation of R2=0.99, sensitivity (slope) of 0.4, and a 4.06% relative standard deviation for reproducibility. The method also correlated well with the popular oxygen radical absorbance capacity (ORAC) assay (r = 0.954, P = 0.05). Three 50% acetone extracts from soft wheat grain, hard wheat bran, and cinnamon were tested with the method and found to have values of 38.78, 74.91, and 3009.85 trolox equivalents per gram respectively. This method may provide researchers in the food and human health fields a simple protocol to rapidly evaluate free radical scavenging capacity of pure antioxidative compounds and natural extracts in vitro against the very reactive hydroxyl radical.
H. F. Yancy, D. E. Farrell, J. D. Washington, C. M. Deaver, R. A. Frobish, CVM, FDA, Laurel, MD
The performance characteristics of two lateral-flow test kits, Neogen's Reveal for Ruminant in Feed™ test and Strategic Diagnostic's Feedchek™ test, designed to detect ruminant or terrestrial animal proteins in feeds, respectively and two ELISA test kits, ELISA Technologies MELISA-Tek™ test and Tepnel's Biokit for (Cooked) Species Identification™ test, designed to detect bovine proteins in animal feed, were evaluated. All kits were evaluated using acceptance criteria developed for selectivity, sensitivity, ruggedness, and specificity. A minimum detection level of 0.1% bovine meat and bone meal (BMBM) was required, consistent with the sensitivity of the analytical methods used by FDA The Reveal test met its' label claim of a 1% sensitivity rate but failed to detect feed fortified with 0.5% to 0.1% BMBM. The Feedchek™ test passed the sensitivity assessment, but failed the selectivity assessment. The sensitivity of the Reveal test was affected by the variations in trace mineral concentrations typically present in feed. The Feedchek™ test was not affected by trace mineral levels. The MELISA-Tek™ test failed the selectivity assessment using the acceptance criteria established by the manufacturer, but passed when a set point criteria was applied to those results. It also failed the sensitivity assessment, detecting samples fortified at the 2% level only. The Biokit™ test failed to detect a single BMBM fortified sample. These results indicate that none of the tests are adequate for routine regulatory use for detection of feeds with 0.1% ruminant proteins and/or identification of samples free of ruminant proteins.C. B. Nochetto, C. S. Cheely, C. Gieseker, R. Reimschuessel, M. C. Carson, CVM, FDA
Background: MS-222 (tricaine methanesulfonate) is an approved anesthetic drug for aquaculture in the U.S. It was approved for use as a handling aid with a 3 week withdrawal time. The drug is rapidly metabolized and excreted, therefore it was approved without a regulatory method. However there are suspicions that the drug is used to sedate fish during transport to slaughter. A regulatory method will enable monitoring for unsafe residues of this drug resulting from extra label use. We present a quantitative analysis method using liquid chromatography (LC) at a target level of 100 ppb for three different species, salmon, tilapia and catfish.
Method: The assay begins with an extraction with acetonitrile, followed by filtration and mixed mode cation exchange solid-phase extraction cleanup of the sample. The extracts are analyzed by reversed-phase LC with UV detection at 320 nm.
Results: The method was validated using incurred fish fillets, control fish fillets, and fish fillets fortified at half the target, target and twice the target level. For all species, Accuracy is > 80% and Precision is < 10%.
Conclusion: The method complies with CVM performance criteria for the analysis of veterinary drug residues and can also be a useful starting point for future studies on the metabolism and depletion of MS-222.
G. O. Noonan, T. H. Begley, G. W. Diachenko, CFSAN, FDA, College Park, MD
Monofluoroacetic acid (MFA) , also referred to as compound 1080 is a rodenticide commonly used in the US and New Zealand and a naturally occurring toxic component of poisonous plants found in Australia, South Africa, and India. The availability, stability and proximity of MFA to agricultural products could potentially lead to an accidental or intentional contamination of food. Therefore a screening and confirmatory method for the detection of MFA in foods was needed. The screening method developed, utilized rapid sample cleanup and liquid chromatography mass spectrometry for detection. This method showed a high degree of specificity, with limits of detection (LOD) of 4 ppb and limits of quantitation (LOQ) of 13 ppb. Spike recoveries were matrix dependent and varied from 81 to 110% with comparable recoveries at low (2 ppm) and high (20 ppm) spiking levels. Reproducibility tests at the low spiking levels, had RSD's of less than 5% for all matrices analyzed. None of the unfortified samples showed detectable levels of MFA.
The qualitative confirmatory method developed is conceptually different from the screening method, ensuring that both methods would not be subject to the same interferences. The method uses the formation of the hydrazide of MFA through derivatization with 2-nitrophenylhydrazine. This derivatization is well established for the detection of carboxylic acids, but this is the first application to the detection of MFA. The derivatization yield was matrix dependent, however the LOD (<1 ppb) was sufficiently sensitive to confirm the presence of MFA in all spiked matrices. Reproducibility tests at the low spiking levels, had RSD's of approximately 6% for all matrices analyzed.
M. Hamed, L. Mello, M. Osman, Bioanalytical R&D, Wyeth Research, Collegeville, PA
Background: HKI-272 is an inhibitor of the Her2/neu receptor tyrosine kinase, thus blocking cell proliferation, and is being developed as a potent, irreversible and selective Her2/neu inhibitor for the treatment of cancer. An LC/MS/MS method for quantification of HKI-272 in human plasma has been evaluated for specificity, precision, accuracy and stability.
Methods: The method utilized 250 mL of human plasma. Following addition of internal standard (d6-HKI-272), samples were processed by protein precipitation with acetonitrile. Following centrifugation, the liquid supernatant was separated and evaporated to dryness. The residue was reconstituted in 0.05 M ammonium acetate buffer: methanol: acetonitrile (3:2:5), transferred to autosampler vials and injected on the LC column. Samples were separated on reversed phase C18 column.
Results: Endogenous components in control human plasma did not interfere with measurement of HKI-272 or the internal standard (D6-HKI-272). The assay was linear over the range of 3.0 ng/mL to 250 ng/mL. The intra-day and inter-day precision (CV) and inaccuracy (bias) for HKI-272 were within 15%. HKI-272 was found to be stable in human plasma for 4 hours at room temperature and after three freeze/thaw cycles. The processed samples were found to be stable at least 32 hours at 4oC.
Conclusions: An LC/MS/MS method for the determination of HKI-272 in human plasma has been validated. Based on a 250 mL plasma sample, the linear range of the method was 3.0 to 250 ng/mL. The method was utilized in the analysis of HKI-272 in human plasma generated from clinical studies in cancer patients.
L. Mello, S. Mathews, M. Khan, M. Osman, Bioanalytical R&D, Wyeth Research, Collegeville, PA
Background: PSI-697 is an inhibitor of P-selectin, a platelet and endothelial adhesion molecule that is critical for platelet and leukocyte recruitment to sites of vascular inflammation and injury. Inhibition of P-selectin offers the advantage of blocking a key initial step in the cell adhesion cascade and thereby preventing subsequent downstream activation and signaling events involved in thrombosis and inflammatory disorders. An LC/MS/MS method for quantification of PSI-697 in human plasma has been evaluated for specificity, precision, accuracy and stability.
Methods: The method utilized 200 mL of human plasma. Following addition of internal standard (d6-PSI-697), samples were processed by protein precipitation with acetonitrile. Following centrifugation, the liquid supernatant was separated and evaporated to dryness. The residue was reconstituted in acetonitrile: HPLC water: triethylamine (50:50:0.1), transferred to autosampler vials and injected on the LC column. Samples were separated on reversed phase C8 column.
Results: Endogenous components in control human plasma did not interfere with measurement of PSI-697 or the internal standard. The assay was linear over the range of 4.0 ng/mL to 1000 ng/mL. The intra-day and inter-day precision (CV) and inaccuracy (bias) for PSI-697 were within 15%. PSI-697 was found to be stable in human plasma for 24 hours at room temperature and after three freeze/thaw cycles. The processed samples were found to be stable at least 96 hours at 4oC.
Conclusions: An LC/MS/MS method for the determination of PSI-697 in human plasma has been validated. The method was utilized in the analysis of PSI-697 in human plasma generated from clinical studies in healthy humans.
S. H. Atwell, P. A. White, S. Z. Wahab, United States Pharmacopeia
Background: Two formulae for glycopyrrolate oral suspensions were approved by the USP Compounding Pharmacy Expert Committee. A stability-indicating assay and appropriate beyond-use-date for both formulae were needed to develop compounded monographs. This study covers the development and validation of an HPLC stability-indicating method for sugar and sugar-free Glycopyrrolate Oral Suspension formulae.
Methods: Sugar and sugar-free suspensions were prepared from glycopyrrolate and Vehicles specified in NF. An HPLC method with UV detection at 222 nm was developed to quantitate the content of glycopyrrolate. An L1 column (Phenomenex Gemini C18, 25 cm x 4.6 mm, 5 µm) with 1.0 mL/min flow rate and 20-µL injection was used. The mobile phase consisted of a mixture of 0.1 M monobasic potassium phosphate, 0.1 M dibasic potassium phosphate, acetonitrile, and methanol (26:28:24:16,v/v/v/v).
Results: Glycopyrrolate peak area responses were linear from 0.019 to 0.0 57 mg/mL (r > 0.999). Recoveries of glycopyrrolate from accuracy preparations were not significantly different from theory. Recoveries ranged between 98.4% to 102.8%. The relative standard deviations for intermediate precision were less than 1.2%. Uniformity of glycopyrrolate in samples from both suspensions was less than or equal to 1.0% RSD for triplicate preparations from different locations of the suspensions.
Conclusion: The HPLC procedure was stability-indicating, specific, precise, accurate, linear, and suitable to monitor the stability of gylcopyrrolate in sugar and sugar-free oral suspensions.
A. Ashley, R. Maheswaran, J. L. Belsky, S. Z. Wahab, United States Pharmacopeia
Background: A stability-indicating assay and appropriate beyond-use-dates were needed for two formulas for compounded trimethoprim oral suspensions. This study covers the development and validation of an HPLC stability-indicating method for regular and sugar-free Trimethoprim Oral Suspension formulae.
Method: Trimethoprim Oral Suspensions of two different formulae were prepared from trimethoprim drug substance and Vehicles specified in NF. An HPLC method with UV detection at 271 nm was developed to quantitate the content of trimethoprim in the compounded preparations. A Phenomenex Luna C18(2), 4.6-mm x 100-mm, 5-mm column with 1.0 mL/min flow rate and a 25 mL injection were used. The mobile phase consisted of a mixture of 0.1% triethylamine in water, pH 5.7 and acetonitrile, (850:150, v/v).
Results: Trimethoprim peak area responses were linear from 70% to 130% of the nominal sample concentration. Recoveries of trimethoprim from accuracy preparations were not significantly different from theory. Uniformity of trimethoprim in samples from both suspensions was < 3.0% RSD for triplicate preparations from different locations of the suspensions.
Conclusion: The HPLC method was stability-indicating, specific, precise, accurate and linear and is suitable to monitor the stability of Trimethoprim Oral Suspension, Formula 1 (regular) and Formula 2 (sugar-free).
R. Maheswaran, P. A. White, S. Z. Wahab, United States Pharmacopeia
Background: Formulas for Flecainide Oral Suspensions were approved by the USP Compounding Pharmacy Expert Committee. Stability-indicating assays and appropriate beyond-use-dates were needed to develop these compounded monographs. This study covers the development and validation of an HPLC stability-indicating method for regular and sugar-free Flecainide Oral Suspension formulae.
Method: Oral suspensions were prepared from Flecainide Acetate tablets and Vehicles specified in NF. An HPLC method with UV detection at 298 nm was developed to quantitate the content of flecainide acetate in the compounded preparations. A Zorbax C8, 4.6-mm x 150-mm, 5-mm column with 1.0 mL/min flow rate and a 10-mL injection were used. The mobile phase is a mixture of water, acetonitrile, acetic acid and 1 N tetrabutylammonium hydroxide, (710:290:10:5, v/v/v/v) with an apparent pH of 5.8. The method was validated for both formulae (10.0 mg/mL strength).
Results: Peak area responses were linear from 70% to 140% of the nominal assay concentration of 1.0 mg/mL. Recoveries of flecainide acetate from accuracy preparations were not significantly different from theory. Uniformity of flecainide acetate in samples from both suspensions was < 3.0% RSD for triplicate preparations from different locations in the suspensions.
Conclusion: The HPLC method was proven to be stability-indicating, specific, precise, accurate and linear and suitable for its intended purpose.
E. Biba, L. R. Strauch, S. Z. Wahab, P. A. White, United States Pharmacopeia
Background: Ginger (Zingiber Officinale Roscoe), a well known dietary supplement, has been used in Asian, Indian, and Arabic herbal traditional medicine. Today it is readily available in many avdifferent forms: extracts, tinctures, capsules and oils. Powdered ginger root is the main ingredient of Ginger Capsules
Methods: Three commercial brands of Ginger Capsules were assayed by HPLC to determine the Ginger Capsules Content of gingerols, gingerdiones, and shogaols USP 29, 2333. Dissolution profiles were evaluated per Ginger Capsules Dissolution using the major gingerol component, 6-gingerol, as a marker.
Results: The total amount of actives, individual weight percentages, and the chromatographic profile of the alcohol soluble extractives were brand dependent. Each brand met the USP requirement of not less than 0.8% for the content of gingerols and gingerdiones. Only one brand had a label value for the content of gingerols. The dissolution profiles differed by brand. One brand which contained excipients failed the USP dissolution test.
Conclusions: The lack of labeling raises an issue for use because of reported dose-dependent side effects. The different dissolution profiles of the tested Ginger Capsules represent another concern for standardization.
G. Deng1 , A. Ashley1 , W. Brown1 , J. Eaton1 , M. Liddell1 , L. Kikwai-Mutua1 , R. Manning1 , J. Munoz1 , B. Ning1 , P. Nithyanandan1 , H. Rowe1 , S. Tan1 , S. Z. Wahab1 , W. Hauck2 , 1United States Pharmacopeia, Rockville, MD, 2Thomas Jefferson University, Philadelphia, PA
Background: USP Prednisone Tablets Lot P are disintegrating type tablets. Each tablet weights about 220 mg with a prednisone content of 10 mg. Dissolution process of prednisone tablets is sensitive to experimental parameters. This study developed an experimental protocol to generate data for statistical variance analysis to evaluate and identify the key dissolution variables.
Methods: Prednisone tablets were tested on five dissolution testers (α, β, γ, δ, ε) by at least four analysts (A, B, C, D, E, F) on each tester. Six to eight tablets were tested in each run. Six dissolution runs were performed by each analyst on each tester. Prednisone release percentages on USP apparatus 2 (rotating paddles) were determined at 30 minutes in deaerated water at 37°C.
Results: A total of 132 dissolution runs were performed with near 1000 prednisone tablets. The overall average prednisone release percentage was 48.3% with a range of 37.8% to 76.9%. Significant variances in prednisone release between testers were observed. On Tester α, the average prednisone release percentages were 53.7% to 61.2%, and the average RSD were 13.8% to 17.5%. On Tester γ, the corresponding ranges were 41.4% to 46.5%, with 3.8% to 4.9% RSD. Further studies by switching vessels on α and γ indicated that vessels were one of the major sources of the varibility.
Conclusions: Prednisone Tablets Lot P showed a broad range of prednisone release. Dissolution tester particularly dissolution vessels were one of the major source of dissolution variance. Variances from tablet, analyst, and analytical procedure were low.
G. Deng, W. Brown, B. Chow, J. Eaton, M. Glasgow, M. Goede, L. Kikwai-Mutua, R. Manning, L. Wang, S. Z. Wahab, United States Pharmacopeia
Background: The USP Prednisone Tablets Lot P were recently developed for USP dissolution Apparatus Suitability Test. The tablet has an average weight of 220 mg with a prednisone content of 10 mg. This work examined the key chemical and physical characteristics of the tablets that can contibute to dissolution variability of the tablets.
Methods: USP Prednisone Tablets Lot P have been examined for their appearance, assay, content uniformities, disintegration, weight variation, hardness and friability.
Results: USP Prednisone Tablets Lot P were round and white in color. There were no observable defects on any of the tablets tested. The average weight was 222.0 mg per tablet (n = 108, SD = ± 2.4). The average hardness was 64 Newtons. The weight loss was 0.04% in the friability test. The average disintegration time was 18 seconds. Five portions of a powder composite of twenty tablets were assayed to determine the prednisone content. The average prednisone content was 96.2% (n = 5, SD = ± 0.8) of the label claim. Thirty tablets were assayed individually to determine the content uniformity. The average content of prednisone was 9.7 mg per tablet (n = 30, SD = ± 0.2). Ten tablets were assayed individually to determine the content uniformity of the disintegrant. The average weight of disintegrant per tablet was 4.5 mg (n = 10, SD = ± 0.1).
Conclusions: Chemical and physical properties of the tablets were well maintained in the manufacture process. Low variations of less than 3% RSD were observed.
J. Eaton, G. Deng, W. Brown, R. Manning, S. Z. Wahab, United States Pharmacopeia
Background: USP Prednisone Tablets Lot P are disintegrating type tablets. This study examined the usefulness of prednisone tablets for performance testing using flow-through cell apparatus.
Methods: Both open system and closed system configurations were used at 3 different flow rates (8, 12, and 16 mL/min) using 22.6-mm cells. Sampling time points were 1, 2, 3, 4, 5, 10, 15, 20, 30 and 45 min. 1-mL samples were collected and analyzed by HPLC.
Results: For open system runs, data was reported as percent of label claim dissolved per minute. For each of the 3 flow rates, the highest percent dissolved per minute value occurred at either 1 minute or 2 minutes and decreased at each subsequent time point. At 30 minutes, the value was less than 1 percent dissolved per minute for each flow rate. An approximation of total percent dissolved yielded values of about 85% dissolved after 45 minutes at 8 mL/min and about 100% dissolved after 45 minutes at 12 and 16 mL/min. For closed system runs, data was reported as cumulative percent dissolved at each time point. At a flow rate of 8 mL/min, the cumulative percent dissolved was 52.9% at 15 minutes. At flow rates of 12 and 16 mL/min, the cumulative percent dissolved was 50.5% and 58.8%, respectively, at 10 minutes.
Conclusions: Prednisone tablets disintegrated very quickly, and the onset of dissolution appears to be immediate. It appears that the very beginning of the test could be critical in obtaining reproducible percentage released values.
J. L. Perry, M. P. Yurawecz, CFSAN, FDA, College Park, MD
Background: Total carbohydrate in foods is currently calculated by difference. The calculation involves subtracting the sum of the crude protein, total fat, moisture, and ash from the total weight of the food. Calculation of total carbohydrate by difference creates problems because of inclusion of non-carbohydrate compounds and a dependence on accurate measurements of other food components. Such problems demonstrate the need for more direct methods of analysis.
Method: Foods with various carbohydrate claims were analyzed to determine the level of sugars and /or sugar alcohols in each. The products were selected to test an improved sample cleanup procedure. The matrices varied from different types of bread and cereals to high fat products such as chocolate cookies, ranch and olive oil salad dressings. All samples were composited and extracted in eighty percent (80 %) methanol. Aliquots were withdrawn, dried, and reconstituted with hydroxylamine HCl in pyridine containing phenyl-B-D-glucoside as the internal standard. The oximes were converted to silyl derivatives with hexamethyldisilazane(HMDS) and trifluroacetic acid(TFA) treatment. The derivatives were then analyzed by gas chromatography (GC) using flame ionization detection.
Results: The analyzed concentrations of sugars and / or sugar alcohols agreed closely with amounts of these components declared on the products' labels. Spike recoveries ranged from 92-100 %. The limit of quantification was 10 ug/g.
Conclusions: The analytical procedures worked well for the foods analyzed. The GC runtime of 57 minutes is somewhat lengthy. Work is in progress to reduce overall analysis time.
B. R. Petigara, A. L. Scher, CFSAN, FDA, College Park, MD
Background: A reversed-phase high performance liquid chromatography (RP-HPLC) method was developed to determine parts-per-million levels of Sudan I, 1-(phenylazo)-2-naphthalenol, the unsulfonated subsidiary color in FD&C Yellow No. 6. The color additive used in food, drugs, and cosmetics is subject to FDA batch certification to ensure that each lot meets the published identity, specifications, and other requirements, including a limit of <10 ppm of Sudan I (21CFR 74.706).
Method: FD&C Yellow No. 6 is dissolved in water and methanol, and the filtered solution is directly analyzed by RP-HPLC using a 150 x 2.1 mm i.d. XTerra RP-18 column with gradient elution. Sudan I is identified by retention time and diode-array spectrum and is quantitated from the peak area at 485 nm. For simplicity, calibrations are performed in the absence of the dye matrix.
Results: Linear calibration curves (R = 0.9999) were obtained in both the presence and the absence of the dye. The limit of determination for Sudan I was 0.4 ppm, and the confidence interval was 10.1+ 0.1 ppm (99% confidence level) at the specification level for Sudan I. FD&C Yellow No. 6 batches from 17 different manufacturers were found to contain Sudan I at undetected levels (16 samples), 0.5-9.7 ppm (11 samples, including the pharmacology batch) and >10 ppm (2 samples).
Conclusions: This RP-HPLC method to determine Sudan I in FD&C Yellow No. 6 by direct injection is simple, fast, and reproducible. The use of a narrow-bore column reduces solvent waste. The results agree with those obtained using a more complex method that requires a preliminary extraction.
B. R. Petigara, A. L. Scher, CFSAN, FDA, College Park, MD
Background: A reversed-phase high performance liquid chromatography (RP-HPLC) method was developed to determine part-per-million to percent levels of Sudan I, 1-(phenylazo)-2-naphthalenol, the unsulfonated subsidiary color in D&C Orange No. 4.The color additive, used in externally applied drugs and cosmetics, is subject to FDA batch certification to ensure that each lot meets the published identity, specifications, and other requirements, including a limit of <3% of subsidiary colors (21CFR 74.1304).
Method: D&C Orange No. 4 is dissolved in water and methanol, and the filtered solution is directly analyzed by RP-HPLC using a 150 x 2.1 mm i.d. XTerra RP-18 column with gradient elution. Sudan I is identified by its retention time and diode-array spectrum and is quantitated using the peak area at 485 nm. For simplicity, calibrations are performed in the absence of the dye matrix.
Results: Linear calibration curves (R = 0.9998-0.9999) were obtained in both the presence and the absence of the dye matrix.The limit of determination for Sudan I was 0.0001%, and the confidence interval near the highest level found was 0.2 + 0.002% (99% confidence level).D&C Orange No. 4 batches from 8 different manufacturers were found to contain Sudan I at undetected levels (8 samples), 0.0005 to <0.005% (3 samples, including a pharmacology batch), 0.005 to <0.05% (9 samples), and 0.18% (1 sample).
Conclusions: This RP-HPLC method to determine Sudan I in D&C Orange No. 4 by direct injection is simple, fast, and reproducible. The use of a narrow-bore column reduces solvent waste.
L. Qi1 , Y. Ito2 , 1ONDQA, CDER, FDA, 2Center for Biochemistry and Biophysics, NHLBI, NIH
Background: Centrifugal Precipitation Chromatography has been introduced to replace the traditional differential ammonium sulfate protein precipitation. Enzymes, such as ketosteroid isomerase and hyaluronidase, have been purified using this method. The purpose of this pilot study is to demonstrate that immuno-affinity centrifugal precipitation chromatography is capable of isolating an antigen by exploiting antigen-antibody binding.
Methods and Results: The separation is initiated by filling the sample channel with antibody (rabbit anti-human serum albumin IgG) solution, followed by elution with 40% saturated ammonium sulfate (40% AS) to precipitate the antibody under centrifugal force field. Antigen (Human Serum Albumin) is introduced into the sample channel at a flow rate of 0.06 mL/min. After an excess antigen and other proteins are eluted out, a releasing agent (0.5 M Glycine, pH 10 in 40% AS) is introduced into the sample channel to harvest the isolated target antigen leaving the antibody inside the channel. The human serum albumin was isolated and identified by SDS-PAGE.
Conclusion: The present method does not require immobilizing the antibody onto a matrix, which is used by the conventional immuno-affinity chromatography. This method ensures full recovery of the antigen and antibody.L. Caputo1 , N. Barnoy1 , J. Kim1 , J. Park1 , K. Senthil1 , P. Stahl1 , P. Delmonte2 , 1U.Md., 2CFSAN, FDA, College Park, Md.
There has been a significant proliferation of dietary supplements in the marketplace in the last 10 years. This has occurred in part because ofincreased interest in alternative medicines and beliefs that use of supplements may help reduce health care costs. Ginseng is a popular supplement which is used as a general health tonic; it is believed to increase energy.Preparations of the dried root are the most common commercially available form. The primary active components are thought to be ginsenosides.The mechanisms of action of these and other componentsarenot completely understood.
Standards for 6 of the ginsenosidesare commercially available, as are analytical methods for their determination. We analysed the content of 6 ginsenosides in 10 ginseng products.Samples of each product were extracted using accelerated solvent extraction and analyzed by HPLC.Extraction conditions were optimized considering different extraction solvent compositions, temperature and pressure.HPLC-PDA was used for the analysis of extracts because of its applicability to routine analysis.
Data from the HPLC analysis will allow the calculation of concentrations of specific ginsenosides.A cost comparison analysis of the 10 products will be conducted to determine whether a correlation exists between costand ginsenoside content (e.g., do higher priced supplements provide more ginsenosides than lower priced products?).Comparisons will utilize information provided on the product labels.Label information will also be used with the analytical data to calculate the amounts of ginsenosides that might be ingested if a consumer followed label directions for use.
J. C. Reepmeyer, J. T. Woodruff, Division of Pharmaceutical Analysis, CDER, FDA, St. Louis, MO
Background: An herbal dietary supplement, marketed as a natural product for the enhancement of sexual function, was purchased covertly over the internet.Preliminary analysis suggested that the product contained a compound related to synthetic phosphodiesterase 5 (PDE-5) inhibitors.
Methods:Standards of the three FDA-approved erectile dysfunction drugs (sildenafil, vardenafil and tadalafil) and an extract of the herbal product were analyzed by liquid chromatography on a reversed-phase C18 stationary phase using a photodiode array detector and a single quadrapole mass spectrometer in tandem and by MSn ion trap mass spectrometry. Sildenafil, vardenafil, and the herbal component were subjected to hydrolysis in order to cleave the sulfonamide bond, splitting the molecule into two pieces, which were analyzed by LC-MS and GC-MS.Results: The unknown compound generated a pseudomolecular ion via electrospray positive ion mode MS at 460 Da.Its UV spectrum was practically superimposable with that of vardenafil.Sildenafil, vardenafil and the unknown each generate a mass spectral product ion with a common mass accompanied by loss of dissimilar neutral fragments. The structure of the compound was tentatively identified as a designer drug of vardenafil in which the N-ethylpiperazine ring had been replaced by a piperidine ring.This structure was unambiguously confirmed by analysis of the hydrolysis products of the unknown ("piperidenafil"), sildenafil and vardenafil.
Conclusion: A component of an herbal product, marketed as a dietary supplement to enhance sexual function, was found to contain a vardenafil designer drug. The dosage level was estimated at 41 mg per capsule.
A. Grinev1 , S. Daniel1 , M. Laassri2 , K. Chumakov2 , V. E. Chizhikov2 , I. K. Hewlett1 , M. Rios1 , 1LMV/DETTD/OBRR/CBER/FDA, Rockville, MD, 2OVRR/CBER/FDA, Rockville, MD
BACKGROUND: Since the first outbreak of West Nile Virus (WNV) in the US in 1999, and in the six subsequent years. To date 7 subsequent epidemics have occurred causing more than 19,000 known human cases, and at least 750 human deaths. WNV is primarily transmitted by mosquito bites, but in 2002 additional modes of transmission were identified, including human-to-human by blood transfusion, breast-feeding, transplacental and by organ transplants. WNV has been a great public health concern and as an attempt to prevent viral spread by blood transfusion, screening for WNV by NAT was implemented under FDA approved INDs in July 2003. The reoccurrence of epidemics could suggest viral adaptation through mutations in the viral genome. In addition, genetic mutations can potentially interfere with the performance of diagnostic and screening assays, viral pathogenesis and potential therapeutic approaches. Therefore, the investigation, identification and detection of variants that may appear in the course of WNV outbreaks are extremely important and can only be achieved by the genomic characterization of new WNV isolates in a timely fashion. METHODS: We have developed a microarray-based assay that can rapidly detect mutations in the WNV structural region (5'NCR, Core, preM, M and Env). Glass slides containing 263 covalently immobilized oligoprobes were produced. The oligoprobes were designed and synthesized based on the reference strain NY99-flamingo 382-99 (AF196835). This new microarray-based-assay was standardized using the reference isolate. WNV amplified products containing the T7 promoter were used to generate fluorescently labeled Cy3 WNV-RNA for hybridization with the microarray, as described in Chizhikov V., et al., J Clin Microbiol 2002; 40:2398-2407. Microchip images were taken using the fluorescent scanner ScanArray 5000 and analyzed using the ScanExpress software. RESULTS: Assay validation was performed with 18 previously sequenced WNV isolates obtained from the last four US epidemics 2002 - 2005. The microarray assay detected unambiguously all mutations previously identified in the structural region of each one of the isolates by traditional sequencing analysis. The assay was sensitive and specific for all isolates tested. CONCLUSION: We have developed a microarray-based assay capable of rapid and simultaneous detection of multiple mutations in a large genomic segment of WNV. The assay is far more suitable for the high throughput analysis needed in large epidemiological studies than other methods (e.g. RFLP or DNA sequencing). We are in the process of expanding the microarray-based assay to cover the entire WNV genome.
J.A. Roach, CFSAN, FDA, College Park, MD
Background
Usnic acid is unambiguously confirmed by tandem mass spectrometry (MS/MS) in tumbleweed shield lichen, Xanthoparmelia chlorochroa, shown to have caused a mass die-off of at least 328 elk within the Wyoming Red Rim wildlife management area in the spring of 2004. The lichen contains 2% usnic acid by liquid chromatography with UV quantification at 282 nm.
Methods
Grind lichen to pass 40 mesh screen. Extract between 20 and 400 mg lichen powder with 2 x 8 mL acetonitrile by vigorous shaking for 20 min. Spin filter to remove solids. Dilute extract to 100 mL with acetonitrile. Mix 100 µL extract portions with aliquots of standard usnic acid solution making total test portion volume 1 mL with acetonitrile. Quantify by method of standard additions by UV at 282 nm. Confirm usnic acid by comparison of MRM data for test portions with MRM data for usnic acid standard.
CAUTION: Usnic acid is a strong hepatotoxin associated with human liver failure.Acetonitrile is a contact and inhalation hazard.
Results
Quantifications by UV and electrospray ionization (ESI) MS/MS disagree.The respective chromatographic data show that usnic acid is the principal compound detected in the extracts by both techniques.The quantification disparity in the data decreases when the extracts are diluted into the linear operating range of the ESI-MS/MS. However, the MS/MS data do not provide a means to cull MS/MS data recorded outside the linear range of the instrument.In contrast, the recorded diode array UV spectra can be used to exclude saturated data points from the UV quantification data.
Conclusions
Quantification by mass spectrometry has its limitations. Quantification by UV with confirmation by mass spectrometry is a more reliable assay for usnic acid in lichen.
W. D. Rowe1 , M. R. Madson1 , J. N. Sofos1 , F. J. Schenck2 , V. A. Vega2 , L. H. Lagman3 , D. M. Altwein4 , 1FDA, Denver, CO, 2FDA, Atlanta, GA, 3FDA, Jefferson, AR, 4FDA, Bothell, WA
Background: FDA uses Laboratory Information Bulletin (LIB) #3461 to monitor for ivermectin (IVR), a potent antiparasitic agent, in raw milk.Lagman, et al. demonstrated in LIB #4155 a faster, more sensitive method for IVR and for three related compounds, doramectin (DOR), abamectin (ABA), and moxidectin (MOX). All four may be administered to cattle.
Method: A modified version of LIB #4155 was validated by a two-laboratory method trial with additional validation by the Denver lab. Method extracts the avermectins from milk with ammonia, ethanol, ethyl acetate and iso-octane. The ethyl acetate - iso-octane layer is evaporated to an oily residue.The oily residue is partitioned between hexane and acetonitrile and the acetonitrile is evaporated to near dryness. The residue is dissolved in acetonitrile and derivatized with trifluoroacetic anhydride in the presence of N-methylimidazole.The derivatized extracts are injected on an LC (C-18 column, fluorescence detector).
Results: Average recoveries are typically greater than 60% with CV's less than 20%.MDL calculates to less than 0.1 ppb. Incurred milk samples were positive for the expected analytes at the expected ranges, 0.5 to 4 ppb.Ruggedness was demonstrated by varying extraction volume (± 20%), extending drying times, and substituting glassware with plasticware.
Conclusion: This method, published as LIB #4347 and LIB #4348, is faster than LIB #3461, sensitive to 0.2 ppb and rugged for the determination of MOX, ABA, DOR and IVR residues in milk.
J. M. Betz1 , L. G. Saldanha1 , M. J. Smith2 , B. J. Cañas2 , Y. Tokiwa2 , L. C. Sander3 , K. E. Sharpless3 , S. A. Wise3 , 1Office of Dietary Supplements, NIH, Bethesda, MD, 2CFSAN, College Park, MD, 3National Institute of Standards and Technology , Gaithersburg, MD
Background: Validated analytical methods and reference materials to ensure the identity, purity, quality, and strength of constituents in dietary supplements are essential.Researchers need them to characterize materials used in safety and efficacy studies and ensure that these materials are of sufficient quality that the studies can be reproduced.Regulators and industry need them in dealing with regulatory, safety, labeling, quality control, and manufacturing issues.In FY02, the U.S. Congress directed ODS to accelerate an ongoing methods validation process.The Dietary Supplements Methods and Reference Materials Program was created and through this program the funding of initiatives at FDA and NIST.
Program Administration: The program is stakeholder driven, with participation by government agencies, non-governmental organizations, academia, and the private sector.The analytical methods validation program is conducted through an Interagency Agreement with FDA to provide funding to AOAC International.The Certified Reference Materials (CRM) development program is through an Interagency Agreement with NIST.
Results: About 38 methods are in various stages of validation at AOAC. Methods for ephedra, ginkgo flavones, beta-carotene, and glucosamine are now available. Suites of Standard Reference Materials (SRM) for ephedra are now available. Suites of SRM for beta-carotene, bilberries, bitter orange, black cohosh, blueberries, cranberries, ginkgo, green tea, oils containing omega-3 fatty acids, saw palmetto, St. John's wort, tocopherol, and multivitamin/multiminerals are in preparation.
Saldanha LG, Betz JM, and Coates PM. Development of the Analytical Methods and Reference Materials Program for Dietary Supplements at the National Institutes of Health.Journal of AOAC INTERNATIONAL, 2004 87:162-65.
R.D. Satzger, FCC, ORA, FDA, Cincinnati, OH
The mission of the Forensic Chemistry Center (FCC) is to develop and maintain the capability to respond immediately to incidents involving adulterated products and illegal or counterfeit products. The threat of terrorist events has placed an even higher level of importance on the ability to rapidly develop approaches that can be used to screen products for the presence of highly toxic chemical adulterants. Similar approaches have been used to identify differences between counterfeit/adulterated products. Scientists at the FCC have developed a series of analytical screening procedures to detect priority chemicals at levels that would pose a significant health threat to the consumer. These procedures have been applied to selected foods and pharmaceuticals.F. J. Schenck, S. E. McMullen, Southeast Regional Laboratory, FDA, Atlanta, GA
Chlorothalonil is a widely used pesticide, which accounts for a large number of violative samples in our laboratory each year.Unfortunately, there are difficulties involved in the analysis of fruit and vegetable samples for chlorothalonil residues.One of the most effective solid phase extraction (SPE) sorbents for the cleanup of fruit and vegetable sample extracts is primary secondary amine (PSA). Using PSA SPE columns, which are used in many multiresidue methods (MRMs), will result in the degradation of chlorothalonil residues.Consequently, chlorothalonil residues in the sample may be destroyed during sample preparation. There is an MRM that uses a buffered acidic extraction, which will stabilize chlorothalonil so that a PSA SPE cleanup can be used. However, this results in a diminished cleanup resulting in chromatographic interferences and the need for greater instrument maintenance. This study showed that simply using binary solvent mixtures containing toluene during the PSA SPE cleanup will stabilize the chlorothalonil.It also showed that the chlorothalonil residues in sample extracts were stable if the extracts were in toluene, but they were very unstable if they were in acetone or acetonitrile.K. J. Shefcheck, J. H. Callahan, S. M. Musser, CFSAN, FDA, College Park, MD
Peanut allergies are a serious public health issue.Peanut-adulterated foods can potentially be very harmful to sensitive individuals.Testing for the presence of peanut allergens in foods is most commonly performed with enzyme-linked immunosorbent assay (ELISA) based analytical methods, but false positives due to cross reactivity are a concern with ELISA.Mass spectrometry has the sensitivity and specificity to be used as a confirmatory method for positive ELISA tests. However, quantitation is difficult using a mass spectrometer since many variables, such as ionization efficiency, can affect the response of the species in question.Isotopically labeled internal standards can reduce this problem since the isotopic standard should have the same response as the sample.We use tryptic digestion products of the peanut allergen Ara h 1 as markers for peanut.Trypsin can also be used to label synthetic peptide standards, using 18O water to make isotopically labeled internal standards.We propose to use standard peptides labeled with 18O to effectively quantitate the peanut allergens in food with mass spectrometry.T. L. Williams1 , R. Singh2 , H. C. Harbottle3 , D. Andrezjewski1 , D. N. Heller3 , R. D. Walker3 , S. M. Musser1 , 1CFSAN, FDA, College Park, MD 20740, 2CVM, FDA, Rockville, MD 20855, 3CVM, FDA, Rockville, MD 20708
Background: Salmonella enterica serotypes cause approximately 1.4 million illnesses each year, with 95% of these illnesses contracted through contamination of food.Given the impact of diarrheal disease in the US, it is imperative to develop effective intervention strategies to ensure the safety of consumers against foodborne diseases. In this study we have developed a method for generating intact protein profiles from the LC/ESI/MS chromatogram of Salmonella Newport, in order to determine unique protein biomarkers indicative of food animal origin.
Materials and Methods: Thirty Salmonella serotype Newport isolates were chosen to represent a variety of animal sources of infection and a variety of PFGE (Pulsed-Field Gel Electrophoresis) subtypes.These isolates were analyzed in a blinded fashion.The method and supporting software translates the chromatographic and multiply-charged protein information into one comprehensive mass versus intensity spectrum.After the data is converted, a number of software tools can be used to compare bacterial protein profiles and monitor changes in protein expression that may be linked to specific traits such as thermal tolerance and pathogenicity.When significant changes in protein expression profiles are identified, the proteins of interest can be purified, sequenced, and examined for alterations in primary sequence composition and/or posttranslational modifications.Differences in protein sequence translates to a difference in nucleotide sequence that can be used to make PCR primers for rapid diagnostic detection.
Results: Two unique proteins were found at a molecular weight of 11236 Da and 11222 Da which corresponded to avian sources of infection (turkey and chicken) and quadruped sources of infection (cattle and swine), respectively.Upon further comparison with PFGE, these unique proteins identified with specific PFGE clusters and did not uphold their identity as an avian or quadruped specific protein.
Conclusions: Identification of bacterial protein biomarkers is an important first step in controlling, identifying, and understanding the processes by which bacterial pathogens are transferred from animal hosts to humans. Data from this study could help FDA determine if the emergence of Salmonella in humans is due to the transfer of these pathogens from particular animal groups, or if they exhibit a weakly clonal population structure with no animal host specificity.
M. L. Smith, D. N. Heller, CVM-OR, FDA, Laurel, MD
Introduction
Aminoglycosides are broad spectrum antibiotics, a number of which are used as veterinary drugs and crop-protection agents. Our goal was to develop a simple feed extraction technique for aminoglycosides that is compatible with hydrophilic interaction liquid chromatography (HILIC) and mass spectrometry. The challenges lie in the fact that aminoglycosides are thermally labile, non-volatile, have strong hydrophilic characteristics, and some are strongly basic.
Methods
A mixture of aminoglycoside standard solutions prepared in water was used for initial chromatography testing. A novel zwitterionic HILIC column (ZIC) was used with a normal phase gradient for polar compounds. Multiple techniques to extract aminoglycosides from animal feeds were employed. Analysis was performed on a Finnigan LCQ Classic ion trap mass spectrometer with electrospray and atmospheric pressure chemical ionization.
Results
ZIC column performance degraded if mobile phase had > 5% acetonitrile. The more basic aminoglycosides bleed off the column rather than being sharp peaks. All aminoglycosides are retained on the ZIC column at neutral pH and low ionic strength (less than or equal to 1 mM ammonium formate). Using a combined pH and ionic strength gradient elutes the amonoglycosides successively with sharp peaks.
Conclusions
The ZIC HILIC column is an alternative to LC with counter ions, and keeps the LC/MS interface clean. Feed extraction methods are being developed that are compatible with ZIC HILIC.
Acknowledgements
This project was supported in part by an appointment to the Research Fellowship Program for the Center for Veterinary Medicine administered by the Oak Ridge Associated Universities through a contract with the U.S. Food and Drug Administration.
R. E. Smith, R. Luchtefeld, FDA
LC-MS has been used to analyze Illicium religiosum and I. verum, or Japanese and Chinese star anise. Hot aqueous extracts of powdered roots were analyzed, because the Chinese form is used to make a tea to treat infant colic. Positive ion atmospheric pressure chemical ionization (APCI) and UV detection were used to provide chromatograms that served as a type of fingerprint of each form of star anise. I. verum was spiked with 0.1-1
S. Satchithanandam1 , M. Ganzera2 , E. Grundel1 , K. D. White1 , J. I. Rader1 , 1CFSAN, FDA, College Park, MD, 2University of Innsbruck, Innrian 52, Austria
Background: Dietary supplements containing Blue Cohosh are available over the counter. Blue Cohosh is a perennial plant whose extracts have been promoted for their estrogen-like benefits, thought to be due to the presence of saponins. Limited information is available regarding its toxicity. Blue Cohosh contains alkaloids which may produce birth defects and neonatal heart failure. This study was conducted to determine the amount of 3 major alkaloids and 3 major saponins in dietary supplements containing Blue Cohosh.
Methods: Fifteen dietary supplement samples were analyzed. Samples of authenticated rhizomes and root cut were powdered. Powdered samples and contents of capsules were extracted in 100% methanol overnight and centrifuged. The supernatants and liquid extracts of roots were diluted 1:10 in methanol and filtered. All samples were analyzed by HPLC with a gradient mobile phase of 10mM ammonium acetate buffer (pH 8.0) and acetonitrile using a Synergi Max-RP 80A column. Alkaloids were monitored with a PDA detector at 310 nm and saponins were monitored with an ELSD.
Results: The alkaloid peaks obtained from HPLC analysis were identified by LC/MS as magnoflorine, N-methylcytisine, and baptifoline and the three saponins were similar to those reported by Ganzera et al. (2003) Phytochem. Anal.14: 1-7, and confirmed by LC/MS.
Conclusions: Total alkaloids and saponins that might be ingested will vary with the form (e.g. roots, capsules, liquid extracts, etc.) and intakes recommended in the labeling. Intakes, based on label information, may range from 0.42 to 75 mg/day for alkaloids and from 2.5 to 434 mg/day for saponins.
P. R. Sundaresan1 , E. Grundel1 , K. D. White1 , E. Mazzola2 , J. I. Rader1 , 1CFSAN, FDA, College Park, MD, 2CFSAN, FDA & Univ. MD, MD
Plants of the Teucrium genus are rich in neo-clerodane diterpenoids. Germander (T. chamaedrys, T.canadense), for example, has been used in traditional folk medicine for its stimulant, diuretic, antipyretic and antiseptic properties. The furano neo-clerodane diterpenoid, Teucrin A (TC-1) present in T. chamaedrys has been implicated in its in vivo hepatotoxicity. Methanol extracts of authenticated powdered plant material were prepared and analyzed by analytical HPLC. Dihydroteugin (TC-2), teuflin (TC-5), teuflidin (TC-7) and teucvidin (TC-6) were tentatively identified in the plant extracts by LC/MS and 1H NMR experiments. A solid-phase-extraction method was adopted using styrene divinyl- benzene sorbent for the separation of TC-1, TC-5 and TC-6 from chlorophyll and phenylpropanoid glycosides. Semi-preparative HPLC yielded TC-1, TC-5 and TC-6 in milligram quantities. One- and two-dimensional NMR experiments have provided conclusive evidence for the existence of the diterpenoid compounds TC-1, TC-5 and TC-6. All contain the distinctive teucrin furan system with proton NMR signals at ca, 7.5 and 6.4 ppm. In addition, the 1H NMR spectra of isolated compounds compare closely with those of authentic standards and /or literature values. The analytical HPLC method for the isolation of selected diterpenoids is simple and sensitive. The method can be used to quantitate major diterpenoids in Teucrium and related plant extracts.
J. X. Tang, E. Vidunas, J. Moran, P. Desai, M. Ruppen, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965
CMD-193 belongs to a class of therapy known as antibody-targeted chemotherapy. Knowledge of calicheamicin conjugation sites and loading distribution will aid the development of CMD-193 and other calicheamicin based immunoconjugates.
For identifications of conjugation sites, the conjugated antibodies were denatured, reduced and alkylated and then digested with trypsin and the digests were subject to LC-ESI-MS, LC-ESI-MS/MS analyses. For determination of calicheamicin loading distribution, intact conjugate and Ab chains were prepared by desalting followed by reduction with dithioltheritol. The samples were immediately analyzed by ESI-MS.
The tryptic digests of CMD-193 and the unconjugated antibody G193 were analyzed by LC-ESI-MS. By comparing the mass spectra of the tryptic digest of CMD-193 with that of the antibody in the same retention time range, the unique mass peak(s) containing the conjugation sites in the CMD-193 digest were identified. The unique peptide ions were then selected for sequence analysis and conjugation site identification by LC-MS/MS. Our preliminary results indicated that the calicheamicin molecule is primarily distributed among 4 primary conjugation sites in CMD-193, as in CMA-676.
MS based methods may provide a more insightful and accurate measure of the calicheamicin loading and distribution. By comparing the mass spectrum of the conjugated heavy chain with that of the unconjugated heavy chain, the distribution of calicheamicin was readily obtained.
Calicheamicin loading and distribution in conjugates can be determined using MS based methods, which may provide substantial improvement to the development and production processes of CMD-193, and other calicheamicin based immunoconjugates in development.
J. X. Tang, R. Tsao, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965
LC-MS is a very powerful technique for analyzing impurities and degradation products. ESI and APCI are the two ion sources commonly used for LC-MS analyses. However, LC-MS sensitivity is very low for low polar pharmaceutical molecules such as steroids and LC-MS has no sensitivity for small volatile organic molecules such as those leaching from the pharmaceutical packaging material. Recent development in atmospheric pressure photo ionization (APPI) technique allows high sensitivity LC-MS analyses for both low polar pharmaceutical molecules such as steroids and for the small volatile organic molecules that are not feasible by LC-MS using ESI or APCI source. We have achieved high sensitivity in LC-APPI-MS analyses of low-level steroidal impurities and degradation products as well as small volatile organic molecules.
In this investigation, we have found that mass spectra obtained with LC-APPI-MS usually exhibit extensive fragmentation as commonly seen in EI mass spectra, although the fragmentation pattern may differ from that of an EI mass spectrum of the same compound. Due to the extensive fragmentation observed in LC-APPI-MS, molecular ion signals of some compounds may be very weak or not even detected in their LC-APPI mass spectra, while they are readily observed in their EI mass spectra. LC-APPI mass spectra of the steroidal and the small volatile organic compounds obtained with LC-APPI-MS using PhotoSprayTM ion source will be compared to EI mass spectra. The instrumental parameters that may affect the degree and characteristic of fragmentation observed in LC-APPI-MS and its utility for structural analysis will be discussed.
S. Tefera, S. Ehling, S. J. Yoo, I. P. Ho, Food Products Association (FPA), Washington, DC
Background: High levels of histamine can be present in certain types of fish due to the decarboxylation of histidine by the bacterial enzyme histidine decarboxylase, resulting in consumer illness. Most methods of analysis of histamine reported to date are quite laborious and involve a derivatization step. Here we report an improved method of analysis for histamine in fish by High Pressure Liquid Chromatography with a Diode Array Detector (HPLC-DAD), without derivatization.
Methods: The mobile phase used for HPLC analysis was also used as extraction solvent (phosphate buffer - methanol - acetonitrile). Two C18 Prodigy HPLC columns (250 mm x 4.6 mm) connected in series were used for analysis at 30°C and 0.5 mL/min. Detection was performed with a DAD detector at 214 nm.
Results: The method eliminates the histamine retention time shift because the pH of the sample and that of the mobile phase are identical. The use of two columns connected in series allows for an interference-free separation of histamine. The method is simple and robust, and does not involve a derivatization step. The limit of detection and limit of quantification based on a signal-to-noise ratio of 3:1 and 10:1 are 1.6 ppm and 5 ppm, respectively, in fish (a guidance level of 50 ppm has been set by the FDA). Spike recoveries of histamine from samples are between 85 - 105%.
Conclusion: An improved method of analysis for histamine in fish by HPLC-DAD with direct detection was developed and validated.
* Author to whom correspondence should be addressed.E-mail: IHo@FPA-Food.Org
E. Yanik1 , L. Lenz1 , C. M. Weaver2 , C. J. Oles3 , M. W. Trucksess2 , 1University of Maryland, College Park, MD, 2CFSAN, FDA, College Park, MD, 3CFSAN, FDA,College Park, MD
Aflatoxins are mycotoxins, secondary metabolites produced mainly by Aspergillus flavus and A. parasiticus.There are four naturally occurring aflatoxins.Aflatoxin B1 is the most toxic and carcinogenic of the these structurally similar compounds.Ten different ginseng herbal supplements were examined for aflatoxins contamination.Powdered ginseng in tablet form was weighed and ground with a mortar and pestle, and capsule of ground ginseng were emptied and the content weighed.Four replicates of each test sample and one test sample to which 2 ng/g aflatoxin B1 was added were analyzed as follows: Each test sample was extracted with 80% methanol by shaking for 30 min.The extract was centrifuged and diluted with water and then passed through an immunoaffinity column.Aflatoxin B1 was eluted from the column with acetonitrile.After evaporation, the residue was redissolved in 50% methanol.The toxin was separated and determined by reverse phase LC post column UV derivatization and fluorescence detection.
M. W. Trucksess1 , C. M. Weaver1 , C. J. Oles1 , J. I. Rader2 , 1CFSAN, FDA, College Park, MD, 2CFSAN, FDA,College Park, MD
Fumonisins are toxins produced mainly by the molds Fusarium moniliforme (F. verticillioides), F. proliferatum, and several other Fusarium species that grow on agricultural commodities in the field or during storage. More than ten types of fumonisins have been isolated and characterized. Fumonisin B1 (FB1), B2 (FB2), and B3 (FB3) are the major fumonisins produced. FB1 is the most prevalent and most toxic.Recently, fumonisins have been found in botanical roots.Fumonisins have produced liver damage and changes in the levels of certain classes of lipids, especially sphingolipids, in all animals studied.
Many analytical methods for determining fumonisins in foods have been published. Among the most common of these methods are liquid chromatographic (LC) separation with fluorescence detection, enzyme-linked immunosorbent assay (ELISA) and LC/mass spectrometry.In our laboratory, the botanical roots of ginseng, ginger, turmeric and kava-kava were extracted with a mixture of methanol and water, followed by cleanup on an immunoaffinity column. FB1 was then derivatized, separated and determined by LC with fluorescence detection. Recoveries of added FB1 to ginseng, ginger, turmeric, and kava-kava roots at levels ranging from 0.25 to 2 ìg/g were >85%.ELISA was also applied to screen these roots for FB1.
S. B. Turnipseed1 , C. M. Karbiwnyk1 , W. C. Andersen1 , S. B. Clark1 , K. E. Miller2 , 1ORA, FDA, Denver, CO, 2University of Denver, Denver, CO
Background: The use of LC-MS for multi-class multi-residue screening has been successfully applied to drug residues in eggs, shrimp and finfish. This work describes the initial method development for a similar procedure for whole milk. Such a method could greatly enhance the Agency's ability to monitor and regulate drug residues in milk. The residues of regulatory interest include ß-lactams, aminoglycosides, sulfonamides, tetracyclines, macrolides, fluoroquinolones, and avermectins.
Methods: An LC-MSn instrument using electrospray ionization with an ion trap detector was used. Reverse phased chromatography with a 0.1% formic acid/acetonitrile gradient separated the over 30 residues included in this assessment.Several simple methods of extracting the drug residues from milk were investigated including the use of ultrafiltration, and bonded (OASIS) solid phase extraction (SPE) after precipitation of milk proteins with acetonitrile.Residues were detected and identified with MS2 and/or MS3 product ion spectra.
Results: Initial results indicate that after concentration of the milk extracts with an OASIS SPE cartridge, LC-MSn allows for the detection and confirmation of many drug residues at levels of 100 ppb and below. Matrix suppression from milk components is a problem for some residues. Additional clean-up to reduce these contaminants and lower the detection to levels of regulatory interest (10 ppb for many drugs) will be ongoing.The aminoglycosides may require alternative extraction and separation methods including mixed mode SPEs and hydrophilic interaction chromatography.
Conclusion: This poster describes the development and optimization of extraction, chromatographic, and MS parameters for a multi-class multi-residue drug residue screen for whole milk.J. R. Urban, D. S. Jackson, US FDA, Forensic Chemistry Center, Cincinnati, OH
The presence of undeclared sulfites in dried foods is a major concern to those allergic to sulfites.Exposure to sulfites can cause headache and stomach cramps; but for an asthmatic, it can cause labored breathing, anaphylactic shock, and sometimes death.Sulfites are used in a number of foods as preservatives, antioxidants, antimicrobials, and color stabilizers.Regulations require foods containing sulfites at concentrations above 10 PPM (Parts Per Million) to be declared on the label.The official method for analyzing sulfite is the Monier-Williams procedure.The Monier-Williams procedure requires several hours to complete a single analysis and does not distinguish between sulfites and many other volatile sulfur-containing compounds.Cleanup can be labor intensive for samples that contain high levels of sugars; and the glassware is fragile and expensive.Ion chromatographic analysis is a technique that offers rapid detection and selectively of anions.Sulfite can be determined using anion exchange ion chromatography with conductivity detection in minutes rather than hours resulting in savings of both time and money.Dried fruit samples were analyzed using both methods to evaluate method comparability.
E. B. Asaju-Adjaye, P. J. Faustino, M. A. Tawakkul, D. A. Volpe, DPQR, CDER, FDA, Silver Spring, MD
Background: Gastrointestinal stability of venlafaxine was evaluated in vitro in simulated gastric (SGF) and intestinal (SIF) fluids using a stability indicating HPLC.
Methods: The method was validated for venlafaxine using a 5 μm Ascentis® C18 column (150' 4.6 mm) and mobile phase consisting of 30% acetonitrile in 20 mM potassium phosphate buffer (pH 6.5) delivered isocratically at a flow rate of 1 ml/min with UV detection at 228 nm.Venlafaxine was added to USP SGF and SIF fluids (0.4 mg/mL) and incubated in a 37°C shaking water bath. Gastric stability samples were assayed at 0, 15, 30 and 60 minutes with sampling for the intestinal stability study at 0, 1, 2 and 3 hours.
Results: System suitability determinations (n=18) gave mean values (%CV) of 1.67 (0.68%), 662.6 (0.5%), 1.23 (3.9%) and 37617( 11.7%) for k′, peak area and tailing factor, respectively.The method was shown to be accurate, precise, specific and linear over the analytical range of 1.0-10.5 µg/mL.Intra- and inter-day precision (CV) was <5%.Forced degradation studies of drug substance in acidic and basic media at 70ºC as well as in hydrogen peroxide for 1 hour did not produce any detectable degradation products. Venlafaxine was stable in SGF (pH ~1.2) and SIF (pH 6.8) with <1.5% relative concentration difference at the designated time points.
Conclusions: This stability experiment in simulated fluids suggests that drug loss in the gastrointestinal tract takes place by membrane permeation rather than a degradation process.
J. T. Piper, J. G. Vostal, CBER, FDA, Bethesda, MD
Clinical performance of a platelet product processed or stored under novel conditions is difficult to predict based on in vitro studies alone.Evaluation of such products involves determination of recovery and survival of radiolabeled platelets in human volunteers as a surrogate endpoint for platelet efficacy.Such human studies pose some risk to the volunteers, are a financial burden on the sponsor, and, in general, stifle innovation in the development of platelet products.The development of an animal model for evaluating the in vivo recovery and survival of human platelets has been limited by rapid, immune-mediated clearance of human cells.In the current studies, severe combined immunodeficient (SCID) mice were used to circumvent the need to block the reticuloendothelial system and prolong circulation of human cells.Human platelets were infused via the tail vein.Mouse whole blood was collected at various time points post-infusion, and human platelets were detected in mouse whole blood by flow cytometry using and anti-human CD41 monoclonal antibody conjugated to FITC.Human platelets were infused into normal and SCID mice, and the recoveries and survival times compared.The survival time in circulation was defined as the t1/2 of the human platelets.Recoveries and survival times were different between normal and SCID mice, with a maximal difference in recovery of 60.27% at 4 hours post-infusion (Control recovery, 11.09%; SCID recovery, 71.36%), and survival times of approximately 6.0 hours and 22.0 hours in normal and SCID mice, respectively.Chemically damaged and aged platelets were used to evaluate the ability of the model to detect differences in control and impaired platelets.Carbonyl 3-chlorophenylhydrazone (CCCP) is a mitochondrial uncoupler which mimics the platelet storage lesion.CCCP treatment of platelets resulted in a decreased agonist induced aggregation response (Control aggregation, 73.25%; CCCP treated platelet aggregation, 13.75%).Recovery of control and CCCP treated platelets were 31.5% and 7.91%, respectively, at 4-hours post-infusion.Although the CCCP treated platelets had lower recoveries, the survival times were not dramatically different (Control, 1.3 hours; CCCP treated platelets, 1.9 hours), and P-selectin expression was minimally increased (2.7% greater in CCCP treated platelets).In platelets stored for 7 days, in vitro platelet parameters were decreased.These changes in in vitro parameters were consistent with the development of the platelet storage lesion.Platelet counts decreased an average of 22.8% + 2.2% between Day 1 and Day 7.All platelet products had good swirl on Study Day 1, but by Study Day 7, none had visible swirl.Platelets stored for 7 days showed a decreased recovery over Day 1 platelets, with a difference of approximately 66.7% at both 2 and 4 hours post-infusion.
The SCID mouse may be a useful model for evaluating the impact of new technologies (apheresis devices, anticoagulants, storage containers, pathogen inactivation systems) on the in vivo efficacy of human platelets.In two different models of platelet damage (chemical and storage induced damage), this model can distinguish between normal and damaged platelets.
H. Wang, N. M. Etz, J. C. May, CBER, FDA, Rockville, MD
There is no doubt of the significant impact of vaccination on the health of the world's population over last 200 years. Vaccination has controlled major diseases such as smallpox, diphtheria, tetanus, yellow fever, pertussis, Haemophilus influenzae type B disease, poliomyelitis, measles, mumps, and rubella. Vaccinations against influenza, hepatitis A, hepatitis B, varicella, pneumococcal and meningococcal infections have made headway against those diseases, although much remains to be done. As more than a dozen countries grapple with newly reported cases of bird flu, influenza virus vaccine is one of the most important tools in the current HHS Pandemic Influenza Plan to be used against the disease.
In two CBER regulated influenza vaccines, the formaldehyde is used as an inactivating agent. Due to the potential carcinogenic effect of excess free formaldehyde on human beings, formaldehyde's content is specified as "no more than 200 ppm". The actual free formaldehyde levels in the two licensed influenza virus vaccines are near 10 ppm formaldehyde or lower. The Hantzsch method has been used to analyze the free formaldehyde for quality control purposes for decades. It is an outmoded method which involves a temperature-control chemical reaction before a time-dependant colorimetric measurement. Evidence has shown that the free formaldehyde level can be held below the limit of quantitation (LOQ) of the Hantzsch method (0.9ppm). Therefore, a new, more sensitive, and fast assay for the free formaldehyde analysis is obviously needed to accurately evaluate the vaccine quality. We have adapted an HPLC method which is under validation for the analysis of formaldehyde in Anthrax Vaccine Adsorbed. Results show that it is applicable to influenza virus vaccine because the method has much lower sensitivity. It also has advantages of using much less vaccine samples, and less human labor due to the use of an auto-sampler.This HPLC method is also found to be practical for analyzing other vaccine products containing free formaldehyde.
Y. Wang, CDER, FDA, Rockville, MD
Background: Different approximation methods for the marginal likelihood of nonlinear mixed effect models remain a mystery for many pharmacokinetic scientists due to its complex statistical and mathematical derivation. This project tends to visualize the core of these methods and clarify their differences with simple pictures.
Methods: A simple one-compartment model with intravenous bolus dosing was used to illustrate the difference among the three main approximation methods, Laplacian (LAP), first-order conditional estimation (FOCE) and first-order estimation (FO), in NONMEM, a commonly used tool in pharmaceutical industry for population pharmacokinetic and pharmacodynamic modeling. Between-subject random effect was assigned to the elimination rate constant (Ke) to demonstrate how the kernels for the approximation of the marginal likelihood looked like on one-dimension scale. The difference among the three methods was summarized both visually and numerically. The similar demonstration was performed on two-dimension scale when both Ke and volume of distribution (V) had between-subject random effects.
Results: The visualized results clearly showed the core difference among the three main estimation methods in NONMEM for models with different complexity. The results indicated that superiority rank of the three methods is generally consistent in terms of approximation accuracy to the true marginal likelihood (LAP>FOCE>FO) even though occasionally FO could be the closest to the true marginal likelihood during the search path.
Conclusion: The complex mathematical and statistical relationship among the different estimation methods in NONMEM was translated into simple visualized concepts.The visualized results can help pharmacokinetic scientists to easily understand the key difference among the different estimation methods in NONMEM without being puzzled by the stunning matrix algebra.C. R. Warner1 , G. O. Noonan1 , N. Sugimoto2 , A. Beisaw3 , W. Hsu3 , G. A. Perfetti1 , T. H. Begley1 , G. W. Diachenko1 , 1FDA, College Park, MD, 2National Institutes of Health Sciences, Tokyo, 3JIFSAN, University of Maryland, College Park, MD
A number of episodes of rapid-onset gastrointestinal illnesses associated with school lunches have been reported and, in the cases under consideration in this report, tortillas supplied by a single manufacturer were implicated.Extensive studies were performed in search of the causative agent(s).Analytical studies were carried out in other laboratories for bacterial toxins, heavy metals, pesticides and mycotoxins.As yet, these studies have not identified the etiologic agent.
Investigations of outbreak associated tortillas in this laboratory revealed unusually high levels of two food additives: bromate and propionate.Bromate levels equivalent to 1 to 3 ppm as potassium bromate were found.The amount of propionate, introduced as an antimycotic in the form of sodium or calcium propionate, ranged from 2.0 to 2.5% as equivalents of calcium propionate.These levels are substantially greater than the 0.13% to 0.6% found in non-outbreak tortillas; and the 0.3%, relative to the weight of flour that is standard practice in the baking industry.Alternatives to the excessive use of the salts of propionate should be investigated.
As the etiologic agent, neither propionate nor bromate was entirely consistent with the epidemiology of the outbreaks; therefore, work was continued with the Bioluminex® screening of the TLC chromatograms of tortilla extracts.This technique is effective for identifying compounds that are toxic to the bacteria on silica gel TLC plates.The Rf values of some interesting substances have been identified, and the extracts of these spots subjected to MS analysis.
S. Zhao1 , P. F. McDermott1 , S. Friedman1 , J. W. Abbott1 , S. L. Ayers1 , A. Glenn1 , E. Hall-Robinson1 , S. K. Hubert1 , H. C. Harbottle1 , R. D. Walker1 , T. M. Chiller2 , D. G. White1 , 1CVM, FDA, Rockville, MD, 2CDC, Atlanta, GA
Background: Salmonella isolates were recovered from a monthly sampling of chicken breasts, ground turkey, ground beef and pork chops purchased from selected grocery stores in 6 FoodNet sites (CT, GA, MD, MN, OR, and TE) in 2002 and 2 additional sites in 2003 (CA and NY).
Methods: In 2002 and 2003, a total of 6,046 retail meats were examined.MICs were determined and interpreted via CLSI broth micro-dilution methods and standards.All isolates were analyzed for genetic relatedness using PFGE patterns generated by digestion with Xba1 or Xba1 plus Bln1.
Results: Overall, six percent of (N=365/6,046) of retail meat samples were contaminated with Salmonella, the bulk recovered from either ground turkey (52%) or chicken breast (39%).S. Heidelberg was the predominant serotype identified (23%), followed by S. Saintpaul (12%), S. Typhimurium (11%), and S. Kentucky (10%).Overall, resistance was most often observed to tetracycline (40%), streptomycin (37%), ampicillin (26%), and sulfamethoxazole (25%).Twelve percent of isolates were resistant to cefoxitin and ceftiofur, though only 1 isolate was resistant to ceftriaxone.All isolates were susceptible to amikacin and ciprofloxacin, however, 3% of isolates were resistant to nalidixic acid and were almost exclusive to ground turkey samples (n=11/12). PFGE fingerprinting profiles showed that Salmonella, in general, were genetically diverse with a total of 175 Xba1 PFGE profiles generated from the 365 isolates. PFGE profiles showed good correlation with serotypes and in some instances, antimicrobial resistance profiles.
Conclusion: Results demonstrated a varied spectrum of antimicrobial resistance and PFGE patterns, including several multidrug resistant clones.
P. Whittaker1 , C. E. Keys1 , E. W. Brown1 , B. D. Tall2 , S. G. Edelson-Mammel1 , F. S. Fry1 , 1CFSAN, FDA, College Park, MD, 2CFSAN, FDA, Laurel, MD
Capillary gas chromatography with flame ionization detection (GC-FID) was used to determine the cellular fatty acid (CFA) profiles of 134 Enterobacter sakazakii strains which were compared to the CFA profiles of closely related Enterobacter and Citrobacter species. For GC-FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 37°C for 24 hours were obtained by saponification, methylation and extraction into hexane:methyl tert-butyl ether. A database for E. sakazakii was prepared using fatty acid profiles from the 134 strains. The CFA profiles for E. sakazakii and E. cloacae show that there are several fatty acids, 14:0, 17:0 ωcyclo 7-8, 18:1 ω7c and summed 16:1 ω7c/16:1 ω6c, that differ significantly between these two species. A principal component analysis (PCA) model based on CFA profiles for E. sakazakii strains clearly shows separation of E. sakazakii from closely related Enterobacter and Citrobacter species. Pulsed field gel electrophoresis (PFGE) was also performed on the E. sakazakii strains to examine strain to strain relatedness, but no correlation between the PFGE results and the fatty acid profiles was found. These results suggest that FAME analysis is not as sensitive as PFGE analysis at the strain level, but is a better and faster screening tool at the species level at which it discriminates between closely related species. Further possible correlations between groups from PFGE and FAME data sets will be explored. In summary, this analytical method provides a confirmatory procedure for the differentiation of E. sakazakii strains from closely related Enterobacter and Citrobacter species.
L. J. Wrisley, L. Zhang, Wyeth Research
Background: Quality by Design (QbD) continues to gain momentum in the pharmaceutical industry.These concepts, crucial to the implementation of flexible, real-time quality assured manufacturing processes, are easily adapted to analytical chemistry.
Methods: Sound, statistically based design of experiments (DOE) provides the basis for development of robust analytical methodology. Design Expert® software has been used to optimize complex methods (e.g. dissolution) where multiple parameters are simultaneously varied. Similarly, the software is used to design and evaluate results of experiments to assess method robustness efficiently. Additionally, the utility of DryLab® is demonstrated as an efficient tool in optimizing and evaluating robustness of chromatographic methods.
Results: Examples illustrating the utilization and clear benefits of multivariate optimization/evaluation tools are presented. DOE optimization of a dissolution method illustrates the benefits of understanding the design space constraints of the dissolution variables.Interaction effects, which cannot be evaluated using a univariate approach, are discovered using a 24 factorial experiment designed to evaluate the robustness of a sample extraction procedure. Finally, the effectiveness of using DryLab® multivariate chromatography optimization software is illustrated. For example, resolution maps provide a clear representation of a multi-dimensional design space and immediate feedback for method robustness.
Conclusions: Analytical chemists can embrace concepts embodied in QbD and gain clear benefits.An understanding of a method's design space and critical attributes is of tremendous value. Data quality and method performance are ensured through the design of effective and efficient methods.
W. H. Wu, C. R. Anderson, PRLNW/SPRC, ORA, FDA, Bothell, WA
Background: Discoloration of tuna product can be effectively prevented when carbon monoxide (CO) is bound to the Fe(II) binding site of myoglobin in tuna muscle. Commercial CO treatment of tuna and a few other finfish species is therefore an increasingly popular practice to improve product acceptance in the commercial market. In the US, tuna treated with CO should display labeling indicative of that process.
Methods: CO contents in tuna and mahi-mahi were measured by a GC/MS method developed in our laboratory.GC/MS analysis was completed by injecting 100 µL from headspace of a mixture of CO-distillated water, after bound CO was chemically released from fish tissue using sulfuric acid (5M).
Results: Using the method described in this study, indigenous CO contents of fresh Ahi tuna and mahi-mahi samples were found to be close to or less than 150 ng/g and 100 ng/g, respectively.Commercially CO-treated, vacuum-packaged tuna from multiple sources consistently showed CO level near or greater than 1 mg/g, while CO level in the only CO-treated frozen mahi-mahi sample was in the 500 ng/g range. Treated specimens appear distinguishable from untreated samples as the difference in CO content between these two categories was in the range of one order of magnitude.
Conclusions: Our findings suggest an easy quantitative and confirmative method for CO in fish using widely available instrumentation. This method may be useful for regulatory purpose, with proper validation, in determining whether a commercially available fishery product has been exposed to CO even if not labeled as such.
J. J. Yin1 , M. P. Yurawecz2 , J. K. Kramer3 , A. R. Eynard4 , L. Yu5 , 1CFSAN, FDA, College Park, MD, 2Retired, 3Agri-Food Canada, 4Universidad Nacional de Córdoba, Argentina, 5University of Maryland
Conjugated linoleic acids (CLA) are a group of octadecadienoic acid (18:2) which are naturally present in food products and may have health beneficial effects. This study was undertaken to determine and compare the high purity c9,t11-CLA, t10,c12-CLA, and LA for their effects on oxygen diffusion-concentration products and depletion in liposome suspensions and ethanol solutions of the soy, egg yolk, and rat brain and heart phospholipids using electron spin resonance spin-label oximetry method.Individual CLA isomers differed in their effects on oxygen diffusion-concentration products or lipid peroxidation in both aqueous liposome suspensions and ethanol solutions, and these effects are dependent on the nature of the testing lipid system and the origin of the phospholipids.This study also examined and compared c9, t11-CLA, t10, c12-CLA, and linoleic acid (LA) for their reactions with the stable DPPH radicals in ethanol solutions and liposomes.PC containing individual CLA isomers and LA differed in their capacities to react with and quench DPPH radicals in both ethanol solution and in liposome, suggesting that both fatty acid composition and lipid system may alter the estimation of DPPH radical-fatty acid interactions. In addition, the effects of the two CLA isomers on phase transition temperature of liposomes were investigated and compared to that of linoleic acid (18:2n-6).Incorporation of LA and CLA isomers into DMPC reduced the phase transition temperature from 23.6°C to 23.1° and 23.3°C, respectively. The results form this study may provide additional information to explain the potential mechanisms involved in the beneficial actions of CLA isomers.
S. J. Yoo, E. Kwok, I. P. Ho, Food Products Association (FPA), Washington, DC
The US Food and Drug Administration (US-FDA) requires the U.S. Food Industry to list trans fat contents in the nutrition labeling of products by Jan. 1, 2006. To assist the Food Industry in dealing with this regulatory requirement, the Food Product Association (FPA) in Washington, DC has been developing in-house capability in analyzing trans fat by gas chromatography (GC) with a flame ionization detector (FID).
Currently, there are two official methods, AOAC Method 996.06 and AOCS Ce 1h-05, available for the quantification of trans fats using GC-FID. Both methods include FAME derivatization and GC separation on a 100m polar capillary column (e.g. SP-2560).
It was found that correct peak identification of trans/cis FAME isomers is an important factor for accurate quantification. In particular, as noted in AOCS Ce 1h-05, some of the positional FAME isomers co-elute with other isomers depending on GC column temperature.
To improve the identification of trans FAMEs, we are building a FAME library of 61 FAMEs including 14 trans FAME isomers on the 100m SP-2560 column by GC-FID. Besides retention time, the library includes retention indices and mass spectra (MS), which provide additional information to resolve co-eluting peaks. In this presentation, challenges in the separation and identification of trans FAME isomers are discussed.
* Author to whom correspondence should be addressed.E-mail: IHo@FPA-Food.Org
R. M. Jacobs1 , P. T. Palmer2 , 1ORA,FDA, Alameda, CA, 2SFSU, San Francisco, CA
Background: Energy dispersive x-ray fluorescence (EDXRF) technology to detect and quantify toxic elements has dramatically improved in the last 25 years. Over the last half decade portable EDXRF (PEDXRF) devices have emerged. These devices have impressive capabilities. In addition to being fully portable hand-held devices, their on-board functions have enabled them to accomplish an assortment of applications. The design of these devices has been focused on the non-scientific user. Some devices include features such as identification algorithms, the ability to display emission spectra, and the ability to quantify elements. Some are able to determine many toxic elements in ppm concentrations in foods. An experiment was conducted to emulate the field examination of food using a PEDXRF device by an investigator. The objective was to test the device's capability to quickly identify acute levels of some toxic elements in foods that were ready to eat.
Methods: Six toxic elements (As, Cd, Cr, Pb, Hg, Tl) were added to two model foods (cranberry juice and mashed potatoes) at acute but sub-lethal levels. The toxic elements at 1x and 0.5x the acute dose were mixed in typical serving sizes (240 mL and 140 g, respectively) of these foods. To emulate true field conditions, the foods were placed in either HDPE plastic bags (potatoes) or in HDPP plastic centrifuges tubes (juice) for testing. A Niton Xli 728e equipped with dual radioactive sources (109Cd and 241Am) PEDXRF was used in "thin mode" to qualitatively identify these elements. This mode also yields a quantitative measure of the element expressed on a µg/cm2 basis. "Bulk mode" software that would allow data to be expressed on a basis of µg/g (ppm) was not loaded on this particular instrument.
Results: While three of the six elements (As, Pb, and Hg) were successfully identified in seconds by the on-board algorithms at both dose levels using the 109Cd isotopic source, Cr in juice was not identified initially by the algorithm in plastic tubes because its relatively low energy x-ray emissions. Its identification was also almost immediate when the juice was placed in a plastic bag and tested. Cadmium's identification required the use of the 241Am isotopic source. The identification of Tl required comparison of experimental spectra to on-board library spectra, as this particular analyzer did not have an identification algorithm for Tl, an option. Semi-quantitative responses (in µg/cm2) of these 6 elements at the 1x and 0.5x levels were proportional.
Conclusions: PEDXRF devices can be used to rapidly identify acutely toxic levels of some element in prepared foods at relatively dilute concentrations in common sample containers. More recent studies (see Palmer et al., Science Forum abstract, 2006) using this device equipped with "bulk mode" software, or more advanced devices equipped with miniature X-ray tubes as an excitation source, have demonstrated the detection of sub-acute levels of many elements far below the levels used here. Elements can not only be detected, but also be accurately quantified in foods at relatively low ppm concentrations. Since little sample preparation is required for most food samples, the XRF device is ideal field use. Moreover the simplicity of the design of many PEDXRF devices lend themselves to be used by non-scientists with minimal training. We anticipate that "positive" findings would require concomitant collection of samples for confirmatory laboratory analysis. PEDXRF and laboratory grade EDXRFs could fill very important niches in FDA's inspectional and laboratory capabilities in detecting toxic elements in foods and Asian patent medicines.
P. T. Palmer1 , R. M. Jacobs2 , K. Yamamoto3 , S. Webber3 , K. Ferguson3 , 1SF District Lab & SF State University, San Francisco, CA, 2SF District Lab, Alameda, CA, 3SF State University, San Francisco, CA
Background: While portable energy dispersive X-ray fluorescence spectrometry (PEDXRF) devices have been routinely used for the determination of lead in paint and elemental analysis of alloys and soils, their use to screen for toxic elements in food and medicines has been surprisingly limited to date.The goals of this study were to determine various analytical figures of merit for three different PEDXRF analyzers and to ascertain their suitability for rapid screening of toxic elements in support of a variety of FDA applications.
Methods: The products chosen for this study included liquid (cranberry juice), semi-solid (yogurt), and solid substances (chocolate).Samples of each product were fortified with up to four different toxic elements (As, Hg, Pb, and/or Se) to give known concentrations on a weight-weight basis.Samples were analyzed "as is" using Niton XLi 728, Niton XLt 793, and Innov-X Alpha PEDXRF analyzers using measurement times of 2 minutes or less.Results provided by the analyzer (elements detected and their concentrations are automatically computed by analyzer software with no additional calibration required) and raw XRF spectra (response versus energy in keV) were evaluated to compute the figures of merit.
Results: Selectivity for each target element was generally good with the exception of As, whose emission lines at 10.5 and 11.7 keV overlapped a Pb line at 10.6 keV and a Se line at 11.8 keV.Limits of detection (LODs) for both analyzers were in the low ppm range for all four target elements.Calibration curves were linear over three orders of magnitude, spanning concentrations from the LODs out to percent levels.Relative errors were typically less than 20%, which demonstrates remarkably good accuracy considering no external calibration procedures were employed. Precision was also quite good with percent relative standard deviations (%RSDs) of 5% or less.Perhaps the most impressive features of this method included minimal sample preparation requirements, short analysis times of 2 minutes or less, ease and simplicity of operation, and field portability of these devices.
Conclusions:For applications where the sensitivity of PEDXRF is sufficient, it offers a number of significant advantages including minimal sample preparation, multi-element detection down to low ppm levels, and estimated throughputs of approximately 60 samples per hour using a device that is hand-held and can be operated by a non-experts in the field.Collectively, these capabilities make PEDXRF a powerful tool for screening of toxic elements in food and medicinal products and rapidly responding to emergency situations that require rapid identification and quantitation of toxic elements.
L.H. Ali, G.A. Perfetti, and G.W. Diachenko, CFSAN, FDA
Coumarin is the chemical 1,2-benzopyrone, C9H6O2, which is found in tonka beans and in the essential oils of a large number of plants. In some countries, it is used as a flavoring agent in some foods and beverages, including vanilla extracts. Because coumarin has been shown to cause liver damage, the FDA prohibited the use of coumarin in food in 1954 (21 CFR part 189.130). An LC-UV method was developed to provide a rapid screening technique for determining coumarin in vanilla extracts.
Conditions for separation and detection of coumarin in vanilla extract were established. A mobile phase composition of 55% acetonitrile/45% water, at a flow rate of 1.0 mL with a detection wavelength of 275 nm, yielded the optimum conditions. The linearity of the detector response for coumarin was determined. Using coumarin standards ranging from 0.1005 to 2.01 µg/mL, regression analyses showed that detector response is linear over this range, with coefficient of determination of about 0.9986. A natural vanilla extract product was used in the recovery study. Test portions were fortified at 250, 500, and 1000 µg/g. The percent recovery was determined using external calibration. Average recoveries of 5 replicates at each level were 100.7, 106.2, and 104.2%, respectively, and the relative standard deviations (CV) were 6.20, 4.40, and 5.90%. Fourteen domestic and imported vanilla extract products purchased locally were analyzed by the method. No coumarin was detected. This method for determining coumarin in vanilla products will enable the Agency to enforce the regulation prohibiting the use of coumarin in food.
H. S. Sharma1 , S. F. Ali2 , 1University of Uppsala, Sweden, 2NCTR, FDA, Jefferson, AR
Background: Blood-brain barrier (BBB) maintain the fluid microenvironment of the brain and spinal cord strictly within a narrow limit (1). Thus, breakdown of the BBB permeability alters the brain fluid microenvironment resulting in abnormal brain function (2). Abnormal cell and tissue reaction occurs following BBB disruption leading to long-term consequences and brain damage depending on the magnitude, severity and duration of the barrier breakdown. Thus, the possibility that stress associated with morphine and amphetamine administration or withdrawal will influences the blood-brain barrier (BBB) and brain dysfunction was examined in a rodent model.
Methods: Repeated daily administration of morphine (10 mg/kg, i.p.) resulted in drug dependence in rats on the 6h day and onwards. Measurement of the BBB permeability to large molecule tracers normally bound to proteins, e.g., Evans blue albumin (EBA) and radioiodine ([131]-Iodine) did not show any leakage on the 12th day of drug-dependence. On the other hand, spontaneous withdrawal of morphine on day 1 resulted in profound stress symptoms.
Results: These symptoms were much more intense on the 2nd day of morphine withdrawal. Alterations in the BBB to protein tracers were seen in several regions of the brain. This increase in BBB to protein tracers was most pronounced on the 2nd day of morphine withdrawal. These rats also exhibited abnormal neuronal, glial and stress protein, the heat-shock protein 72 kD (HSP 72 kD) response. On the other hand, acute administration of methamphetamine (40 mg/kg, i.p.) in mice resulted in marked extravasation of endogenous serum protein as seen with increased expression of albumin immunohistochemistry.
Conclusion: These observations suggest that psychostimulants and associated stress are capable to influence the brain function, probably through modifying the BBB function.
S. F. Al-Khaldi1 , S. Courtney2 , M. E. Mossoba1 , T. S. Hammack1 , C. E. Keys1 , 1CFSAN,FDA, College Park, MD, 2JIFSAN,FDA, College Park, MD
Background: In order to design and confirm a method to identify virulence genes of Salmonella Typhimurium using DNA microarray, a protocol was developed to label the isolated bacterial DNA directly and to use PCR amplification of limited numbers of genes to confirm the hybridization signals.
Methods: A DNA Microarray chip of 71 virulence genes of S. Typhimurium was developed and evaluated using ten isolates. Each gene was represented by 65 bp oligonucleotide probes (oligoprobes) and immobilized on the surface of chemically modified slides. Whole DNA genomes were digested with Hinf1 and Sau3AI, labeled with a fluorescent tag of Cy3 and then hybridized. The presence of virulence genes in ten strains of S. Typhimurium was established by measuring a fluorescent signal above the background noise of the chip.PCR amplification of ten genes (orgA, ORF319, ttrB, rmbA, misL, spi4F, spi4H, spi4N, 16s rRNA, and purR ) of S. Typhimurium was used as a standard strategy to verify the confidence level of the DNA microarray chip.
Results: When DNA microarray hybridization data differed from PCR amplification, this resulted in a reduction in the confidence level of the chip. However, when the PCR data supported and confirmed the hybridization data, the rest of the microarray hybridized genes showed high signal intensities and reproducibility leading to a high confidence level of the chip.
Conclusions:Using PCR amplification of some of the genes in the DNA microarray chip to increase the confidence level of the chip was proven to be very reliable strategy.
J. R. Ammann1 , M. A. Mabud1 , E. A. Garber2 , 1Alcohol and Tobacco Tax and Trade Bureau, US Treasury Dept., Beltsville, MD 20705, 2CFSAN, FDA, College Park, MD 20740
Background:
Prior to bottling, many wines typically possess a suspension of particles usually comprised of polyphenols such as tannins.Since tannins are known to impart a bitter taste and are usually undesirable in the finished product, winemakers add substances to clarify or fine the wine before marketing.Although fining agents such as bentonite, agars, synthetic polymers and silicon dioxide are sometimes used by winemakers, proteins are frequently the agents of choice. Egg white is an example of a commonly used fining agent due to the affinity of egg protein for polyphenols at wine pH.The fining agent is removed from the wine before consumption; however, trace amounts of soluble egg protein may have a deleterious effect for individuals with allergies against eggs.To establish the safety of alcoholic beverages for the allergic consumer, we have evaluated methods to detect residual egg protein in wine.
Method:
The evaluation utilized commercially available ELISA-based kits including R-Biopharm's RIDASCREEN® Ei/Egg Protein, Tepnel Biosystems' Egg Assay Kit, and Neogen Corporation's Veratox® Quantitative Egg Allergen Test.A total of ten wines were obtained from the manufacturer and analyzed. Eight of the wines (red) were fined with egg products, two of the wines (one red, one white) were unfined.
Results:
A total of ten commercially available wines were used in the evaluation.Two of the wines were known to be unfined and used as a control.The controls were spiked with a known amount of Henningsen Foods spray-dried egg reference material (RM).Although all three kits detected egg protein in the spiked wine controls, each kit responded differently to the RM.For a 10 ppm spiked control, the R-Biopharm kit yielded a response of 15.5 ppm (155% recovery).The Neogen and Tepnel kits showed responses of 11.1 ppm (111% recovery) and 3.9 ppm (39% recovery), respectively.None of the kits detected egg protein in the wines known to be fined with eggs.The lower limit of quantitation of the kits ranged from 0.5 ppm-2.0 ppm.
Conclusions:
Our evaluation demonstrated that commercially available ELISA-based kits detect egg protein in control wine matrices spiked with spray-dried RM (10 ppm).None of the kits detected egg protein in the wines samples fined with eggs (<2 ppm).
U. S. Babu1 , D. W. Gaines1 , H. Lillehoj2 , R. B. Raybourne1 , 1FDA, Mod I, Laurel, MD, 2USDA, Beltsville, MD
Background: While there have been in vivo comparisons of mouse and chicken Salmonella experimental infection, no studies have specifically focused on comparing Salmonella enteritidis (SE) and Salmonella typhimurium (ST) interaction with mouse and chicken macrophages which are known to play a major role in eliminating these bacteria.Therefore, our objective was to compare the uptake and killing of Salmonella serovars by murine and avian macrophage cell lines.
Methods: We used Salmonella enterica serovars Enteritidis (SE338) and Typhimurium (SR11) for this study.Uptake of green fuorescent protein-labeled bacteria was measured using flow cytometry. Bacterial killing by macrophages was assessed by plating the sorted viable infected cells.Nitric oxide was assessed in macrophage supernatants using Griess reagent and superoxide was measured by superoxide dismutase (SOD) inhibitable reduction of ferricytochrome C. In experiments to determine the impact of recombinant chicken interferon-ã (rchIFN-ã) on bacterial killing and NO production, HD11 cells were incubated in the presence of a 1:50 v/v dilution of rchIFN-ã or control supernatant for 18 hours, followed by washing to remove IFN.
Results: Bacterial clearance was significantly better with J774A.1 compared with HD11 cells. HD11 cells produced significantly higher amounts of nitric oxide (NO) than J774A.1 cells upon infection with SE338 and SR11, whereas J774A.1 cells exhibited greater superoxide production with SR11. Treatment of HD11 cells with recombinant chicken interferon gamma in the absence of bacteria enhanced NO production but did not induce increased levels synergistically with bacteria. Interferon treatment did not influence phagocytosis or increase bacterial killing by HD11 cells.
R.W. Bennett, CFSAN, College Park, MD
Studies were conducted with an automated enzynme-linked immunosorbent (ELISA) based method, (Vidas®, SET-II), exhibiting an antibody capture which had undergone modification by removal of the Fc fragment on the antibody.Conversely, most ELISAs as well as other serological systems utilize the whole antibody which has not undergone modification for detection of the staphylococcal enterotoxins (SET).The use of the whole antibody has, on occasions, produced false-positive results due to non-toxin protein reactivity with antienterotoxins containing both the F1 ab and Fc fragments.In this study, the polyvalent (serotypes A-E) configured automated enzyme-linked fluorescent immunoassay (ELFA) containing the modified antibody (F1ab fragment) was compared to ELISAs (polyvalent configured manual and automated) which uses the whole antibody (F1ab + Fc fragments) to capture the proteins.The food matrix studied was raw liquid (mixed or separated) or dried eggs containing a non-toxin protein with an attraction to the staphylococcal antienterotoxins as determined in previous ELISA studies.Negative controls were eggs purchased in retail outlets not containing the non-toxin egg protein.Egg extracts were subjected to preassay treatment (e.g., heat, pH and CHCl3) to study the general characteristics of the non-toxin egg protein.The fate of the egg protein was monitored intermittently from 0 to 62 days.Prior to ELISA testing, the eggs were homogenized and extracts collected by centrifugation.Studies showed that regardless of the ELISA-based method used, (manual and/or automated), which employed the unmodified antibodies with both the F1ab + Fc fragments intact that positive (false positive) ELISA responses occurred with fertilized egg yolks and fertilized whole liquid or dried eggs.Conversely, when modified (F1ab) antibodies were used, the automated SET-II ELFA was negative.Similarly, when observations were conducted on the general characteristics of the protein, unmodified antibodies produced positive (false positive) responses while modified antibodies were unresponsive to the egg protein producing negative serological reactions.The use of modified antibody to the staphylococcal enterotoxins represents significant improvement in antibody quality thus ensuring a greater degree of specificity without sacrificing the sensitivity of ELISA-based methods for the detection of SET in foods.
S. M. Bodeis-Jones, D. G. White, T. NARMS Working Group, FDA, CVM, Office of Research, Laurel, MD
Background:Enterococci exhibiting high-level aminoglycoside resistance (HLAR, MIC > 500 µg/ml) have been reported as a cause of nosocomial infections in the U.S. and are usually mediated by aminoglycoside-modifying enzymes. Although earlier studies have reported the isolation of HLAR enterococci in food-producing animals, little information is available on the prevalence of HLAR enterococci among retail foods of animal origin.
Materials and Methods:HLAR enterococci (n=568) were previously isolated from meat samples purchased from grocery stores in 4 participating FoodNet states (GA, MD, OR, TN) via the NARMS retail meat program in 2002.The polymerase chain reaction (PCR) was utilized to examine each isolate for the presence of six different genes associated with aminoglycoside resistance in enterococci.
Results: The majority of HLAR enterococci were either E. faecium (n=338) or E. faecalis (n=214).The aac(6')-Ie-aph(2")-Ia (n=113) and aph(3')-IIIa genes (n=108) were the most common aminoglycoside resistance genes identified among recovered enterococci. The aac(6')-Ie-aph(2")-Ia gene was most often identified in E. faecalis isolates (n=102/113) and from ground turkey samples (n=69/113).Whereas, the aph(3')-IIIa gene was found in both E. faecalis (n=60) and E. faecium (n=45) isolates primarily from ground turkey samples (n=62/108).None of the isolates contained the aph(2")-Ic or aph(2")-Ib genes.
Conclusions:This study shows the presence of a reservoir of HLAR enterococci among retail foods of animal origin, predominately ground turkey.Additionally, the finding that many HLAR enterococci failed to generate a PCR product for any of the genes tested suggests the presence of new and unidentified aminoglycoside resistance genes.
D. Wray-Cahen1 , R. P. Brown1 , A. I. Steen2 , H. Baskar3 , M. E. Stratmeyer1 , 1MOD2, CDRH, FDA, Laurel, MD, 2ODE, CDRH, FDA, Rockville, MD, 3University of Maryland
Background: The FDA Critical Path Initiative calls for a "new product development toolkit" containing more predictive and clinically relevant animal models. Critically ill patients with endotoxemia (a common condition in the ICU) have an increased risk of developing adverse effects following exposure to drugs and toxic compounds, compared to healthy individuals. Our objective was to develop a predictive and clinically relevant animal model of endotoxemia that could be used for preclinical and post-market biological evaluations of medical devices used in critically ill patients, with a special focus on the pediatric population. Our target survival period was six hours or more.
Method: Intravenous (IV) administration of low doses (1 µg/ml) of lipopolysaccharide (LPS) to anesthetized (Isoflurane) juvenile pigs resulted in the rapid onset on hypotension, apnea, and death. We attempted to slow the development of symptom onset to create a more stable model by administering a moderate LPS dose (5 µg/kg) Intraperitoneally (ip). Vital signs were monitored for 8 hours. Venous blood was collected hourly for clinical chemistry and hematology; arterial blood was collected for blood gas analysis.
Results: The ip injection of 5 µg/kg of LPS to juvenile swine resulted in a ≥ 6 hr survival of treated animals with development of characteristic signs of endotoxemia, including febrile response, tachycardia, hypotension, hyperlactatemia, and hypoglycemia. The model was sufficiently stable to allow comparisons of responses to exogenous compounds.
Conclusion: A juvenile pig model of endotoxemia was developed that may be useful for toxicity testing of compounds released from medical device materials.
R. P. Brown1 , D. Wray-Cahen2 , A. D. Lucas1 , A. I. Steen3 , H. Baskar4 , M. E. Stratmeyer1 , 1CDRH, FDA, Silver Spring, MD, 2CDRH, FDA, Laurel, MD, 3CDRH, FDA, Rockville, MD, 4University of Maryland, College Park, MD
Background: Many medical devices are sterilized using ethylene oxide; patients can be exposed to its hydrolytic breakdown product, ethylene glycol (EG).Last year we presented feasibility work examining acute hemodynamic and hemolytic effects of intravenously (IV) administered EG.We examined effects of EG concentration and osmolarity in the present study.
Methods: Anesthetized pigs were instrumented with blood flow probes around renal and carotid arteries. EG (0, 12.5 to 125 mg/kg) was administered IV over 2 minutes at increasing EG concentrations (0, 2.5 to 100%).Blood was collected before injecting EG and at 5 minute intervals following injection. Plasma hemolysis (plasma free hemoglobin) and EG levels (using gas chromatography) were determined.Mean arterial pressure was monitored. In vitro hemolysis was determined following incubation of washed pig red blood cells with increasing concentrations of EG.
Results: Administration of EG at 125 mg/kg and at concentrations ≥5% produced hemodynamic changes (decreased blood pressure and flow).Renal blood flow was more sensitive than carotid.
Plasma concentration of EG for first recorded hemolysis and hemodynamic changes in vivo ranged from 651-2325 µg/ml (n=6).This was approximately 100-fold less than that required to produce equivalent effects in vitro.
Conclusions:Both concentration and dose may be important in responses to EG.The differential vascular response to EG may reflect greater risks to certain organs (kidneys). Caution should be exercised when using in vitro hemolysis assays to assess the potential for EG-induced intravascular hemolysis to occur in vivo due to the lower sensitivity of the in vitro assay.
R. P. Brown1 , A. D. Lucas1 , V. M. Hitchins1 , M. D. O'Hara1 , T. L. Murray2 , W. H. Cyr1 , M. E. Stratmeyer1 , S. F. Ali3 , M. Srivatasan4 , 1CDRH, FDA, Silver Spring, MD, 2CDRH, FDA, Laurel, MD, 3NCTR, FDA, Jefferson, AR, 4Arkansas State University, Little Rock, AR
Background: The proposed short-term Allowable Limit (AL) for residual ethylene oxide (EO) remaining on EO-sterilized medical devices is 4 mg/device, per the draft ISO 10993-7 standard.However, due to difficulties in achieving this AL for oxygenators used in cardiopulmonary bypass, some manufacturers have asked for an exemption from the proposed AL, seeking a 20 mg/device limit. As part of the safety evaluation of this proposed exemption, a battery of cytotoxicity studies was conducted on extracts from an EO-sterilized oxygenator.
Methods: An oxygenator was gas sterilized with EO; following aeration, the blood path was filled with RPMI-1640 containing 10% FBS. The device was extracted for 24 hours at 20°C.Extracts from a non-sterilized oxygenator, and from EO-sterilized and non-sterilized 4x4 gauze sponges, served as controls. The cytotoxicity of the extracts was examined in L929 fibroblasts, RAW 264.7 macrophages, mouse bone marrow cells, washed pig RBCs, Jurkat human lymphoma cells, and primary rat dorsal root ganglion (DRG) cells.Methods of determining cytotoxicity varied according to the cell type used. The concentration of EO in the extracts was determined using gas chromatography.
Results: No cytotoxic effects were seen in any of the established cell lines, the washed RBCs, or primary bone marrow cells incubated with the sterilized (143.05 µg EO/ml) or control (0.48 µg EO/ml) oxygenator extracts or the 4x4 extracts; however, exposure of DRG cells to the sterilized oxygenator extract resulted in morphological changes and changes in cell adhesiveness.
Conclusions: Extracts from EO-sterilized oxygenators may be neurotoxic in vitro.
H. Baskar1 , A. I. Steen2 , R. P. Brown3 , 1University of Maryland, College Park, MD, 2CDRH, FDA, Rockville, MD, 3CDRH, FDA, Silver Spring, MD
Background: Exposure to residual disinfectants remaining on reprocessed medical devices can result in adverse effects (e.g., hemolysis, burns) in patients being treated with these devices. A rapid, in vitro biological screening assay would be useful for detecting levels of residual disinfectants that may cause these adverse effects. A preliminary study was conducted to determine the cytotoxic effect of the enzymatic cleaner, SurgiSoak® and the disinfectant, Cavicide® on various cells lines. These data were compared to the ability of these agents to induce hemolysis in washed bovine RBCs.
Methods: L929 mouse fibroblast, RAW 264.7 mouse macrophage, and Jurkat human lymphoma cell lines (5 x 105 cells/ml) were incubated with increasing concentrations of SurgiSoak® and Cavicide® for 24 hours at 37°C. Cell survival was determined following staining with trypan blue and manual counting with a hemocytometer.Hemolysis studies were conducted by co-incubating SurgiSoak® and Cavicide® with 50 ul of washed bovine RBCs (Hct 30%) in Dulbecco's PBS (1 ml total vol) for 24 hours at 37°C. Percent hemolysis was determined by comparing absorbance (540 nm) of the supernatant from treated vs. negative (PBS/RBCs) and positive (water/RBCs) controls.
Results: The relative sensitivity of L929 fibroblast, RAW 264.7 macrophage, and Jurkat lymphoma cells lines and bovine RBCs to SurgiSoak® was comparable; however, bovine RBCs were about 3-fold less sensitive to the cytotoxic effects of Cavicide® than cultured cells.
Conclusions: Hemolysis may be an appropriate endpoint for a rapid, in vitro biological screening assay for some residual cleaners and disinfectants on reprocessed medical devices.
G. S. Bushar1 , R. Shively2 , S. C. Wood1 , M. M. Lightfoote1 , 1CDRH, FDA, Silver Spring, MD, 2CDRH, FDA, Rockville, MD
Introduction
"Strep Throat", a common childhood disease, is caused by bacteria from Streptococcus Group A.While the disease can be treated readily with antibiotics, when left untreated, it Qualitative Analysis of Sensitivity of Streptococcus Group A Rapid Antigen Detection could cause serious consequences.In the past, cultivated bacteria in a blood agar plate was the only method, but that assay could take 24 - 48 hours to develop.Currently, there are many Rapid Antigen Detection Test (RADT) kits on the market.The purpose of this project is to provide an objective analysis of the sensitivity of five test kits.
Material and Method
We chose three test kits with dip sticks and two with cassettes from different manufacturers.We purchased the same strain of Streptococcus Group A culture that all five test kits use as positive control as our common antigen.We followed the package inserts from each manufacture for the testing method.All measurements were read by BioDot strip reader.
Results
All five test kits have clear and easy to follow instructions.We had found that visual observation is about as sensitive as machine reading, and all five test kits have about the same sensitivity within three fold of each other in titration.
Future Study
We plan to compare these test kits using the purified carbohydrate antigen to get a more quantitative analysis of the sensitivity.
N. Cadieux, V. Dheenadhayalan, M. Parra, M. J. Brennan, CBER, FDA, Bethesda, MD
Mycobacterium tuberculosis and other pathogenic mycobacteria contain many related genes coding for the highly homologous PE and PE_PGRS proteins of unknown function.Since the non-pathogenic M. smegmatis contains none of those genes, it was used as a host to study the role of one of them, PE_PGRS33.Recombinant M. smegmatis strains containing the vector alone, the PE domain of PE_PGRS33 or the full length PE_PGRS33 were used to infect bone marrow macrophages (BMMO) and CFUs as well as cytokines released in the culture supernatant were measured at different time points.The strain expressing PE_PGRS33 persisted longer in BMMO and induced reduced levels of IL-12 and nitric oxide but greater levels of IL-10 when compared to the other two strains. This pattern of cytokine responses, induced by expression of a PE_PGRS protein, could modulate the usually effective Th1 cellular immune response against this pathogen and conversely, induce a Th2-type response.Interestingly, when given as a DNA vaccine in mice, PE_PGRS33 induced a Th2-type immune response characterized by low levels of IFNα, higher levels of IL-10 (upon stimulation of splenocytes with PE_PGRS33 protein) and a strong antibody response of the IgG1 subtype.Moreover, in the mouse aerosol model of TB infection, PE_PGRS33 was not protective, whereas DNA vaccines constructed from PE genes induced a Th1-type immune response and were protective against Mtb challenge.The data presented here suggests that expression of PE_PGRS genes may aid the pathogen resist an effective immune response by inducing responses that help it persist in the host.
A. Chandrasekaran, S. Ahmad, W. DeMaio, T. Hultin, R. Talaat, J. A. Scatina, Department of Biotransformation, Wyeth Research, Collegeville, PA
Background: Bazedoxifene (BZA) is a selective estrogen receptor modulator being developed for the prevention and treatment of postmenopausal osteoporosis.
Methods: Incubations of 50 µM [14C]BZA in human liver, kidney and duodenal microsomes and cDNA expressed human UDP-glucuronosyltranferases (UGTs) were conducted with NADPH or UDPGA. Metabolite profiles were determined by HPLC with radiochemical detection, and the metabolites were characterized by LC/MS.
Results: Little or no CYP mediated metabolism of BZA was evident. However, BZA was extensively metabolized via glucuronidation. There are two hydroxy groups (4' and 5) on BZA where glucuronidation can occur. Liver microsomes produced predominantly BZA-4'-glucuronide, in contrast to the major circulating metabolite, BZA-5-glucuronide. Kidney and duodenal microsomes formed both glucuronides. The rate of glucuronidation in liver for the 4'-glucuronide was about two-fold and ten-fold greater than in kidney and duodenum, respectively. However, kidney and duodenal microsomes had 5-times higher activity toward BZA-5-glucuronidation than liver microsomes. UGT1A1 and UGT1A10 were the most active isoforms involved in the glucuronidation of BZA. The turnover rate for glucuronidation at the 4' position was about 105 pmol/min/mg for both the isozymes, and 4 and 27 pmol/min/mg, respectively for BZA-5-glucuronidation.
Conclusions: Extrahepatic metabolism may play a significant role in the disposition of BZA in women, since intestinal and kidney microsomes produce the major circulating metabolite, 5-glucuronide at a higher rate than liver microsomes. UGT1A10 may contribute to a greater extent than other isoforms in the metabolism of BZA, since this isozyme produces the major in vivo metabolite and it is expressed in the intestine.
Y. X. Chen1 , W. Zhang2 , S. J. Knabel1 , 1The Pennsylvania State University, University Park, PA, 16801, 2Illinois Institute of Technology, Summit, Illinois, 60501
A multi-virulence-locus sequence typing (MVLST) strategy was recently developed and yielded higher discriminatory power than PFGE when applied to a set of genetically diverse isolates. To evaluate the epidemiologic relevance of MVLST, 85 isolates including 58 isolates from fourteen listeriosis outbreaks and 27 isolates not associated with outbreaks were analyzed. MVLST successfully identified 4 epidemic clones of L. monocytogenes with epidemic clone I, epidemic clone II and epidemic clone IV belonging to lineage I and epidemic clone III belonging to lineage II. A recent multistate listeriosis outbreak in U.S. in 2002 was identified as belonging to ECII. The three lineages identified by MVLST agreed with previously identified lineages and epidemic clones of L. monocytogenes. In addition, a new epidemic clone was identified, which we have termed ECIV. Twelve single nucleotide polymorphisms (SNPs) in 4 virulence genes identified all epidemic clones and outbreak strains. Virulence genes appear to be excellent molecular markers for investigating the epidemiology of L. monocytogenes.
V. M. Chenault1 , M. G. Hodges2 , C. P. Schnupp1 , P. W. Miles3 , V. B. Solberg3 , 1FDA, 2NIH, 3WRAIR
Background: Psammomys obesus is a common rodent in the Middle East. Sand rats (SRs) have been identified as the major reservoir host that transmits Leishmaniasis. Phlebotomine sand flies (SFs) bite humans or other vertebrates to obtain blood proteins for the production of eggs, a necessary step in the transmittal of viruses, bacteria, and protozoa to humans. The volatile outbreak of Leishmaniasis in US troops deployed in Afghanistan and Iraq has caused 28 days of costly treatment for patients. There are no preventive vaccines or prophylactic drugs and the approved healing device is just over 70% effective. Personal Protective Measures have not worked efficaciously. Spraying of insecticides has not controlled SFs. Further, SRs are selective eaters in the wild. Thus, novel adult, pupal and larval control methods must be investigated. These studies were conducted to determine effective endectocide treatment and control methods.
Methods: SRs were fed ivermectin and doramectin treated sand rat chow for 2 weeks. Blood, feces and hair samples were collected to measure the amount of the active ingredients. Body weights were measured to assess food palatability.
Results: The results suggest that endectocide treated food was palatable at levels presumably high enough to kill immature SFs.
Conclusions: Unique precision targeting of rodent reservoir hosts with insecticides applied topically (future studies), or used as endectocides (current study) will provide unprecedented investigations of parasite control. In subsequent studies, treated SRs will be anesthetized and challenged with SFs. Ultimately, therapeutic devices or drugs will be used to investigate treatments.
E. Cherkasova1 , E. Korotkova2 , V. Agol3 , K. M. Chumakov1 , 1CBER, FDA, Rockville, MD, 2A. N. Belozersky Institute of Physical-Chemical Biology, Moscow State University, Moscow, Russia, 3M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, Moscow, Russia
Attenuated strains of Oral Poliovirus Vaccine rapidly undergo genetic changes upon replication in human organism.The changes include point mutations and recombination between three serotypes and occasionally with other enteroviruses.The significance of recombination for viral replication and pathogenicity is not completely clear and requires further studies.To characterize intertypic recombinants we have developed a rapid microarray-based procedure (MAVR) that allowed us to map several dozens of intertypic recombinants between all three types of poliovirus.In addition, we have analyzed all recombinants for which the complete genomic structure was published.The majority of known recombinants belong to serotypes 2 and 3, suggesting that Sabin 1 strain may be under a smaller pressure to recombine with other viruses.Individual genomic parts of recombinants were derived from preferred donors.For instance, 2C-3A regions were rarely or never derived from Sabin 3 poliovirus while the distal part of 3D gene was heavily biased against Sabin 2 sequences.The non-random origin of individual parts could be explained by bias in their relative fitness.The recombination that easily occurs among all enteroviruses leads to selection of those combinations of individual parts that ensure better replicative capacity and allow attenuated virus to get rid of deleterious (attenuating) mutations.The role of several mutations identified in this analysis will be discussed.The intertypic recombinant with maximized fitness may contain P1 and P3 regions from Sabin 1 and P2 region from Sabin 2.
P. Cullen, S. L. Friedman, J. W. Abbott, L. L. English, A. A. Stearns, P. F. McDermott, S. Zhao, D. G. White, R. D. Walker, FDA, Laurel, MD
Background: Campylobacter spp. are ubiquitous in the animal production environment and are a leading cause of foodborne illness. The objective of this study was to investigate the prevalence and genetic relatedness of Campylobacter spp. recovered from retail meats. Methods: In 2004 ten NARMS FoodNet sites sampled 4,699 retail meats, including chicken breasts (n=1,172), ground turkey (n=1,165), pork chops (n=1,176) and ground beef (n=1,186) for Campylobacter spp.. Isolates previously identified to a species level (517 C. jejuni ; 204 C. coli) were characterized by pulsed-field gel electrophoresis (PFGE). Results: A total of 721 Campylobacter isolates were recovered, with the highest recovery rate occurring in chicken breasts (60.2%, n=706), followed by ground turkey (<1%, n=12) and pork chops (<1%, n=3). No Campylobacter was found in ground beef. SmaI restriction yielded 214 unique C. jejuni patterns and 103 C. coli patterns. Isolates with indistinguishable SmaI patterns (C. jejuni=93 patterns; C. coli=41 patterns) were further analyzed with KpnI. Analysis of SmaI in conjunction with KpnI identified several clonal profiles, including 12 C. jejuni recovered from 4 states (CA, CT, MN, and OR) over a three month time span. Isolated DNA from three C. jejuni resistant to SmaI digestion were successfully cut with KpnI, yielding identical patterns. Conversely, ten isolates resistant to KpnI digestion generated 4 distinct patterns when cut with SmaI. Conclusions: While substantial genomic diversity exists within Campylobacter spp., the use of two enzymes in performing a PFGE analysis allows for the detection of clones dispersed throughout various temporal and geographical ranges.M. R. Darnell, D. R. Taylor, CBER, Office of Blood Research and Review, FDA, Rockville, MD
Background: SARS-CoV has been detected in the blood of infected individuals, and may have the potential to contaminate donated blood and plasma-derived products in the event of a future outbreak. Effective methods for inactivating the SARS-CoV in protein solutions are described.
Methods: Heat, UV irradiation, octanoic acid, and solvent/detergent methods were tested individually for their ability to inactivate SARS-CoV in protein solutions appropriately mimicking blood-derived products. Treated samples were tested for inactivation in a tissue culture growth assay.
Results: Viral inactivation by heat treatment at 60°C required 15 to 30 minutes to inactivate the SARS-CoV. UVC efficiently inactivated SARS-CoV in 40 minutes, while UVA required the addition of psoralen to enhance inactivation of the virus. The presence of BSA limited the ability of UVC and UVA to inactivate SARS-CoV. Solvent/Detergent treatment required two, four and up to 24 hours for Triton X-100, TWEEN® 80, and sodium cholate inactivation, respectively. Octanoic acid treatment does not reduce the infectivity of SARS-CoV-spiked protein solutions.
Conclusion: Heat, UVC irradiation, and Solvent/Detergent treatments effectively inactivate SARS-CoV, while octanoic acid treatment is insufficient for inactivation of the virus.
N. Lee, S. Gannavaram, A. Selvapandiyan, A. Debrabant, CBER, FDA, Bethesda, MD
Leishmania and Trypanosoma cruzi (T. cruzi) cause life threatening chronic human diseases (leishmaniasis and Chagas disease) that can be transmitted by blood transfusion. Many endemic areas are frequented by US travelers and the deployment of military personnel to these areas can result in even greater exposure (over 1,000 cases of leishmaniasis in the troops in Iraq and Afghanistan). To date, there is no effective vaccine to prevent Leishmania or T. cruzi infections and these parasites show increasing resistance to the current drug treatments. Programmed cell death (PCD) is an essential part of cell biology involved not only in the regulation of growth and development of multicellular organisms but also in population control of unicellular organisms such as Leishmania and T. cruzi. Caspases are key effector molecules of PCD of higher eukaryotes but are not found in trypanosomatids. However, we identified "caspase-like" genes (metacaspases) in these parasite genomes. We characterized the Leishmania metacaspases and showed that they are indeed involved in Leishmania PCD. Since metacaspases are not present in the human host, they represent new targets for the development of novel anti-parasitic drugs that would specifically trigger the parasite PCD. Such studies also contribute to our better understanding of the biology and pathogenesis of trypanosomatid parasites which is essential for making informed regulatory decisions with regard to these infectious agents and other related blood borne pathogens.S. Dhruvakumar, PETA
Every batch of vaccines is tested for potency and safety, currently consuming approximately 10 million animals per year (an estimated 10% of all animal use in biomedical experimentation globally). Animal potency testing involves a pathogenic challenge, and thus causes much suffering. Animals can be entirely replaced in potency testing by physico-chemical methods of quantifying antigens (the part of the vaccine that stimulates the protective response) if they are known, but for most currently used vaccines which were developed empirically, the protective antigens are not known. This is problematic because poorly characterized vaccines are difficult to test in ways other than black-box animal studies, but animal potency tests are often unpredictive due to species differences as well as non-biological routes of exposure (e.g., rabies inoculation through intracerebral injection in the NIH test). Antigen quantification systems have been devised for some older vaccines, but resources may be better spent focusing on new vaccines and novel methods of vaccine development. As researchers deduce the sequences and structures of pathogenic proteins and develop a detailed knowledge of their roles, they can purposefully design vaccines with defined components in order to maximize effectiveness and minimize safety concerns. Computational immunology can help predict which epitopes are likely to be the best targets. Fortuitously, rational vaccine design by its nature goes hand-in-hand with non-animal testing since designing vaccines to contain defined antigens enables the direct measurement of antigenic content, thus rendering the use of animals in potency testing obsolete.
S. G. Edelson-Mammel1 , R. C. Whiting2 , S. W. Joseph3 , R. L. Buchanan4 , 1DDES,OPDF, FDA, College Park, MD, 2OPDF, FDA, College Park, MD, 3CBMG, UMD, College Park, MD, 4OSCI, FDA, College Park, MD
Listeria monocytogenes F4258 was initially grown in acidogenic TSB + 1% glutamine + 1% glucose (TSBG+G) and non-acidogenic TSB + 1% glutamine + 0% glucose (TSBG-G) to produce cells that were and were not pre-adapted to a moderately acidic environment, respectively. The thermal resistances of the cultures were determined by transferring the cells to BHI adjusted to different combinations of pH (3.0 - 8.5) and aw (0.960 - 0.987), then heating at 58°C for specified time periods using a submerged coil heating apparatus. Survivor curves were determined by plating on BHIA (injured and non-injured cells) and VJA (non-injured cells only). The survivor data were fitted to a 2-phase log-linear inactivation model, and TL, D, and T4D values were calculated. Comparison of T4D values demonstrated that the microorganism was most thermally resistant over the range pH 5.0 to 7.0, with thermal resistance reduced substantially at pH values less than or equal to pH 4.5. Water activity had less impact. The acid adapted cells were more thermally resistant in the lower pH heating menstrua, while the non-adapted cells were more heat resistant in the pH 4.5 - 7.5 range. T4D values based on the BHIA data were typically 1.5 to 2.5 greater than T4D values based on the VJA data, indicating that the cells go through an injury phase before becoming non-viable.
E. Enache, Y. Chen, Food Products Association, Washington, DC 20005
Background: Juice concentrates like cranberry, lemon or lime juice may create a hostile environment for bacterial survival because of the intrinsic concentrate properties (low pH, high titratable acidity, antimicrobial compounds etc).
Methods: Survival of E. coli O157:H7, Salmonella spp. and L. monocytogenes was monitored in cranberry juice concentrates to determine if a 5-log reduction might be achieved without other treatments. Inactivation at 0°C in concentrates with different °Brix levels was determined for a five-strain composite of the individual pathogens in two physiological states.
Results: In concentrates at 18-46 °Brix (pH 2.2-2.5), all three pathogens (stationary phase or acid-adapted cells) showed at least 5-log reduction after 6 or 24 hr incubation. At 14 °Brix (pH 2.5), a greater than 5-log reduction was obtained for L. monocytogenes and Salmonella spp. after 24 hr incubation, but for E. coli O157:H7, 96 hr incubation was needed to constantly obtain a greater than 5-log reduction. All three pathogens in stationary phase survived longer than in acid-adapted phase under the same conditions. The most resistant was stationary phase E. coli O157:H7 and the most sensitive was acid-adapted L. monocytogenes. The rate of pathogen destruction increased with increasing °Brix level of the juice concentrate, which suggests that concentrated acids and/or some intrinsic compounds may play an important role in the bactericidal effects of cranberry juice concentrates.
Conclusions: Results from this study may provide an alternative to heat treatment for cranberry juice processors to comply with the juice HACCP performance standards and ensure product safety.
P. Espandiari1 , T. J. Miller1 , J. Zhang1 , A. D. Knapton1 , P. L. Goering2 , R. P. Brown2 , V. S. Vaidya3 , A. M. Johnson3 , J. V. Bonventre3 , B. A. Rosenzweig1 , K. L. Thompson1 , P. S. Pine1 , S. Schnackenberg4 , R. D. Beger4 , E. H. Herman1 , J. L. Weaver1 , J. P. Hanig1 , 1CDER, FDA, Silver Spring, MD, 2CDRH, FDA, Silver Spring, MD, 3Harvard Medical School, Boston, MA, 4NCTR, Jefferson, AR
The search for early biomarkers of pediatric drug toxicity should be initiated at each major stage of growth because of the rapidity of developmental changes. Gentamicin was used as a tool to identify pediatric vs. adult nephrotoxicity biomarkers. Male SD rats, 25, 40 and 80 days old (as models of human toddlers, young & mature adults, respectively) were dosed with 0, 50 & 100 mg/kg/day s.c. for 6 or 14 days. Urine samples were collected (for metabonomic analysis) at 0, 8, 24, 48 and 72 hrs after initial dosing & blood & tissues at sacrifice (7 or 15 days)(for clin chem., histopathological and genomic analysis).Eighty day old rats given the highest dose showed diminished rate of growth; increased serum creatinine (Cr) & BUN and decreased serum ALB, ALP, total protein & amylase (Amy), as well asincreased levels of urine kim-1 (a biomarker of nephrotoxicity) after 48 hrs. In comparison, high dose 40 day old rats, had increased levels of serum BUN and Cr whereas in 25 day old rats, decreased levels of serum Amy was the only change found. The order of age-dependent sensitivity to gentamicin nephrotoxicity was 80>40>25 days. The magnitude of change in gene expression level of a set of nephrotoxicity biomarkers correlated with the extent and severity of histopathology which was age & dose dependent.The levels of urinary bioindicators/biomarkers of nephrotoxicity & evaluation of metabonomic data (using principle component analysis) indicated a distinctly separate & unique pattern for each of the different age groups. These findings indicate that multi-age animal models could be used as a means for predicting pediatric drug safety in preclinical studies.
H. Fang, L. Xu, T. Chen, J. Cyr, D. M. Frucht, DMA,OBP,CDER,FDA
Purpose: Survivors of the anthrax bioterrorism attacks of 2001 developed robust neutralizing antibody responses against the receptor-binding anthrax protective antigen (PA) component of anthrax lethal toxin (LT). We addressed whether B cells are, in turn, targets for anthrax LT. Methods: B cells were purified from murine spleens or elutriated lymphocytes from human donors, through negative selection (mouse) or positive selection (human) using immunomagnets and were subsequently assessed for toxin binding using flow cytometry with FITC-labeled PA. The effects of anthrax LT on MAPKK signaling, proliferation and Ig production in vitro and in vivo were then assessed by Western blotting, thymidine incorporation and ELISA, respectively. Results: Anthrax PA directly binds primary B cells, resulting in the cleavage of the MAPK kinase (MAPKK)s and disrupted signaling to downstream MAPK targets.Anthrax LT treatment causes severe dysfunction of B cells, greatly reducing proliferative responses to IL-4-,anti-IgM-, and/or anti-CD40-stimulation. Moreover, B cells treated with anthrax LT in vitro or isolated from mice treated with anthrax LT in vivo have markedly diminished proliferation and IgM responses to toll-like receptor (TLR)-2 and TLR-4 ligands. Suppressive effects of anthrax LT on B cell function occur at low doses in vitro, and at sub-lethal doses in vivo. Conclusions: B cells are direct targets for anthrax LT, in vitro and in vivo, leading to proteolysis of MAPKKs and disrupted downstream signal transduction. Anthrax LT blocks MAPKK-dependent B cell proliferation and IgM production at low/sublethal concentrations, representing a mechanism through with the toxin could attenuate protective humoral immune responses.
K. H. Fields1 , F. Schwartzkopff1 , R. Hu2 , H. Schmeisser2 , J. Bekisz2 , C. R. Lankford1 , K. Zoon2 , K. A. Clouse1 , 1CDER, FDA, Bethesda, MD 20892, 2NIAID, NIH, Bethesda, MD 20892
Interferon‑α (IFN-α) is an anti-viral and anti-neoplastic drug approved by FDA for the treatment of HBV, HCV, and Kaposi's sarcoma. Although IFNα was able to decrease viral load in some patients and potentiate activity of RT nucleoside analogs when studied in early stage HIV-1 disease, it proved ineffective at controlling virus replication in late stage disease. The concept that IFNα may have limited application in HIV-1 disease is disappointing, since previous in vitro studies in human macrophages or lymphocytes suggested that IFNα would be a potent HIV-suppressive cytokine. To determine why IFNα could be effective early, but not late, in HIV-1 disease, we studied the effect of IFNα on HIV-1 replication in primary human monocyte derived macrophages (MDM) in parallel with peripheral blood lymphocytes (PBL). Our in vitro studies reveal that IFNα can have dichotomous effects on HIV-1 replication, which are dependent on both the cell type and virus tropism. IFNα could effectively inhibit replication of CCR5-tropic (R5) or CXCR4-tropic (X4) HIV-1 isolates in human MDM and could moderately inhibit replication of R5 HIV-1 in PBL. However, IFNα either failed to inhibit replication of X4 HIV-1 isolates in PBL or, in some cases, potentiated virus replication.These in vitro findings reflect the in vivo scenario in which effectiveness of IFNα diminishes with progression of infection from macrophages to T-cells and evolution of virus co-receptor use from CCR5 to CXCR4, and may provide insight into the cause of failed IFN-α therapy.
T. J. Flynn, C. Y. Kelly, CFSAN, FDA, Laurel, MD
Background: The utility of cultured cryopreserved human hepatocytes as an in vitro model for assessing hepatotoxicity has been limited by their poor attachment to tissue culture plates. Poor attachment limits cell viability and does not allow for the cells to proliferate and/or maintain differentiated function.
Methods: Cryopreserved human hepatocytes were cultured in 96-well plates coated with various cell matrix proteins. Medium components were varied to assess the combined role of matrix protein and growth factors in cell attachment, proliferation and differentiation. Cell proliferation was monitored using the resazurin reduction assay, and cell differentiation was assessed using cell-specific antigenic markers and biochemical functions.
Results: Cells attached to the plate with very low plating efficiency (10-20%). Cells were quiescent for 5-7 days, then began proliferating to confluence by 14-21 days. There appeared to be no difference in cell growth on any of the subcellular matrices examined. Cells survived for at least 38 days. Antigenic markers suggested that several liver cell types were present including: hepatocytes, cholangiocytes, stellate cells, and myofibroblasts. Induceability of CYP1A2 with β-naphthoflavone was not maintained in serum-free medium formulations examined.
Conclusions: Our findings suggest that it may be possible, starting with cryopreserved human hepatocytes, to obtain long-term cultures that attach to the culture plate, proliferate, retain differentiated function, and contain a mix of cell types that are characteristic of intact liver. This may prove to be a useful model for examining issues in hepatotoxicity such as the marked gender differences in sensitivity and the role of inflammation.
S. Gannavaram, A. Debrabant, Laboratory of Bacterial Parasitic and Unconventional Agents, Division of Emerging and Transfusion Transmitted Diseases, CBER, FDA, Bethesda, MD 20892
Apoptosis is a physiological process critical for normal cellular development and tissue homeostasis in multicellular organisms but also in population control of unicellular organisms such as Leishmania and Trypanosoma which are responsible for a variety of human parasitic diseases (e.g. leishmaniasis, Chagas disease, African sleeping sickness). Although evident in several protozoan organisms, programmed cell death (PCD) pathway is not adequately exploited for developing strategies to control these important human parasites. We sought to identify components of protozoan PCD pathway as a means of parasite elimination. One of the hallmarks of apoptosis is the cleavage of chromatin into oligonucleosomal-sized fragments. Biochemical and genetic studies have identified at least two endonucleases involved in mammalian DNA fragmentation during apoptosis, the DNA fragmentation factor and endonuclease G (EndoG). We identified homologues of mammalian EndoG in Leishmania and Trypanosoma genomes. Overexpression of EndoG in Leishmania resulted in increased DNA damage, leading to parasite death. In addition, parasites overexpressing EndoG displayed heightened sensitivity to H2O2 induced PCD. In contrast, selective depletion of EndoG mRNA by RNA-interference in Trypanosoma brucei rendered the parasites more resistant to H2O2 induced PCD. We conclude that EndoG is an important component of protozoan PCD pathway. Understanding of the PCD pathway may contribute to better understanding of the biology and pathogenesis of trypanosomatid parasites and also in devising control strategies against these blood borne pathogens.
M. P. Gelderman-Fuhrmann1 , O. Simakova2 , L. B. Carter1 , J. Simak3 , 1FDA, Rockville, MD, 2USUHS, Bethesda, MD, 3DFA, Rockville, MD
Introduction: We previously demonstrated the presence of cell membrane microparticles (MPs) derived from platelets, blood-, and endothelial cells in apheresis platelet units (APU) on day 6 of storage. Seven day storage of platelets recently became a reality in transfusion management. Therefore, we investigated the impact of extended storage on MP-immunophenotype and activity profiles in APU on day 6 and 8 of storage.
Methods: We analyzed MPs in APU supernatants (n=28) on day 6 and 8 using a three-color flow cytometry assay and several functional assays. Plasma from healthy volunteers (HVP, n=38) was used for comparison.
Results: When comparing APU on day 6 and 8, platelet MPs (CD41+), endothelial MPs (CD144+), RBC MPs (CD235a+), and proinflammatory CD154+ MPs did not change significantly. In contrast, a significant decrease in pH, and a significant increase in leukocyte MPs, including leukocyte MPs exposing tissue factor (TF, CD142) was observed on day 8. All studied MP-phenotypes were significantly higher in APU vs. HVP for both days. TF activity of washed MPs increased (day 6 vs. 8) and correlated with CD142+ counts. The results of assaying intracellular free Ca2+ indicated that APU MPs induced extracellular calcium influx. Conclusions: We found that several APU MPs exhibited various pathophysiological activities in vitro, and that there was a correlation between certain MP-phenotypes and APU MP-activities in these units. However, seven day storage of effectively leukoreduced APU should not have a dramatic impact on a potentially pathogenic MP-content. Next, we will investigate a possible correlation between our findings and APU transfusion related adverse events.M. Kulka, B. B. Goswami, T. A. Cebula, D. Ngo, M. Ayodeji, CFSAN, OARSA, FDA, Laurel, MD
Background: Hepatitis A virus (HAV) is a human pathogen transmitted via the fecal-oral route. Because of its exceptional stability in the environment, contaminated food and water are frequently associated with localized outbreaks.Direct demonstration of the presence of infectious virus in foods is difficult, however, due to the absence of sensitive cell culture methods.
Methods: Cultures of FrhK4 cells were infected with a cytopathic apoptosis inducing strain of HAV or transfected with dsRNA. Cellular functions related to apoptosis were investigated by Western blotting, agarose gel electrophoresis, and RT-PCR.
Results: The cytopathic strain of HAV as well as dsRNA, were found to activate RNaseL, an enzyme normally associated with innate immunity of cells and organisms to virus infection. The virus activated RNaseL directly without inducing synthesis of interferon, which is observed in dsRNA treated cells. Activation of RNaseL was linked to activation of caspase 3, a key enzyme involved in apoptosis, by a mechanism not involving activation of initiator caspases 8 or 9, but possibly involving caspase 12 and ER stress. A full-length cDNA of RNaseL from FrhK4 cells was cloned and sequenced. Comparisons with human and mouse enzymes revealed numerous changes in amino acid sequence.
Conclusions: The cytopathic strain of HAV activates the apoptotic pathway by a mechanism independent of interferon or dsRNA. RNaseL from FrhK4 cells is directly activated by an as yet unidentified viral product. Identifying this viral product is critical to establishing a more sensitive cell culture system for the detection of HAV in contaminated foods.M. A. Grant, R. M. Trump, Pacific Regional Laboratory, Northwest, FDA, Bothell, WA
Background: FDA recently issued guidelines for fresh-cut produce after outbreaks of food-borne illness were traced to lettuce and other produce. Detection of pathogenic E. coli is difficult in these foods and methods may need modification.
Methods: Lettuce samples were examined for epiphytic microbial populations to determine whether they might adversely affect detection of E. coli O157:H7 (EHEC). Samples were also spiked with EHEC and detection efficiency compared between the FDA Bacteriological Analytical Manual (BAM) method and an experimental acid-shock method.
Results:Lettuce samples contained larger populations of Gram negative bacteria than other food matrices preciously reported to have caused EHEC outbreaks. Although total coliform populations were relatively low, other enterics, as well as Pseudomonas spp., were present at levels up to 1.5 X 106 gm-1. Lettuce samples were spiked with EHEC at approximately 1 CFU gm-1 and recovery was examined using Sorbitol-MacConkey Agar with cefixime and tellurite (TCSMAC) as well as a standard USDA medium, Rainbow agar with tellurite and novobiocin (RTN). EHEC detection was more effective using the acid enrichment method than the BAM method, with target populations ranging from 6 to over 100-fold higher on TCSMAC and 8 to over 100-fold higher on RTN. Growth of interfering non-target bacteria on TCSMAC was also reduced using the acid enrichment method by as much as 3 orders of magnitude. Detection of diagnostic stx1 and stx2 genes by real time PCR mirrored the improved enrichment by the acid method. Immunomagnetic separation and column purification of templates did not substantially improve Ct values beyond a simple centrifugation clean up step.
N. Gupta1 , W. Wang2 , E. Desmezieres2 , R. Vassell1 , Y. He1 , P. Wingfield3 , C. D. Weiss2 , 1CBER,FDA,BETHESDA,MD, 2CBER,FDA,BETHESDA,,MD, 3NIH,BETHESDA,MD
HIV envelope glycoprotein (Env) mediates infection by fusing virus with cellular membranes. Fusion inhibitors, a new class of antiretroviral drugs, inhibit HIV infection by binding to gp41 to form a peptide-gp41 6HB that is fusion-incompetent. To understand resistance mechanisms to fusion inhibitors, we generated an escape-mutant virus against an N peptide inhibitor. We found that two mutations in gp41, one each in the N- and C- heptad repeats, confer early resistance to the N peptide. These same mutations also confer resistance to a C peptide inhibitor, suggesting an indirect mechanism of resistance. Interestingly, the N mutation alone or in combination with the C mutation increases sensitivity to sCD4 and confers increased growth kinetics, syncytia formation, and infectivity in cells with low levels of CD4. The resistance mutations also conferred better exposure of the CD4-induced epitopes. Together these findings suggest improved receptor usage and kinetics of entry. Our previous data with thermal denaturation studies had showed that the N mutation improved the energetics of the viral 6HB, without affecting the energetics of the 6HB formed with the inhibitor and C peptides. Thus, our results demonstrate a complex resistance pathway that involves global changes to the envelope affecting receptor activation and possibly thermodynamic factors, which regulate virus entry to reduce the ability of fusion inhibitors to bind Env. This indirect method of escape from peptide is distinctively different from previous reports of escape from C peptide inhibitors, which directly reduce peptide-binding affinity for the endogenous heptad repeat. The work was supported by NIH intramural IATAP grant.
T. S. Hammack, A. P. Jacobson, W. H. Andrews, CFSAN, FDA, College Park, MD
The sensitivity of the BAM Salmonella culture method, for the recovery of Salmonella Typhi from orange juice, bottled water, lettuce, raw shelled oysters, and mamey pulp was examined.In the first phase, 1 mL aliquots, from decimally diluted day old S. Typhi culture suspensions (strains ISP 1820 and ATCC 9993), were added to duplicate flasks containing 25 g (mL) portions of food/beverage and 225 mL of preenrichment broth.The BAM method detected S. Typhi at a level of ca 1 cfu/25 g (mL) in orange juice, lettuce, and water.For raw oysters, the BAM method detected S. Typhi at levels 102 and 105 cfu/25 g for strains 9993 and 1820, respectively.For mamey pulp, S. Typhi was not detected at levels as high as 107 cfu/g.
In the second phase, the effect of sample preenrichment on the effectiveness of the BAM method was examined.Bulk quantities of mamey (ca 3,000 g) were inoculated with S. Typhi and allowed to age for 4 days at 2 - 6ºC.The mamey was then preenriched in lactose broth, buffered peptone water (BPW), and Universal Preenrichment (UP) broth.UP broth was significantly more productive (p < 0.05) than either BPW or lactose broth for the recovery of both strains of S. Typhi from mamey.S. Typhi isolates were not recovered from either BPW or lactose broth.On the basis of the above results, it is recommended that UP broth replace lactose broth for the detection of S. Typhi in mamey.
H. C. Harbottle1 , D. G. White2 , S. Zhao1 , R. D. Walker1 , P. F. McDermott1 , 1FDA, CVM, OR, DAFM, Laurel, MD, 2FDA, CVM, OR, Laurel, MD
Background: Multi-drug resistant phenotypes of Salmonella enterica serotype Newport (MDR-AmpC) have emerged as a major public health problem.Multi-locus sequence typing (MLST) is a technique used for analyzing relationships between bacteria.In this study, MLST and antimicrobial susceptibility profiling (ASP) was used to show that over the past 4 decades, S. enterica serotype Newport has diversified genetically and is less susceptible to antimicrobial agents.
Materials and Methods: The relatedness of 79 S. enterica serotype Newport human isolates from historical collections (1958-1979) and 81 isolates from 2001-2003 collections of human and animal isolates was determined using MLST of genes aroC, dnaN, hemD, hisD, purE, sucA, and thrA.Antimicrobial susceptibility patterns (ASP) were tested in accordance with CLSI standards using 17 antimicrobial agents.
Results:The historical isolates were 75% pan-susceptible with 25% resistant to >1 antimicrobial.Fourteen percent were resistant to >3 antimicrobials, and the most common phenotype (8%) exhibiting resistance to five of the antimicrobials that contribute to the MDR-AmpC phenotype.ASP of the 2001-2003 collections identified 48% as MDR-AmpC and 40% pan-susceptible.MLST identified 15 sequence types (ST) in the historical collection, mostly comprised (28%) of ST118.Thirteen ST were identified in the 2001-2003 collections with 62% ST45, a hepta-locus variant of ST118.
Conclusion: The S. enterica serotype Newport population appears to be diversifying into genetically different populations over time, shown by the replacement of the predominant ST118 to a hepta-locus variant, ST45.Resistance phenotypes doubled in the intervening 48 years with MDR-AmpC phenotypes increasing more than 30 fold.
E. C. De Martinis, R. E. Duvall, A. D. Hitchins, CFSAN, FDA, College Park, MD
Background: Enumerating foodborne pathogens at <100 cfu/g relies on the most probable number (MPN) method, which takes 4 days or more. A real time polymerase chain reaction assay (PCR) to accelerate MPN enumeration of foodborne Listeria monocytogenes (Lmo) was developed.Methods: Foods were spiked with Lmo (70-110 cfu/g) and triplicate sub-portions (0.0001-1g) were selectively enriched (48h; 30°C). For regular MPN enumeration, enrichments were subcultured on Oxford agar (48 h: 35°C to isolate Listeria.For PCR MPN, the Lmo cells in same enrichments were washed and resuspended in 2 ml sterile water. DNA was extracted by boiling for 10 min. The DNA in the supernatant was targeted with published oligonucleotide primers for amplifying a Lmo specific sequence of 16S rRNA genes. Amplification was continuously monitored using SYBR Green and the amplicon characterized by melting temperature.
Results: The Lmo-specificity of the primers was confirmed with Lmo (15 strains), L. innocua (11 strains), and 1 strain each of L. welshimeri, L. seeligeri, L. ivanovii and L. grayi. Quantitatively spiked milk, lettuce, smoked salmon, brie, ice-cream, pork pâté, salami, ready-to-eat shrimp, raw ground beef, and fresh soft cheese were enumerated by both the regular and PCR MPN methods. The paired results from the two MPN methods agreed well. For some foods, 1-g samples required a decimal dilution for a positive test result, suggesting concentration dependent interference with the PCR.
Conclusion: This PCR method reduces the time necessary for MPN enumeration of foodborne Lmo from 4 to 2 days.
N. V. Gopee1 , R. D. Edmondson1 , S. Thyparambil1 , R. C. Jones1 , J. T. Taylor1 , W. G. Wamer2 , P. C. Howard1 , 1NCTR, Jefferson, AR, 2CFSAN, College Park, MD
In response to a paucity of data on tattoo pigment toxicity, we have examined inguinal and axillary lymph node protein expression in female SKH-1 hairless mice following: no tattooing; tattooing with vehicle (10% aqueous glycerol); tattooing with either 20% w/v cadmium sulfide (CdS) or Pigment Red 22 (PR22) in vehicle.Mice were then held for 2 weeks and exposed for 13 weeks to 1.4 SED/day simulated solar light. The mice (3/group) were sacrificed and the axillary lymph nodes removed, combined, frozen, and pulverized in liquid-nitrogen.Duplicate samples from each group were separated using polyacrylamide gel electrophoresis, the entire gel lane was excised into 30 bands, and each band subjected to in-gel trypsin digestion. The resulting peptides were analyzed using an automated nano-HPLC MS/MS system, and proteins were identified using the Mascot database search engine.ProteinTrack software (developed in-house) was used to filter the protein results. In this first report of murine lymph node proteome more than 1700 proteins were identified.Hierarchical analysis of the total protein expression demonstrated that the non-tattooed and control (aqueous glycerol) proteomes were significantly different (p<0.05).Protein expression in mice tattooed with PR22 or CdS were different from each other and from the vehicle control.Analysis of the altered genes as a result of tattooing with PR22 or CdS suggests there are changes in pathways involved in cell proliferation and inflammation.These results suggest (a) tattooing induces persistent changes in regional draining lymph node protein expression, (b) tattooing with PR22 and CdS induced changes above those detected with vehicle control.
N. V. Gopee1 , V. G. Desai1 , B. J. Miller1 , J. C. Fuscoe1 , W. Tong1 , H. Fang1 , W. G. Wamer2 , P. C. Howard1 , 1NCTR, Jefferson, AR, 2CFSAN, College Park, MD
Tattooing is increasing in popularity with more than 45 million Americans having at least one tattoo. This study explores the global gene expression profile in skin of female SKH-1 hairless mice following tattooing. Mice were: not tattooed; tattooed dorsal, longitudinally with 10% aqueous glycerol (vehicle); tattooed with 20% w/v cadmium sulfide (CdS) or Pigment Red 22 (PR22) in vehicle. Two weeks later, the mice were exposed for 13 weeks to 1.4 SED/day simulated solar light (SSL) (one-half the mice), then sacrificed and the tattooed skin removed and frozen. Gene expression was determined using an in-house oligonucleotide microarrays (20,000 genes). Gene expression data were analyzed using the FDA microarray data management, analysis and interpretation software, ArrayTrack. There was not a significant light effect on gene expression, and as a result the data from no-SSL and SSL treated animals were combined for the tattooed groups. A total of 109 genes were differentially expressed >1.5-fold for PR22 tattooed mice, of which 35 were upregulated. In CdS tattooed mice, 561 genes were differentially expressed >2-fold, of which 388 were upregulated. Only 29 of the differentially expressed genes were common to both tattoo pigments, 11 of which were upregulated. Analysis of the altered genes suggests there are changes in pathways involved in immunological and inflammatory responses, carcinogenesis, cell proliferation and cell death. The results suggest microarray analysis of RNA from tattooed skin can be an effective tool for investigating the impact of tattooing pigments on the skin and immune system.
P. C. Howard1 , N. V. Gopee1 , A. R. Warbritton2 , N. J. Walker3 , W. W. Yu4 , V. L. Colvin4 , P. Webb1 , R. L. Bronaugh5 , M. E. Kraeling5 , D. W. Roberts1 , 1NCTR, Jefferson, AR, 2Toxicol. Pathology Assoc., Jefferson, AR, 3NIEHS, Research Triangle Park, NC, 4Rice University, Houston, TX, 5CFSAN, Laurel, MD
We are currently examining the dermal penetration and distribution of nanoscale material in pig and human skin in vitro and mouse skin in vivo using nanoscale fluorescent CdSe quantum dots (QD; 621 nm emission). We report the preparation of fluorescence standards of QD homogeneously distributed in a stable glycol methacrylate-based hydrophilic polymer that under standard conditions can be used to quantify QD-based fluorescence in tissues. The QD-polymer standards were sectioned at 4 microns or used to fill chambered slides of known thickness. Fluorescence was determined using a Leica® DM RA2 photofluoromicroscope with UV illumination and a SPOT® high resolution digital imaging system and appropriate excitation, dichroic, and emission filters. Tissues and standards were photographed at exposure times from 1 msec to 15 sec and fluorescence determined for each pixel. Considering only exposure/concentration combinations that did not result in pixel saturation, the average percent coefficient of variation (CV) was 2.66% for the mean pixel intensities of 21 random 400 x 400 pixel squares on images of standards ranging in concentration from 3.3 to 380 nM of QD particles at a QD-polymer thickness of 20 microns. For these same concentrations, the mean pixel intensities (each n=3) ranged from 1.4 to 4022.9. The increase in pixel value as a function of exposure time for QD-polymer standards was linear until pixels approached saturation (functions of concentration and exposure time). This quantitative methodology and the use of pixel-saturation as a secondary approach are well-suited to detect QD-based fluorescence in tissues.N. V. Gopee1 , D. W. Roberts1 , P. Webb1 , C. Cozart1 , P. Siitonen1 , A. R. Warbritton2 , N. J. Walker3 , W. W. Yu4 , V. L. Colvin4 , P. C. Howard1 , 1NCTR, Jefferson, AR, 2Toxicol. Pathol. Assoc., Jefferson, AR, 3NIEHS, Research Triangle Park, NC, 4Rice University, Houston, TX
There is a paucity of toxicology data on nanoscale materials, especially regarding dermal penetration. As part of a National Toxicology Program efforts examining the health risks of nanoscale materials (http://ntp.niehs.nih.gov/index.cfm?objectid=7E6B19D0-BDB5-82F8-FAE73011304F542A) we sought to determine nanoscale material: (a) distribution following intradermal administration, and (b) penetration of intact or dermabraded mouse skin. CdSe-based quantum dots (QD) that were nail shaped and coated with polyethylene glycol (PEG; 37 nm diameter) were used. Female SKH-1 mice were intradermally injected with 5 µL of 19 µM QD. Following sacrifice, tissues were removed and analyzed for Cd and Se content using inductively coupled plasma mass spectrometry (ICP-MS). Cd was detected at the site of application and in the following tissues: liver > regional lymph nodes > kidney > spleen > heart. These results support the use of liver and regional draining lymph nodes as sentinel organs for assessment of systemic distribution after dermal exposure of QD. QD were suspended in an oil-in-water emulsion at approximately 9 µM and applied at 2 mg/cm2 to the dorsal skin of untreated mice, mice tape-stripped to remove the stratum corneum, or mice following dermabrasion. Using ICP-MS of sentinel organs, QD could not be detected in sentinel organs of untreated or tape-stripped mice, but were detected in liver and lymph nodes of mice with dermabraded skin. These results suggest that an assessment of the safety of topically applied nanoscale materials needs to take into consideration the vehicle used, and a variety of skin conditions that may be present in humans.A. P. Jacobson, T. S. Hammack, W. H. Andrews, FDA, College Park, MD
Alfalfa seeds used in sprout production are often the source of Salmonella organisms on sprouts.Test portions of alfalfa seeds, artificially contaminated with S. Stanley, S. Poona, or S. Muenchen, were used to compare 5 methods of sample preparation for recovering Salmonella organisms: 1) wet blend method- test portions were blended for 2 min with lactose pre-enrichment broth; 2) dry blend method- test portions were blended for 30 s and inoculated into lactose pre-enrichment broth; 3) soak method- test portions were inoculated into lactose pre-enrichment broth; 4) rinse method- test portions were rinsed with lactose broth by swirling flasks 100 times clockwise and 100 times counterclockwise; the media were then decanted and incubated; and 5) rinsed seed method- the rinsed test portions were inoculated into lactose pre-enrichment broth.
The soak method of pre-enrichment was most effective.Of a total of 120 test portions examined, the numbers of Salmonella positive test portions given by each of the 5 methods were as follows: soak, 70; rinsed seed, 66; dry blended, 57; wet blended, 53; rinse, 18.On the basis of these results, the soak method of pre-enrichment is recommended for the analysis of alfalfa seeds for the presence of Salmonella organisms.
C. E. Keys, P. Whittaker, F. S. Fry, E. W. Brown, CFSAN, FDA, College Park, MD
Enterobacter sakazakii is a potentially dangerous foodborne pathogen particularly among neonates where it is linked to severe and sometimes fatal meningitis in infants and children.Previous studies of conserved gene sequences note the presence of several phylogenetically heterogeneous clusters of E. sakazakii.Such observations confound development of identification and discrimination strategies. To further assess the genetic diversity of E. sakazakii, we examined genotypic variation across an ecologically diverse group of 125 strains isolated from food, clinical, and environmental sources. Molecular subtyping was done using PFGE (pulsed-field gel electrophoresis) across three enzymes.XbaI analysis yielded 85 unique PFGE types of which 57 (67%) appeared only once (i.e., within a single strain) indicating substantial polymorphism for the species. Analysis with BlnI and SpeI yielded results similar to XbaI with 58 out of 84 (69%) unique patterns and 63 out of 89 (71%) unique patterns appearing only once, respectively.Cluster analysis of XbaI patterns revealed extensive diversity among PFGE types, with several clusters associated more closely to other Enterobacter spp. than to more distantly related E. sakazakii groups. This find is consistent with the taxonomic disparity noted in previously described 16S rDNA analyses.Despite substantial diversity, fatty acid profiles were almost unanimous in confirming E. sakazakii species identity for these strains underscoring its value in identity confirmation for this pathogen.Taken together, these data suggest that, like other Enterobacter spp. (e.g., E. cloacae), E. sakazakii represents a widely divergent group at the genetic level.
E. W. Brown, C. E. Keys, CFSAN, FDA, College Park, MD
The importance of PFGE (pulsed-field gel electrophoresis) as a strain discriminator is well established, however, it remains unclear in which instances PFGE-based strain clusterings reiterate evolutionary history. This question is of paramount importance when attempting to trace origin among outbreak strains. Here, phylogenetic utility of PFGE is assessed among three populations of Salmonella enterica—the SARA, SARB, and SARC reference collections, each representing a distinct level of taxonomic diversity. Comparisons of resultant PFGE trees with MLEE (multi-locus enzyme electrophoresis) and mdh (malate dehydrogenase) gene phylogenies, both of which reflect the evolutionary relationships of Salmonella strains, revealed substantial incongruence for all three strain collections indicating that PFGE phylogenies deviate considerably from actual strain evolution. For example, SARC sibling strains representing four of the eight S. enterica subspecies were broken into disparate locales on the PFGE tree. In the PFGE SARB tree, representing S. enterica subspecies I, five of six clades comprised strains from at least two different MLEE and mdh lineages. The PFGE tree representing the highly homogeneous SARA strains, consisting of S. Typhimurium and its four closest serovars, also appeared evolutionarily obscured. Four of five clades retained strains from diverged MLEE lineages while three clades comprised strains from diverse mdh lineages. These data suggest that, in Salmonella, the reconstitution of evolutionarily meaningful strain groupings may be a largely intangible task for PFGE. Moreover, our findings underscore previously noted pitfalls in using single-enzyme PFGE analysis to obtain accurate genetic relationships. Currently, we are exploring combinations of enzymes for improved phylogenetic accuracy.
C. R. Kiessling1 , M. B. Buen1 , W. M. Kiessling1 , E. W. Laster1 , M. H. Loftis1 , J. N. Sofos2 , 1FDA, 2Colorado State University
During 2005, a total of 7,083 samples from FDA regulated products (domestic and import) were collected and analyzed for the presence of Salmonella. A total of 316 samples (4.5%) were found to be Salmonella positive. Of the 316 positive samples, a total of 300 isolates (264 from foods and 36 from feeds/miscellaneous products) were submitted for antimicrobial susceptibility testing. This paper presents the results of positive samples analyzed in 2005 and compares them with previously reported data. The frequency of isolates exhibiting multiple resistance (two or more antimicrobial agents) was 9.1% (n=24) compared to previous rates of 15.0 % (n=166; April 1999 thru July 2003 and 5.6% (n=22; August 2003 thru 2004). Of the 36 isolates recovered from feeds, animal treats and miscellaneous items, 69.4 % (n=25) were found susceptible to all antimicrobial agents tested compared to previously reported findings of 37.0% (n=104), and 94.2% (n=97), respectively. The frequency of isolates exhibiting multiple resistance (against 2 or more antimicrobial agents) was 13.9% (n=5) as compared to 34.2% (n=81) and 1.9% (n=2), respectively.The most frequently isolated serotypes were Thompson 11.1% (n=4), Infantis and Mbandaka 8.3% (n=3), and Agona, Anatum, Cubana, Enteritidis, and Typhimurium 5.6% (n=2). The leading serotypes reported from feeds during previous years were Enteritidis and Infantis; however, the number of isolates received for the previous years was greater (n=281) and n=103; respectively) compared to only 36 for 2005.
M. Kioi1 , J. Han1 , E. Michishita2 , T. Shimamura1 , R. K. Puri1 , 1CBER, FDA, Bethesda, MD, 2NCI, NIH, Bethesda, MD
Background: Interleukin (IL)-13 is a hormone which exhibits antitumor activity in some solid tumor cell lines while it displays mitogenic activity to Hodgkin's lymphoma cells. IL-13Rα2 chain, one of the two chains of IL-13 receptors, binds IL-13 with high affinity and is overexpressed in 60~80% of glioblastoma multiforme (GBM) tumors; however, the significance of IL-13Rα2 expression in cancer cells is not known.
Methods: To investigate the role of IL-13R, we knocked down IL-13Rα1 and IL-13Rα2 genes by RNAi and studied gene expression profiles, cellular and tumor growth, invasion, and tumorigenicity in vivo.
Results: Retroviral vector-mediated RNAi delivery inhibited IL-13Rα2 expression and proliferation of some GBM cell lines (U251, U373, and U87); however, PM-RCC renal cell carcinoma cell line was not affected. The IL-13Rα2 knockdown tumor cells showed lower tumorigenicity compared to control tumors in immunodeficient mice. Decreased tumorigenicity was specific to IL-13Rα2 knockdown, as knockdown of IL-13Rα1 did not affect tumor growth. In sharp contrast, when U373 cells were engineered to overexpress IL-13Rα2, they formed subcutaneous tumors in mice while same number of cells transfected with mock control failed to form tumors.
Conclusions: These studies begin to elucidate the role of IL-13Rα2 in tumor biology and identify pathways that regulate tumor growth and tumorigenesis. These studies also provide insights into development of potency and identity tests for cancer vaccines and identification of biomarker for tumor response to cancer vaccines or other cancer therapeutics.
H.S. Ko, CBER, FDA, Rockville, MD
Background. Data have been accumulated attesting to the safety of immune globulin products, and preclinical studies in support of clinical trials of such products are not routinely conducted. Depending on the source of immune globulin and the condition to be treated, however, animal studies may be important for assuring safety of human use in clinical investigations.
Methods. Original IND submissions of immune globulin products were reviewed. The products included immune globulin intravenous (human) (IGIV) from various manufacturers (8), and polyclonal human and heterologous immune globulin products with activities against bacteria and viruses or their toxins (10), or targeting human antigens/cells (1). The following were looked for: safety data from animal studies on excipients, blood-borne transmissible agents, hypersensitivity and use in pregnancy; and animal data on the pharmacologic effect of the product or in vivo efficacy.
Results. Out of the 20 INDs reviewed, safety data on excipients were provided to support product use in humans in 14 of them, transmission of blood-borne agents in 1, hypersensitivity in 2, and pregnancy in none. Evidence of pharmacologic effect demonstrated in in vivo animal studies was provided in 8 submissions; some of these studies included in vivo assays for potency. However, there have not been data from adequate and well-controlled animal studies designed to show efficacy in support of investigational use of an immune globulin product in these submissions.
Conclusions. Despite familiarity with the general safety profile of immune globulin products, preclinical studies are still important to assure quality and potency, and evaluate special toxicities before initial use in humans. Where human field trials are unfeasible, they may also have utility in support of efficacy.
A. Kornhauser1 , R. R. Wei1 , J. C. Hubinger2 , C. N. Barton2 , K. Kaidbey3 , S. A. Miller4 , S. G. Coelho5 , J. Z. Beer5 , B. Z. Zmudzka5 , V. J. Hearing6 , Y. Yamaguchi6 , 1FDA, College Park, MD, 2FDA, College Park, MD, 3Ivy Laboratories, PA, 4FDA, Rockville, MD, 5FDA, Rockville, MD, 6NCI, Bethesda, MD
Background: Sunburn cells (SBCs) are keratinocytes undergoing apoptosis. Cyclobutyl pyrimidine dimers (CPDs) are a type of DNA damage.Both are induced in human skin by exposure to UV radiation, primarily UVB, and provide quantifiable measures of UV-induced cellular damage. They are also implicated in the process of photocarcinogenesis.
Methods: Seven healthy volunteers were recruited for this segment of the study. A test site measuring 10x5 cm was marked at the mid-back of each subject and the minimal erythema dose (MED) was measured. A single dose of 1 MED of UV radiation from a solar simulator was delivered to the test sites of each subject. One subsite was selected to serve as an unirradiated control. Shave biopsies (4 mm) were obtained immediately post UV for CPD determinations and 24 hours post UV for SBC assays. Biopsies were fixed in formalin and embedded in paraffin. CPDs were analyzed by immunofluorescence using a monoclonal antibody specific for CPD. SBCs were counted in specimens stained with hematoxylin-eosin, and in addition were assayed by in situ end labeling with terminal deoxynucleotidyl transferase (TUNEL assay).
Results: UV exposure resulted in erythema and CPD formation in all subjects. No significant SBC increase above the control level was observed, as documented by SBC count and TUNEL assay.
Conclusions: To our knowledge, published data to date report that erythema and CPD formation result in generation of SBCs. Based on our present study in progress, we suggest that a dose effect, or a dose rate effect, might play a role in SBC development.
M. E. Kraeling, R. L. Bronaugh, Office of Cosmetics and Colors, CFSAN, FDA, Laurel, MD
Background: This study was conducted to measure the rate of salicylic acid (SA) absorption from cosmetic creams in hairless mouse (HM) skin, in support of a SA photocarcinogenicity study conducted at the National Center for Toxicological Research (NCTR). SA absorption in human skin was also determined.
Methods: SA penetration was determined from 2% and 4% [14C]SA creams applied to excised viable HM skin (full thickness) and human skin (200 µm thick) in flow-through diffusion cells perfused with a physiological buffer. The formulations remained on the skin for either 4 or 24 h. Four hours after application of SA cream, half of the skin was exposed to UVB radiation (16 mJ/cm2); unexposed skin was the control. The amount of SA that penetrated into skin layers and receptor fluid was measured and expressed as the percent of applied dose penetrated.
Results: At both concentrations, SA was rapidly and extensively absorbed into HM skin in 24 h. Absorption of SA through skin into receptor fluid was 87-91% with 2-3% remaining in skin. UVB treated and control skin showed similar absorption. In the 4 h studies, absorption of SA into receptor fluid was rapid at 37-39%. At the time of UV exposure, 3-5% of the SA applied resided in HM skin. Human skin absorption studies were conducted for 24 h. SA absorption was less extensive (33-42%) with 7-12% remaining in skin.
Conclusion: SA rapidly penetrated HM skin and was found in skin at the time of irradiation. SA human skin penetration was less rapid.
M. Laassri1 , A. Pletnev2 , K. M. Chumakov1 , 1CBER, FDA, Rockville, MD, 2NIAID, NIH, Bethesda, MD
The most serious manifestation of WNV infection is a fatal inflammation of the brain in Human. The frequency of WNV outbreaks in humans has increased in recent years, and since 1999, the geographical distribution of WNV has expanded to the Western hemisphere. Such viruses are of great public health concern. Currently there is no WNV vaccine for human. The main goal of this study is to develop simple, sensitive and reliable methods for rapid screening of emerging mutations and evaluation of genetic stability of Flavivirus vaccines, and to monitor consistency of their production to accelerate development and evaluation of the WNV and other Flavavirus vaccines.
We created an oligonucleotide microarray for rapid analysis of genetic stability of WNV - Dengue chimeras.The microarray consisted of 1250 overlapping oligonucleotides covering the entire genome of the virus.Each oligonucleotide was spotted four times on each microarray producing redundant information that was used to assess statistical significance of the results.The microarray was hybridized with cDNA prepared from serially propagated samples and from the reference DNA obtained by PCR amplification of homogeneous cDNA clone.Validation included analysis of known mutations and several serial passages (up to 12 passages) of the recombinant WNV virus in Vero cells.Several independent lineages were prepared to ensure that the mutant selection process is consistent. cDNA from the passaged virus was analyzed by microarray hybridization and the profiles were used to reveal unstable genomic loci. The microarray showed high sensitivity for detection of point mutations. The WNV-Dengue chimera was found to be genetically stable after 12 passages in Vero cells.
Use of the oligonucleotide microarray method may facilitate the analysis of the genetic diversity of RNA viruses at the levels of genomic recombination and nucleotide sequence heterogeneity. This approach produces a snapshot of genomic heterogeneity analysis of the entire virus population without the need to use the labor-intensive techniques. The method is simple and it does not require cloning of the test samples. It can also open the possibility of large-scale full-genome screening of viral isolates needed for thorough epidemiological surveillance and vaccine quality control.
S. K. Lappalainen, S. V. Gomatam, V. M. Hitchins, FDA, CDRH, Rockville, MD 20850
Background: Many single-use devices (SUDs) are reprocessed between patient uses. In the past, a visual determination was accepted as the endpoint of "clean". However, it is not possible to visually determine "clean" on opaque, narrow lumened devices.
Methods: To determine a quantitative endpoint for "clean", we measured residual total protein (TP) and total organic carbon (TOC) levels on GI biopsy forceps before and after reprocessing with commercially available cleaners. We simulated worst-case clinical use by inoculating 15 numbered devices with a 3-protein test soil and left them to dry prior to extracting proteins from the devices. TP and TOC were measured from each device prior to and after cleaning. This inoculation and cleaning procedure was repeated 5 times to simulate up to 6 clinical uses. Each device was extracted separately and tested for TP, using Bradford's reagent, and TOC, using a direct digestion method.
Results: Data for pre- and post-cleaned devices were generated for all 5 cycles. The highest amount of TP and TOC found in pre-cleaned device extracts were 61.8 ug/cm2 and 39.1 ug/cm2, respectively. The highest amounts of residual TP and TOC on post-cleaned device extracts were 4.0 ug/cm2 and 2.2 ug/cm2, respectively. All post-cleaned devices were visually clean.
Conclusions: Our TP and TOC results on post-cleaned SUDs are comparable to those found by others after cleaning reusable devices. Visual detection alone does not allow one to detect residual bioburden that may remain on cleaned devices. Our results provide quantitative TP and TOC acceptance criteria for cleaned GI biopsy forceps.
S. K. Lappalainen, S. V. Gomatam, V. M. Hitchins, FDA, CDRH, Rockville, MD 20850
Background: Single-use cardiac catheters are being reprocessed between patient uses. In the past, visual determination was accepted as the endpoint of "clean" prior to sterilization. However, it is not possible to visually determine "clean" on opaque steerable devices.
Methods: To determine quantitative endpoints for "clean", we measured residual total protein (TP) and total organic carbon (TOC) levels on 15 bidirectional steerable cardiac ablation catheters before and after reprocessing with commercially available cleaners. We simulated worst-case clinical use by inoculating each numbered device with a test soil containing 50% 3-protein test soil and 50% porcine whole blood which was then left to dry prior to washing. Inoculation and cleaning were repeated 5 times to simulate up to 6 clinical uses. Each device was extracted separately and tested for TP, using Bradford's protein reagent, and for TOC, using a direct digestion method.
Results: The highest amount of residual TP and TOC found on pre-cleaned device extracts was 230.8 ug/cm2 and 106.5 ug/cm2, respectively. The highest amount of residual TP and TOC on post-cleaned device extracts was 4.0 ug/cm2 and 10.4 ug/cm2, respectively. All post-cleaned devices were visually clean.
Conclusions: Our TP and TOC results for cleaned devices are comparable to those found by others for cleaned reusable devices. Visual detection underestimates residual biosoil remaining on devices after reprocessing. Our results provide quantitative TP and TOC acceptance criteria for cleaned single-use bidirectional steerable cardiac devices.
C. H. Lee, W. C. Kuo, CBER, FDA, Rockville, MD
Background: Administration of multi-valent (combination) vaccines has become a trend due to economic and logistic advantages and better patient compliance. Prevnar (Wyeth Lederle) and Menactra (Aventis Pasteur) are examples of multi-valent conjugate vaccines formulated with individually synthesized component polysaccharide (PS) conjugates. To simplify the synthesis, we investigated the preparation of multi-valent conjugate vaccines by conjugating a mixture of PS to a carrier protein, tetanus toxoid (TT).
Methods: An aldehyde- or cyanate-activated PS mixture (from Haemophilus influenzae type b, Neisseria meningitidis and Streptococcus pneumoniae) was reacted with hydrazide-activated TT to form a mixture of conjugate products followed by sodium borohydride reduction and dialysis. The conjugate products were characterized by size-exclusion HPLC and PS-specific antibody-detected ELISA. The immunogenicity of the conjugate products was tested in mice.
Results: Formation of high molecular weight species upon conjugation was indicated by a shift of protein signal in gel filtration HPLC profiles. The conjugated PS in HPLC fractions were detected by ELISA with respective PS-specific antibody and their profiles superimposed with that of the conjugated protein. This is indicative of the presence of PS and protein conjugate complexes. The conjugate products were immunogenic, shown by antibody induction against component PS in mice.
Conclusion: Our results have demonstrated the feasibility of performing combined synthesis of multi-valent conjugate vaccines yielding immunogenic products. The simplified combined synthesis of multi-valent conjugate vaccines may reduce the cost of vaccine manufacturing, and promote public health.
J. N. Lozier, F. d'Agnillo, CBER, FDA, Rockville, MD
High dose adenovirus vector administration in vivo has been associated with toxicity toward many cell types, including endothelial cells. Some of the prominent pathological features of an adenovirus vector death in a gene therapy trial included capillary leak syndrome and disseminated intravascular coagulation (DIC). We investigated the hypothesis that activated protein C (APC) might have a protective effect on primary human microvascular endothelial cells exposed to a first-generation adenovirus vector. We exposed primary human endothelial cells to a first-generation (E1, E3 deleted) adenovirus vector, AVC3FIX5 at concentrations ranging from 103 to 105 vector particles per cell and showed dose-dependent cell death as early as 6 hours (40% cell death at the highest dose). Phase contrast and immunofluorescence microscopy revealed that some cells died rapidly by primary necrosis while others died by apoptosis over a longer time course. By 40 hours, only 40% of the cells were viable. We then tested the effect of pretreatment of endothelial cells with APC concentrations ranging from 1 nM to 100 nM. Dose-dependent protection was seen in which cell death was reduced to 9 and 2 % at APC concentrations of 50 and 100 nM, respectively. We also tested the effect of timing of the APC treatment and showed that 1 hour pre-treatment or concurrent APC treatment were protective, but APC administered one hour after the adenovirus exposure was substantially less protective. This suggested that APC exerts its protective actions on endothelial cells either by interfering with early steps in the interaction of the vector with the cells, (e.g., vector entry) or by modulating death signaling pathways. It has been proposed that APC protects against cell damage in sepsis by interaction with the endothelial cell protein C receptor (EPCR) and protease activated receptor 1 (PAR1) on the endothelial cell surface to induce MCP-1 and other immunomodulatory genes by proteolytic signaling (Riewald et al., Science 296:1880-1882, 2002). Other investigators have shown protective effects of APC for endothelial cells subjected to hypoxia through normalization of levels of p53, Bax, and Bcl-2 gene expression (Cheng et al., Nat Med 9:338-342, 2003). The APC concentrations in our experiments that were maximally protective (50-100 nM) were of the same order of magnitude as was shown to be protective in vitro by these investigators. If APC can be shown to have a protective effect against adenovirus-induced endothelial cell toxicity and DIC in vivo, this may be a useful therapeutic strategy to explore as treatement of gene therapy vector toxicity.
A. D. Lucas, D. Wray-Cahen, R. P. Brown, S. K. Lappalainen, CDRH, FDA Silver Spring MD
Background: Hyperthermia is known to increase the sensitivity of cells to the cytotoxic effect of chemicals and drugs in vitro. Although cytotoxicity testing of extracts from medical device materials is typically conducted at 37°C (per ISO 10993-5 standard), it may be more clinically relevant to screen extracts from device materials for in vitro cytotoxicity at a temperature that is more representative of that found in febrile patients. To address this issue, the cytotoxicities of selected chemicals, drugs, and medical device extracts were compared in Jurkat cells (a human lymphoma cell line) following incubation at either normothermic (37°C) or mildly hyperthermic (39oC) conditions.
Methods: Jurkat cells were initially grown and incubated at 37°C. About 12 hours before adding a compound or extract, the cells were split and half continued to be incubated at 37°C; half were incubated at 39oC. The cells were then incubated with different solvents, metals, drugs, and device extracts at 37 or 39°C for 48 hours. Cytotoxicity was assessed (using flow cytometry) at 4, 24 and 48 hours following addition of the compound or extract.
Results: The cytotoxicity of solvents, metal salts, and some extracts in Jurkat cells at the 24 and 48 hours was significantly greater under mild hyperthermic conditions
Conclusions: Many exogenous compounds are more cytotoxic under hyperthermic conditions. In vitro cytotoxicity testing of chemicals or extracts from medical devices at elevated temperatures may be more clinically relevant than testing under normothermic conditions for febrile patients exposed to these chemicals or extracts.
S. M. Madson1 , E. W. Laster1 , M. Z. Thomas1 , K. A. Watts1 , J. N. Sofos2 , 1FDA Denver District Laboratory, Denver, CO 80225, 2Colorado State University, Fort Collins, CO 80523
Shigella is a pathogen of concern due to its presence in produce, and because a low dose can cause infections. Detection of this organism in produce by FDA laboratories uses a PCR procedure and a cultural method involving aerobic incubation (FDA Domestic and Import Produce Assignments). While PCR detection is quite sensitive, it is extremely difficult to isolate a culture from samples that are positive for the ipaH gene, possibly due to background flora overgrowth. Pathatrix is a new technology patented by Matrix Microscience for the concentration of cells of various food pathogens through the use of immunomagnetic capture beads. Cationically charged beads for the capture ofcells from foods are also available. Since no immunomagnetic beads specific for Shigella were available, in this study, we evaluated the cationic beads for potential use in the detection of Shigella in cantaloupe rinses. A S. sonnei culture containing a green fluorescent protein (GFP) gene was used to inoculate cantaloupe rinses at various levels. The Pathatrix concentration step with or without pre-enrichment was followed by cultural detection after aerobic or anaerobic enrichment. The results of this preliminary testing indicated that the cationic beads used were able to capture Shigella, but since they are not specific for Shigella, they also captured and concentrated other background microorganisms which outgrew Shigella during enrichment. Use of specific Shigella immunomagnetic capture beads in combination with anaerobic pre-enrichment and/or enrichment should enhance detection of the pathogen.
R. K. Mahajan, A. Vilalta, J. Hartikka, T. Martin, D. Jones, V. Bozoukova, D. Rusalov, P. Lalor, K. Hall, M. Sawdey, A. Rolland, D. Kaslow, Vical, Inc.
Numerous plasmid DNA (pDNA)-based vaccines have been evaluated for safety and efficacy in humans. A unique concern raised for DNA vaccines is the theoretical potential for integration into host genomic DNA (gDNA). Although the absence of detectable integration of phosphate-buffered saline (PBS)-delivered pDNA in non-clinical studies is well documented, novel formulations and/or devices to enhance delivery of pDNA-based vaccines have triggered requirements for further evaluation of the potential risk of integration.
Persistence/integration studies were conducted to determine levels of pDNA remaining in injected muscle after intramuscular injection of cationic lipid- or poloxamer-formulated pDNA, and electroporation (EP)-delivered pDNA.Residual pDNA copy number (PCN) in muscle gDNA collected early (2-5 days), one month, and two months post-administration was measured by quantitative polymerase chain reaction. To assess potential integration of PCN remaining in muscle two months post-administration, total DNA was fractionated by column agarose gel electrophoresis (CAGE) to separate extrachromosomal pDNA from high molecular weight (HMW) gDNA.
Significantly different PCN clearance kinetics were observed for the three groups analyzed.PCN for electroporation delivery exhibited a rapid (~6-log) decline by Day 5. At one month, PCN for the lipid and poloxamer-formulations had declined by approximately four logs. By two months, PCN in all three groups were similar.CAGE fractionation of total gDNA from Day 60-64 muscles showed that remaining pDNA fractionated from HMW gDNA to non-quantifiable levels.
These data demonstrate that the integration-related profile of pDNA-based vaccines is not adversely affected by cationic lipid or poloxamer formulations or by delivery of pDNA using electroporation.
K.D. Mainigi, CDER, Silver Spring, MD
O. A. Maximova1 , I. Bacik1 , K. L. Pomeroy1 , J. Cervenak1 , I. Vasilyeva2 , O. Yakovleva2 , L. Cervenakova2 , P. Piccardo1 , D. M. Asher1 , 1CBER, FDA, Rockville , MD, 2The Jerome Holland Laboratory, American Red Cross, Rockville, MD
Background: There is a theoretical risk that TSE agents (prions) might accidentally contaminate cell substrates used to prepare FDA-regulated biological products. Susceptibility of cultured cells to TSE infection cannot be predicted from tissue of origin or level of expression of normal cellular prion protein (PrPC). However, recent studies successfully infected two murine fibroblast cell lines with scrapie agent. We are investigating the ability of cell lines more relevant for manufacture of biologics to propagate TSE agents.
Methods: We inoculated filtrates of four TSE Reference Agent inocula: BSE agent (Swiss cow brain), sporadic Creutzfeldt-Jakob disease (WHO sCJD human brain), variant CJD (WHO vCJD human brain), and normal bovine and human brains into several cell lines: SH-SY5Y human neuroblastoma derivatives overexpressing wild-type or mutant PrP; Vero; CHO; WI-38, EBTR, MDCK and Rab9. We used immunodetection assays (Western blot, cell blot, dot blot, and immunocytochemistry) to test for normal PrPC and abnormal TSE disease-related PrP (PrPTSE).
Results: We detected normal cellular PrPC in all cell lines using antibodies to various PrP epitopes but no PrPTSE in cells exposed to any TSE inoculum tested at passage levels up to 30.
Conclusions: To date, the search for PrPTSE in cell cultures exposed to TSE agents has been negative. We are developing additional methods to detect PrPTSE with increased sensitivity. To assure safety further, we will inject cell lysates into transgenic mice and squirrel monkeys—generally accepted as the gold standard bioassay to detect infectivity.
S. K. Hubert, S. L. Ayers, A. Glenn, E. Hall-Robinson, P. F. McDermott, L. A. Walker, T. Proeschodt, S. Zhao, S. L. Friedman, J. W. Abbott, R. D. Walker, D. G. White, CVM Office of Research, Laurel, MD
Background: We report the results of the first 3 years of Salmonella testing from the Retail Meat component of the National Antimicrobial Resistance Monitoring System (NARMS). Methods: A monthly sampling of chicken breasts, ground turkey, ground beef and pork chops from grocery stores in 6 FoodNet sites (CT, GA, MD, MN, OR, TN) in 2002 and 2 additional sites each in 2003 and 2004 (CA and NY; CO and NM, respectively). Salmonella were serotyped and tested for susceptibility to common antimicrobials. Results: Salmonella contamination of ground turkey decreased slightly in 2004 to 12.3% from 13.3% in 2003, which was higher than the 11.5% contamination seen in 2002. Salmonella in chicken breasts increased from 9.7% to 13.4% between 2002 and 2004.S. Heidelberg and S. Saintpaul were the predominant serotypes in ground turkey for all 3 years, with a large increase in S. Schwarzengrund in 2004. In 2002, S. Kentucky was the primary serotype in chicken breasts, whereas in 2003 and 2004, S. Typhimurium predominated.The most common resistances among ground turkey isolates during 2002, 2003, and 2004 were to streptomycin (38%, 46%, and 34%), tetracycline (55%, 40%, and 56%), and sulfamethoxazole/ sulfisoxazole (20%, 33%, and 28%), respectively.The dominant resistances among isolates from chicken breasts were ampicillin (17%, 35%, and 31%), sulfamethoxazole/sulfisoxazole (17%, 15%, and 29%) and tetracycline (33%, 29%, and 46%).Resistance to ceftiofur was mainly in chicken breasts for all 3 years. Conclusions: Three years of retail poultry testing in NARMS showed comparable data among Salmonella antimicrobial susceptibilities.
P. F. McDermott1 , S. K. Hubert1 , J. Tang2 , L. Quesada2 , R. D. Walker1 , 1CVM Office of Research, Laurel, MD, 2American Type Culture Collection, Manassas, VA
Background:Long term trends in antimicrobial susceptibility are often difficult to determine. In this study we tested banked clinical isolates of E. coli isolated from food animals and humans between 1950 and 2002 to assess their susceptibility ampicillin and gentamicin. Methods: A total of 1734 E. coli, dating back to 1950, were acquired from various sources. Isolates originated from humans (n=987), chickens (n=139), cattle (n=322) and pigs (n=286). Minimum inhibitory concentrations were determined via broth microdilution and interpreted according to CLSI standards. Results: Ampicillin resistance was present in <5% of isolates before 1970, prior to it's introduction to clinical use. Between the 1970s and 1990s ampicillin resistance increased from 22% to 31%, and further increased to 53% among the 2000-2002 isolates. Overall, ampicillin resistance was higher in animal isolates (34%) versus human isolates (16%). Gentamicin was introduced into clinical practice in the early 1970s. The first detectable resistance among our E. coli occurred in isolates collected in the 1980s with 39% being found to be resistant. This decreased to 12% resistance in the 1990s but increased to 26% in 2000-2002. As with ampicillin resistance, gentamicin resistance was higher in animal isolates (~15%) versus human isolates (<1%). Conclusion: Results demonstrated an increase in ampicillin and gentamicin resistance over the past 3-5 decades in clinical isolates of E. coli, with resistance being more common in isolates from food animals.
A. Mukherjee, K. L. McCutchan, D. Roberson, J. E. LeClerc, T. A. Cebula, CFSAN, FDA, Laurel, MD
Background: Genetic and biochemical markers are useful in identifying foodborne enteric pathogens. Enterohemorrhagic (EHEC) E. coli O157:H7 utilizes sucrose but not D-serine, whereas commensal E. coli K-12 strains utilize D-serine but not sucrose. The difference is due to a D-serine operon and absence of sucrose-utilizing genes in K-12, whereas O157:H7 has a sucrose regulon, which is inserted into the D-serine operon causing its partial deletion.Here, we investigated the sucrose and D-serine phenotypes in isolates of EHEC, enteropathogenic (EPEC), and extraintestinal pathogenic (ExPEC) strains of E. coli.
Methods: Screening for growth on sucrose or D-serine was done in a Biolog phenotypic microarray system and confirmed by plating on minimal medium containing either sucrose or D-serine. PCR assays were used to probe the genetic architecture of the regulons and to determine if their structure is similar to the O157:H7 or K-12 strains.
Results: All 120 strains of E. coli O157:H7, 33 other EHEC strains, and 19 EPEC strains examined utilized sucrose, but not D-serine, and harbored a sucrose regulon like that in the sequenced strain of O157:H7, EDL933. Among 23 ExPEC strains, 16 (70%) grew on D-serine, but not sucrose, and had a D-serine operon. The remaining seven isolates showed the opposite phenotype and had a genetic architecture like EDL933.
Conclusion: All 172 diarrheagenic E. coli examined had a sucrose+ D-ser- phenotype, but the phenotypes among ExPEC strains were more diverse. Such diversity may reflect the selection pressures that ExPEC strains experience in establishing and populating their varied extraintestinal niches in human infection.
E. Job1 , G. Wagley2 , S. F. Al-Khaldi2 , S. L. Foley3 , D. E. Farmer2 , M. O. Walderhaug2 , K. Kerdahi2 , C. E. Cerniglia2 , R. Nayak2 , 1Brigham Young Univ., 2FDA, 3Marshfield Clinic Research Foundation
Background: The emergence of antimicrobial resistance and virulence phenotypes in Salmonella pose a serious threat to the public health world wide.
Methods: We investigated antimicrobial resistance and virulence phenotypes of 21 strains of Salmonella (Enteritidis, Heidelberg, Miami, Anatum, Tallahassee, Newport, Iverness, Manhattan, Florida and Give) associated with Roma tomatoes outbreak and 17 strains isolated from chicken egg houses. The strains were mapped by XbaI-PFGE restriction profiles, tested for patterns of antimicrobial susceptibility testing profile and for the presence of virulence genes.
Results: The PFGE-dendrogram grouped the outbreak strains into two groups (>70% homology between genotypes), while the strains from egg houses were grouped (>80% homology) in two separate, but distinctly different from the outbreak clusters. All strains tested were found to be susceptible to each of the 15 antimicrobial agents in the NARMS drug panel. Salmonella strains were found to possess a complex pattern of virulence genes that allow these bacteria to enter, internalize and survive the host epithelial cells (invA, spi4N, spi4F, spi4H, misL and rmbA), and regulate the biochemical processes (aceK, orgA, purR and ttrB)
Conclusions: Salmonella strains are widely distributed in the food production environment and pose a serious threat as a pathogen. The presence of virulence genes contributes to the ability of this pathogen to cause serious illnesses and a biological agent of accidental and/or deliberate outbreak. Lack of antimicrobial resistance in the strains tested suggests that either no antimicrobials were used in the sampled environment or these strains were not exposed long enough to select for antimicrobial resistance.
A. A. Neverov1 , M. A. Riddell2 , W. J. Moss3 , D. V. Volokhov1 , P. A. Rota4 , L. E. Lowe4 , D. Chibo2 , S. B. Smit5 , D. E. Griffin3 , K. M. Chumakov1 , V. E. Chizhikov1 , 1CBER, FDA, Rockville, MD, 20852, 2Victorian Infectious Diseases Reference Laboratory, Melbourne, Victoria, Australia, 3051, 3Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, 21205, 4National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, 5National Institute for Communicable Diseases, Johannesburg, South Africa, 2131
The approach based on the use of pattern recognition for viral genotyping, could be easily adapted for application in other assays where discrimination of closely related sequences is required. It eliminates the need to design strictly single genotype-specific oligoprobes for all known genotypes, and therefore allows many other oligoprobes that bind more than one genotype to be used to produce interpretable genotyping information.
C. E. Allen1 , R. Lieber1 , E. Plum1 , S. Garfield2 , S. Wincovitch2 , J. M. Newell1 , C. Kimchi-Sarfaty1 , 1CBER, FDA, Bethesda, MD, 2NCI, NIH, Bethesda, MD
ADAMTS13, the Von Willebrand Factor (VWF) cleavage protease, is a secreted protein 37 kb in size that is most highly expressed in liver cells. Several studies (reviewed by Levy et al., 2005) have suggested that the C-terminus of the ADAMTS13 protein is not necessary for cleavage of VWF.In this study, we traced the pathway and expression pattern of endogenous and synthetic ADAMTS13 and we established expression and function.Experiments were performed in kidney and various liver cell lines, using confocal microscopy, Fluorescence Activated Cell Sorting (FACS) and Western blot.The transduced protein function was confirmed by a FRETS-VWF assay. Using several antibodies, we showed that the transduced ADAMTS13 can be recognized, within 5 hours post transduction near the nucleus. Confocal microscopy shows that the protein progresses through the Golgi to the Endoplasmic Reticulum (ER), where it remains detectable for 5-6 days post-transduction. These results were confirmed by FACS and Western blots showing high cellular expression five days post transduction. Pep4, a specific monoclonal antibody that binds to the C- terminus of the molecule, revealed a nuclear localization of the protein in all the liver and kidney cells tested. This nuclear localization was verified as a 41 kDa band on Western blots of the nuclear lysates and cytoplasm preparations from the transfected cells. These results suggest that an isoform of the ADAMTS13 protein might have a function in the nucleus which is different from cleavage of Von Willebrand Factor.
J. M. Newell1 , C. E. Allen1 , A. M. Calcagno2 , Z. E. Sauna2 , C. Kimchi-Sarfaty1 , 1CBER, FDA, Bethesda, MD, 2NCI, NIH, Bethesda, MD
ADAMTS13 is the metalloprotease responsible for cleaving Von Willebrand Factor (VWF). Inhibition of ADAMTS13 or the lack of functional ADAMTS13 leads to the accumulation of ultra-large Von Willebrand multimers (ULVWM) in the microvasculature leading to thrombotic thrombocytopenic purpura (TTP). Studies have reported that cyclosporine A (CsA), an immunosuppressant, can both help and hinder TTP treatment. Transplantation patients sometimes develop TTP due to CsA treatment, as it has toxic effects on endothelial cells, causing an overwhelming release of VWF. Also, discontinuation of CsA treatment leads to an increase in ADAMTS13 activity. However, CsA has been used successfully to treat refractory idiopathic TTP. In such cases, severe TTP patients show an increase in ADAMTS13 activity during CsA treatment. Regarding the action of CsA, we seek to elucidate the exact effect of CsA on TTP patients and their ability to manage ULVWM. Specifically, we have monitored in vitro mRNA levels, protein expression, and function of ADAMTS13 in CsA treated liver and kidney cells. RT-PCR results indicated that CsA does not affect mRNA levels. Rather, expression measured by FACS analysis and a functional FRETS assay showed that CsA may influence ADAMTS13 at the protein level. In particular, CsA may affect functionality by inducing a conformational change in ADAMTS13. A time course of varying CsA concentrations revealed different trends in ADAMTS13 expression and function. Future studies will examine similar parameters in recombinant VWF and ADAMTS13. These results may help to explain discrepancies in the literature regarding the effect of CsA treatment.
M. T. Niu, M. Knippen, L. Simmons, L. Holness, CBER, FDA, Rockville, MD
Background: Bacterial sepsis is the third most commonly reported cause of transfusion-related death.Between October 1, 1995 and September 30, 2004, there were 665 transfusion deaths.Eighty-five (13%) were due to bacterial sepsis, of which 58 (68%) were due to Gram-negative organisms.The most common Gram-negative organism was Klebsiella pneumoniae.
Methods: Retrospective case series of transfusion-transmitted Klebsiella pneumoniae deaths reported to the FDA, 1995-2004.
Results: There were 12 deaths due to K. pneumoniae infection.Eleven deaths were caused by transfusion of platelets and one death attributed to contaminated red blood cells.7 of 12 (58%) cases occurred in 2002.Review of these 7 fatality reports did not identify a common (shared) lot for items used during collection or processing of the blood product.The median age of cases was 61 years (range = 33-79 years); 6 were males and 6 females.67% of cases were recipients of split apheresis units.The median time from transfusion to death was 2 days (range=1-14 days), and the median age of platelets was 5 days (range=3-5 days).On donor follow-up, 90% were healthy at the time of donation, 40% had blood cultures drawn (all of which were negative), and 56% were frequent plasmapheresis donors.
Conclusions: In cases of suspected transfusion-transmitted septicemia, broad-spectrum antibiotic coverage including gram-negative coverage should be considered.Strict adherence to infection control measures while collecting, processing, and handling blood and blood components should be employed.Further development of simple and effective test procedures for detecting bacteria in the blood is needed.
J. E. Groves, D. G. Nyachuba, C. W. Donnelly, Department of Nutrition & Food Sciences, University of Vermont, Burlington, VT 05405
Background: Conventional methods for L. monocytogenes detection require 48+ hours enrichment. The 3MTM PetrifilmTM Environmental Listeria (EL) Plate method is a no-enrichment system providing results within 29+2 hrs.
Methods: We inoculated 4-100 cm2 environmental surfaces including brick, dairy board, epoxy resin, and stainless steel with L. monocytogenes CWD 2102 at 6.8 x 105 CFU/ml. Surfaces were air-dried, then sampled by 3 methods (sponges, 3M™ Quick Swabs, and the Microbial-Vac™ (M-Vac) system). The USDA (USDA/FSIS), International Organization for Standardization (ISO), modified USDA/FSIS (mUSDA) and the 3M Petrifilm EL Plate methods were used to detect Listeria on the surfaces. The three sampling devices were evaluated for recovery of Listeria and the four detection methods were compared.
Results: Of the initial population, only 8.2, 4.3, 1.3x101, 1.5x101 CFU/100 cm2 was recovered from the brick, dairy board, epoxy resin, and stainless steel, respectively, indicating sublethal injury or death resulting from drying. The mUSDA/FSIS method detected 100% of the contaminated samples. No significant difference (p<0.05) was found between the 3M Petrifilm EL Plate (76.4%) and USDA-FSIS methods (83.3%). The ISO method was the least sensitive (54.2%). There was no significant difference (p<0.05) between sponges (87/96) and swabs (81/96) in recovering Listeria from the contaminated surfaces. The M-Vac recovered 60% of the positive samples.
Conclusions: The data indicate that sublethal injury is a factor in recovery of Listeria from environmental surfaces. The 3M Petrifilm EL Plate and USDA/FSIS methods are statistically equivalent for environmental detection of L. monocytogenes sampled by sponges, swabs, or the M-Vac system.M. Paul-Satyaseela, S. Gudlavalleti, M. S. Blake, C. E. Frasch, M. C. Bash, CBER, FDA, Rockville, MD USA
Background: Completion of bacterial genomes, together with technological advancement in chemistry has opened unparalleled opportunities for the development of new anti-infective compounds and vaccines. We focus on developing human cell-based in vitro toxicity assays necessary for evaluating vaccines developed by the "reverse-vaccinology" approach for serogroup-B Neisseria meningitidis. Human coronary arterial (HCAEC) and brain microvascular (HBMEC) endothelial cells were used to assess the toxicity of variations of outer membrane vesicle (OMV) vaccines to establish the assay methods. Methods: Native-OMV (NOMV) and detergent-treated-OMV (DOMV) were prepared (Meth. Mol. Med-66:81-107, 2001). Endotoxin controls included purified N.m lipooligosaccharide (LOS) and lipopolysaccharide (LPS) from E. coli. OMV preparations and controls were dialyzed, adjusted to final concentrations of 10, 1.0, and 0.1 μg/ml protein (OMVs), and 1.0, 0.1, and 0.01 μg/ml 2-keto-3-deoxyoctonate (KDO) (LPS and LOS). NOMV contained 2.4, 0.24, and 0.024μg/ml KDO respectively. HCAEC and HBMEC were exposed to the preparations for 24 hrs. Cells were observed microscopically, and supernatants collected for TNF-α determination by ELISA. Results & Conclusion: NOMV stimulated detectable levels of TNF-α in a dose dependant manner, while DOMV and endotoxin controls did not. At the highest levels of NOMV, cytotoxic effects were observed morphologically. These findings indicate that meningococcal LOS may be only a minor factor involved in the observed in vitro cytotoxicity of endothelial cells. LOS-independent elements fractionated during DOMV preparation may play a more critical role. Studies to elucidate the meningococcal outer membrane component(s) that lead to the observed cytotoxic effects are ongoing.
*Correspondence: Margaret.Bash@FDA.hhs.gov
L. Sheng, R. Omeir, M. Yacobucci, M. Klutch, A. Pal, A. Lewis, K. Peden, CBER, FDA, Bethesda, MD
Background: SV40 was present in poliovirus vaccines from the late 1950s to early 1960s. These vaccines were manufactured in kidney cells from rhesus monkeys infected with SV40. Over the last decade, SV40 sequences have been found in certain human tumors. Because many of the individuals with these tumors were born after 1963 and did not receive SV40-contaminated vaccine, the origin of the SV40 in these tumors is unknown. If SV40 is circulating in the human population, then there should be serological evidence of its presence. Neutralization assays are commonly used to demonstrate the presence of viral infection. Because humans are infected with BKV and JCV, we have developed neutralization assays for these polyomaviruses as well as for SV40. If SV40 was introduced into humans via vaccination, then the strains present in the human tumors should be those present in vaccines. Unfortunately, SV40-contaminated vaccines are not available. As an alternative, we have isolated SV40 from adenovirus and poliovirus stocks that were prepared in primary rhesus monkey kidney cells at the same time as when contaminated poliovirus vaccines were manufactured reasoning that the SV40 strains endemic in US primate colonies at that time would likely be the ones present in the vaccines.
Methods: Reporter viruses for SV40, BKV, and JCV were prepared and shown to be infectious for a single round. Infection and neutralization were monitored by luciferase assay. The neutralization assay was characterized using various sera and monoclonal antibodies. SV40 was isolated from adenovirus and poliovirus stocks that had been propagated in primary rhesus monkey kidney cells between 1957 and 1961. The adenovirus or poliovirus was inactivated by heating, and SV40 was obtained after infection of CV-1 cells. The SV40 genomes were cloned in a plasmid vector, and, after demonstrating that they were infectious, the genomes were sequenced.
Results: The single-cycle reporter virus neutralization assay has been developed for the three primate polyomaviruses. In the case of SV40, antibody neutralization titers with several sera and monoclonal antibodies agreed well with titers obtained using the classical plaque neutralization assay. Neutralizing antibodies generated against BKV also neutralized SV40, demonstrating that these viruses share epitopes. Using the assays with nine human sera demonstrated that all nine had antibodies directed against BKV and JCV, while some individuals had SV40-neutralizing antibodies. With respect to the isolation of SV40 strains, several new strains were identified by their polymorphisms in the C-terminal region of the T-antigen gene; a number of recovered strains were identical to previously characterized strains, including the common SV40 strains 776 and 777. None of the viruses had two complete copies of the 72-bp element. The polymorphism did not extend to the other genes, as the sequences of these were identical in all nine strains and perhaps in all published infectious genomes. Because the same strains have being isolated by different laboratories, it is likely that the number of SV40 strains in the US might be lower than expected.
Conclusions: A single-cycle reporter virus neutralization assay has been developed that is capable of detecting and quantifying neutralizing antibodies to the three primate polyomaviruses (SV40, BKV, and JCV) in human serum. The assay should help determine whether SV40-specific antibodies exist and whether SV40 is circulating in humans. Because there is limited heterogeneity in VP1 proteins, it is likely that SV40 is a single serotype. Also, because of the limited number of SV40 strains circulating, assignment of the origin of the SV40 in tumors might not be possible.
E. P. Plant1 , J. D. Dinman2 , D. R. Taylor1 , 1CBER, Office of Blood Research and Review, FDA, Bethesda, MD, 2CBMG, University of Maryland, College Park, MD
M. Rios1 , S. Daniel1 , S. L. Stramer2 , I. K. Hewlett1 , 1LMV/DETTD/OBRR/CBER/FDA, Rockville, MD, 2American Red Cross, Gaithersburg, MD
| Specimen | RBC | STD | Plasma | STD |
| ARC01.04 | 2.30E+03 | ± 903.8 | 1.10E+03 | ± 900.8 |
| ARC02.04 | 1.30E+03 | ± 145.1 | 1.20E+02 | ± 51.6 |
| ARC03.04 | 1.70E+03 | ± 885.2 | 2.10E+02 | ± 216.5 |
| ARC04.04 | 1.60E+03 | ± 1125.9 | 1.30E+02 | ± 123.6 |
| ARC05.04 | 2.90E+03 | ± 597.7 | 1.80E+02 | ± 115.1 |
| ARC06.04 | 3.20E+03 | ± 626.1 | 2.40E+02 | ± 35.5 |
| ARC07.04 | 3.40E+03 | ± 563.6 | 2.70E+02 | ± 162.3 |
| ARC08.04 | 2.90E+03 | ± 381.4 | 8.80E+01 | ± 63.3 |
| ARC09.04 | 2.40E+03 | ± 744.0 | 8.70E+01 | ± 45.1 |
CONCLUSION: These observations reinforce the notion that future improvements in assay development for blood screening should include sample preparation methods for whole blood to enhance sensitivity of detection and further reduce the residual risk of transmission of the infectious agent by transfusion
.M. Rios1 , S. Daniel1 , S. L. Stramer2 , S. Caglioti3 , O. Wood1 , I. K. Hewlett1 , 1LMV/DETTD/OBRR/CBER/FDA, Rockville, MD, 2American Red Cross, Gaithersburg, MD, 3Blood Systems Laboratory, Tampe, AZ
Blood units identified as WNV nucleic acid test (NAT) positive can be classified into 5 different stages of infection: I. early phase with very low viral load only detected sporadically positive by individual-NAT (ID-NAT) and IgM negative. II. Low viral load consistently detected by ID-NAT but negative for minipool-NAT (MP-NAT) and IgM; III. High viral load detectable by MP-NAT and IgM negative. IV. Decreasing viral load only detectable by ID-NAT and IgM positive; V. Convalescent phase with low viral load, negative by MP-NAT sporadically ID-NAT positive and IgM/IgG positive. Retrospective studies led to the identification of WNV transmission by transfusion. There are 30 cases of WNV transmission by transfusion, and retention samples from these donations all tested WNV ID-NAT positive and IgM negative. In addition, it was observed that most WNV low level RNA donations (MP-NAT negative and ID-NAT positive) also had IgM. That raises the question of whether donation ID-NAT and IgM positive but MP-NAT negative are infectious (transmits WNV to recipients). Currently there is no data on infectivity of such units. Animal models currently available do not have the capability of clarifying this issue. We investigated whether viremic plasmas containing IgM and/or IgG could infect susceptible cells in vitro. Vero cell and human monocyte-derived-macrophage (MDM) culture systems were used for infectivity studies of 48 WNV positive plasma samples to address this question. Cells were observed for cytopathic effect (CPE) on a daily basis. MDM cultures were used to test 19/48 plasmas and infectivity in MDM was assayed by RT-PCR-TaqMan for the WNV 3'NC region because WNV infection in MDM does not result in CPE. In the Vero cells infectivity assay 33/48 plasmas were CPE-positive by day 7, including 7 plasmas containing antibodies (3 IgM+ IgG-; 1 IgM- IgG+ and 3 IgM+ IgG+). RT-PCR-TaqMan results showed that 17/19 plasmas infected MDM cultures, including 4 plasmas that did not infect Vero cell
| Serology Negative | Cell culture positive/total | |||
|---|---|---|---|---|
| Vero cells | MDM | Combined | ||
| NAT | MP+ ID+ | 27/30 | 9/9 | 27/30 |
| NAT | MP+/- ID + | 0/2 | 2/2 | 2/2 |
| Serology Positive | Cell culture positive/total | |||
|---|---|---|---|---|
| Vero cells | MDM | Combined | ||
| NAT | MP+ ID+ | 4/9 | 4/4 | 6/9 |
| NAT | MP- ID+ | 2/7 | 2/4 | 4/7 |
In conclusion, WNV positive plasmas containing IgG and/or IgM are infectious for Vero cells and/or human MDM in vitro. Although, in vitro system does not represent in vivo situations the issue needs to be properly addressed. Therefore, non-human primate studies are needed to clarify the relevance of this finding in vivo.
A. Grinev1 , S. Daniel1 , S. L. Stramer2 , S. Caglioti3 , I. K. Hewlett1 , M. Rios1 , 1LMV/DETTD/OBRR/CBER/FDA, Rockville, MD, 2American Red Cross, Gaithersburg, MD, 3Blood Systems Laboratories, Tempe, AZ
This study aimed to investigate potential genetic variation of WNV over time during 2002 to 2005 epidemics in the US. WNV first appeared in the US in 1999 and since has reoccurred in the following 6 consecutive years, and in 2002 caused largest meningoencephalitis outbreak in the Western hemisphere and the largest WNV outbreak ever reported. Immeasurable efforts have been directed to the study of WNV, and information about its potential for genomic variation is desirable and important to understand viral evolution. METHODS: Twenty-five WNV-NAT-positive plasmas collected during the 2002 (n=7), 2003 (n=9), 2004 (n=7) and 2005 (n=2) epidemics, were used for virus isolation in Vero cells. RNA was extracted from the isolates, reverse transcribed and the cDNA used for sequence analysis. Five isolates (FDA-Hu2002, Hu10-02, Hu05-03, HuTX1-05 and HuTX2-05) were completely sequenced, while the 23 other isolates had only viral structural regions (5'NTR, Cap, preM, M and Env) sequenced. Sequence results were analyzed using Vector NTI software and compared to genetic sequences from the prototype WN-NY99 isolate. RESULTS: The isolate FDA-Hu2002 (from 2002) had 20 nucleotide (nt) mutations and one nt insertion (T at position 10497); Five of the 20 mutations were associated with amino acid (aa) substitutions (M22T; A52V; V449A; N684S; S2301G). The isolate Hu10-02 from 2002 had 22 nt mutations 3 of which resulted in aa substitutions (V449A; V2213A; E2826G). The isolate FDA-Hu05-03 from 2003 had 37 nt mutations 8 of which resulted in aa substitutions (V449A; I662V; I798V; I1169V; A1480T; P1971S; K2842R; A2997T). The isolate FDA- HuTX1-05, from 2005, had 41 nt mutations 3 of which resulted in aa substitutions (V449A; L895F; P3056S). The isolate FDA- HuTX2-05, from 2005, had 56 nt mutations 17 of which resulted in aa substitutions (G122R; N298S; V449A; V1611A; M1688T; R1690K; G1702D; F2193L; A2209T; L2261M; G2755R; N2787S; S2820L; K2842R; R3104C; S3132R; M3191T).The Env gene region from 22 of the 23 other isolates had 2 common mutations (1442T>C [V449A] and 2466C>T [silent]). One isolate from 2002 had two silent mutations (1487T>C and 1773C>T) in the envelope gene that were not found in the other 22 isolates. The mutation 660C>T in the prM region was present 19/25 isolates (2 from 2005, 7 from 2004, 7/9 from 2003 and 3/7 from 2002). Isolates from 2004 shared 2 silent mutations in the envelope gene (1320A>G in all 7 and 1974C>T in 6/7) not observed in the 18 isolates from previous years. CONCLUSION: The small number of nucleotide mutations in the envelope gene and the single conservative amino acid substitution in the envelope variable region suggest the absence of strong selective pressure and limited evolution of West Nile virus from 2002 to 2005. However, some mutations lead to aa substitutions that could affect viral pathogenesis and changes in nt and aa sequences that could potentially affect the sensitivity of screening and diagnostic assays.M. G. Robl1 , P. L. Wiesenfeld1 , L. H. Garthoff1 , T. J. Sobotka1 , J. K. Suagee2 , 1OARSA, CFSAN, FDA, Laurel, MD, 2University, of Maryland, College Park, MD
Background: Acute oral toxicity of COL, gender differences and pre-exposure to LPS in rats was investigated (details in Wiesenfeld et al. poster). Histopathology examination of selected tissues was performed to facilitate understanding the gender related toxicity and lethality of COL and interaction with LPS.
Methods: Following a gross necropsy evaluation of most of the rats, tissues were saved and fixed in formalin. Selected tissues from the high dose (30 mg/kg COL of BW) group were processed into H and E stained slides and light microscopic evaluation performed.
Results:Even though more females than males died in rats dosed with 30 mg/kg of BW COL during the first 5 days post dosing (PD), necrosis of bone marrow cells, hepatocytes and lymphocytes was observed in both sexes. The tissues were near normal in surviving rats at 10 days PD. Necrosis of gastrointestinal epithelial cells and/or blunting of the mucosal tissue were found in several rats that died within PD-5. Necrosis of seminiferous tubular epithelium occurred in a few male rats.
The combination of COL (30 mg/kg of BW) and LPS (83 µg/kg of BW) was lethal to most rats. Moderate to severe necrosis of bone marrow cells, hepatocytes and lymphocytes were observed in both sexes. The lesions were minimal and tissues near normal in the few surviving animals at PD-10.
Conclusion: Rats of both sexes that died within 5 days of a single oral dose of 30 mg/kg of BW COL had necrosis of bone marrow cells, lymphocytes and hepatocytes. Necrosis of gastrointestinal and seminiferous tubular epithelial cells also occurred in a few rats. Most of the lesions were similar but more severe when COL and LPS were co-administered. Minimal abnormalities were observed in surviving animals at PD-10. In order to further evaluate COL and LPS gender related differences in lethality, future work may include examination of tissues saved from mid and low dose COL treated rats.
M. G. Robl1 , P. L. Wiesenfeld1 , L. H. Garthoff1 , T. J. Sobotka1 , J. K. Suagee2 , 1OARSA, CFSAN, FDA, Laurel, MD, 2University of Maryland, College Park, MD
Background: Acute oral toxicity of colchicine (COL) in rats is not clearly defined. A modification of 2001 OECD 425 Guideline (Up-and-Down Procedure) was used to establish a maximum experimental dose. Histopathology and evaluation of target tissues was used to ascertain acute toxicity effects.
Method: Nine young adult female S-D rats were given a single oral dose of COL (range - 10 to 50 mg/kg of BW). Clinical findings were made and rats euthanized at post dosing days (PD) 1 - 7. Representative tissues were saved in formalin, processed and evaluated by light microscopy.
Results: Minimal microscopic effects were observed in single rats dosed with 10 or 15 mg/kg of BW COL at 2 days PD but the rat @ 15 mg/kg/ of BW had minimal necrosis of lymphocytes and bone marrow cells. Similar changes were observed in rats dosed with 15 mg/kg at 4 and 7 days PD. One rat dosed with 20 mg/kg of BW at 1 day PD had hepatocellular necrosis, lymphocyte necrosis in multiple tissues, and marked necrosis of bone marrow cells. These changes were less severe at 2 and 7 days PD in other rats dosed with 20 mg/kg of BW. One rat dosed with 25 and another at 50 mg/kg of BW had hepatocellular necrosis, marked necrosis of lymphocytes in multiple tissues and marked necrosis of bone marrow cells at 4 and 1 day PD, respectively.
Conclusions: By modification of OECD 425 and using light microscopy, we identified liver, lymphocytes, and bone marrow as targets of COL toxicity. The information obtained using only 9 female rats enabled us to select dosage levels of 10, 20, and 30 mg/kg as an operative range for a short-term (10 day) oral COL toxicity study.
P. L. Wiesenfeld1 , L. H. Garthoff1 , T. J. Sobotka1 , J. K. Suagee2 , C. N. Barton3 , 1OARSA, CFSAN, FDA, Laurel, MD, 2University of Maryland, College Park, MD, 3OSAS, CFSAN, FDA, College Park, MD
Background: The potential health hazards of toxic substances accidentally or deliberately added to foods depend not only on their intrinsic toxicity, but also on the effects of various food matrices. In addition, previous exposure to inflammatory environmental toxins, such as LPS, may also modulate their toxicity.
Methods: The oral toxicity of COL, a single administration of 10, 20, or 30mg/kg body weight, was determined in young, age-matched male and female rats. The effect of food matrix on COL toxicity was evaluated by administering COL in either saline or Half & Half (H&H). The influence of LPS pretreatment was determined by administering COL 1 hr after animals received a minimally-toxic ip dose of LPS, (0111:B4, 1 x 10 6 EU/mg) from E. coli (83 µg/kg body weight).Body weight, feed intake and clinical observations of behavior were monitored daily for 10 days postdosing.
Results: Colchicine induced a progressively more severe dose-related toxicity including anorexia, decreased body weight, and mortality. Although there were various gender differences in the severity of the toxic response to COL, the most notable sexually-dimorphic difference was in the lethality of COL. Female rats (LD50 = 26 mg/kg) were significantly more susceptible to the lethal effects of COL than males (LD50 = 48 mg/kg).The use of H&H as vehicle did not affect COL toxicity.LPS as a pretreatment resulted in a significant potentiation of COL lethality in females (LD50 = 15 mg/kg) and males (LD50 =20 mg/kg). The use of H&H did not affect this LPS potentiation of COL lethality.
Conclusion: This study dramatically demonstrated that pre-exposure to the ubiquitous environmental contaminant, LPS, can potentiate toxicity. A single oral dose of COL induced gender-dimorphic acute toxicity in young sexually mature rats. Female rats were considerably more suceptible to the oral, acute toxicity of COL compared to male rats. Pretreatment with LPS potentiated the oral toxicity of COL in both sexes, but male rats were more sensitive to this potentiation effect of LPS. The use of H&H delivery vehicle did not affect oral COL toxicity.
M. Ross, C. Leonard, K. C. Klontz, J. Sanders, J. Sheehan, R. Childers, D. Street, CFSAN, FDA, College PArk
Background: Raw milk (no bactericidal/bacteriostatic treatment, apart from cooling) is a potentially high-risk food for all persons, particularly for pregnant women and young, elderly or infirmed persons.
Methods: We describe episodes of raw milk-associated illnesses in the U. S. reported to the FDA from 2000-2005, as well as an episode of raw milk consumption that did not cause illness but required significant public health efforts to prevent illness. We include episodes related to consumption of raw milk or cheeses made from raw milk.
Results: Sixteen raw milk episodes involving >400 persons in 15 states were reported to FDA during the study period. The pathogens responsible for causing illness included Campylobacter sp., Salmonella sp. E. coli O157:H7, Listeria monocytogenes and Mycobacterium bovis. Many of those affected were children, pregnant women, and/or persons of Mexican origin. Morbidity was significant, involving hospitalizations, miscarriages, premature births, invasive infections, and death. Additionally, intensive prophylactic treatment was administered to consumers of raw milk that was potentially contaminated with rabies virus.
Conclusions: Raw milk consumption continues to cause intrastate and interstate outbreaks of illness in the U.S. Emphasis has previously been placed on the importance of bacterial pathogens. However, given that cattle are susceptible to rabies and that milk can potentially be contaminated with rabies virus, FDA must now consider viral pathogens to be a significant concern with respect to raw milk. Continuous, rigorous educational campaigns are required to inform consumers about the high risks inherent to drinking raw milk and consuming cheeses made from raw milk.
C. J. Sauder1 , C. X. Zhang1 , K. M. Vandenburgh1 , P. Duprex2 , K. M. Carbone1 , S. A. Rubin1 , 1FDA, Bethesda, MD, 2The Queen's University of Belfast, Belfast, UK
Mumps virus is a neurotropic agent causing meningitis in up to 10% of clinical cases. Vaccination using live attenuated mumps viruses is highly efficacious; however, some vaccine strains (e.g., Urabe AM9) have caused meningitis. Understanding the genetic basis of mumps virus neurotoxicity is therefore important for ensuring vaccine safety. To this end we investigated the contribution of the two viral surface proteins, hemagglutinin-neuraminidase (HN) and fusion (F), to neurotoxicity in a rat model. Using reverse genetics, the HN and F genes of the non-neurotoxic Jeryl Lynn (JL) vaccine strain were replaced (individually or together) with the corresponding genes from the Urabe AM9 strain and a neurotoxic wild type clinical isolate (88-1961). When the resulting chimeric viruses were rescued and inoculated into the brains of newborn rats a 3-4 fold increase in neurotoxicity score was noted relative to the non-chimeric JL virus. Further analyses showed the increase in toxicity to specifically be due to the Urabe AM9 F gene (in a JL clone consisting of the Urabe AM9 F gene and the JL HN gene) and the 88-1961 HN gene (in a JL clone consisting of the 88-1961 HN gene and the JL F gene). Notably, neurotoxicity scores were considerably lower as compared to those observed with non-chimeric Urabe AM9 or 88-1961 viruses, suggesting that additional genomic regions contribute to neurotoxicity. These studies are valuable for future development of molecular biology-based neurotoxicity tests for live attenuated mumps vaccines.
A. Selvapandiyan1 , P. Kumar2 , J. C. Morris3 , C. C. Wang2 , H. L. Nakhasi1 , 1CBER, FDA, Bethesda, MD, 2UCSF, San Francisco, CA, 3Clemson University, Clemson, SC
Background: Leishmania donovani, is a causative agent of fatal Visceral disease in humans. At present there is no vaccine for such disease. To develop an effective vaccine, one needs to know the pathogenesis of this parasite. We have characterized several genes which play important roles in parasite growth. One such gene is centrin. Recently, we identified several isoforms of this gene in Leishmania. In the present study we have undertaken to analyze the function of centrin isoforms in a related trypanosomatid, T. brucei using RNA interference (RNAi) methodology.
Methods: RNAi is a mechanism, where introduction of homologous double stranded RNA specifically targets a gene product to make a null phenotype.
Results: Depletion of all the five centrin gene products by RNAi in the procyclic form of T. brucei, revealed, that out of five centrins, only the knockdown of centrins 1, 2 and 3 affected the growth of the parasite in vitro. Microscopic observation revealed that these centrin deficient procyclic cells became enlarged in size and contained multiple basal bodies, kinetoplasts, Golgi, nuclei and detached flagella. These multiple organelles were, however, closely clustered together, indicating duplication without segregation in the absence of centrins. This failure in organelle segregation constitutes the likely cause of cytokinesis inhibition, which in turn results in growth inhibition.
Conclusions: This study suggests a unique role for centrin in the segregation of multiple organelles in the procyclic-form of T. brucei and therefore plays an important role in parasite cell division and growth.This study further demonstrates the importance of centrin genes in trypanosomatid pathogenesis.
D. R. Taylor1 , M. Puig2 , K. Mihalik3 , E. M. Silberstein1 , S. M. Feinstone3 , 1DETTD, CBER, FDA, Rockville, MD, 2DTP, CDER, FDA, Rockville, MD, 3DVP, CBER, FDA, Rockville, MD
Background: While many clinical hepatitis C virus infections are resistant to interferon alpha (IFN) therapy, subgenomic in vitro self-replicating HCV RNA (HCV replicons), are characterized by marked IFN sensitivity. IFN treatment of replicon-containing cells results in a rapid loss of viral RNA via translation inhibition through PKR and also through a new pathway involving RNA editing by adenosine deaminase that acts on double-stranded RNA (ADAR1).
Methods: To measure the effect of IFN on translation, we used a dual-Luc reporter. Replicon-containing cells were cotransfected with either PKR or well-characterized PKR inhibitors, and the Luc reporter. Luciferase activity was measured in cell lysates after IFN-αtreatment. HCV RNA was monitored by real-time RT-PCR. Evidence for ADAR1 dsRNA editing in replicon-containing cells was examined by sequencing of RT-PCR products from cytoplasmic extracts and conversion of radiolabeled AMP to IMP.
Results: We show that inhibition of PKR in replicon-containing cells does not limit the negative effects of IFN. However, inhibition of both PKR and ADAR1 stimulates replicon expression and reduces the amount of inosine recovered from RNA in replicon cells. Small inhibitory RNA, specific for ADAR1, stimulated the replicon 40-fold indicating that ADAR1 has a role in limiting replication of the viral RNA.
Conclusions:This is the first report of ADAR's involvement in a potent antiviral pathway and its action to Replicon-containing cells specifically eliminate HCV RNA through adenosine to inosine editing. These results may explain successful viral clearance by IFN in vitro and may provide a promising new therapeutic strategy to counteract HCV as well as other viral infections.
L. Smith, G. Jimenez, A. Geall, P. Lalor, R. Vahle, R. Planchon, Q. Wei, D. Rusalov, A. Rolland, D. Kaslow, Vical Incorporated
Background: Vical Incorporated, with NIH grant funding, is developing plasmid DNA (pDNA)-based vaccines against H5N1 avian influenza viruses posing the greatest pandemic threat. The vaccines encode HA and internal conserved antigens to stimulate broad T- and B- cell immunity. H3N2 and H1N1 challenge studies were conducted to identify the optimal antigen combination and vaccine formulation to provide protection against lethal challenge.
Methods:BALB/c mice were vaccinated with pDNA encoding HA, NP and/or M2 on days 0 and 21 and challenged on day 42 with an LD90 dose of the A/HK/8/68 (H3N2) or the A/PR/8/34 (H1N1) strain. Survival and weight loss were monitored for 21 days. In preparation for H5N1 challenge studies, pDNA encoding H5 from the A/Vietnam/1203/2004 isolate was combined with NP + M2 pDNA and tested for immunogenicity in mice.
Results:Complete protection was provided by HA pDNA alone or NP + M2 pDNA. Vaxfectin™ formulations provided the greatest protection at low pDNA doses. Complete protection against heterosubtypic challenge was also demonstrated with Vaxfectin™-formulated NP + M2 pDNA. The H5 + NP + M2 pDNA vaccine was shown to be immunogenic.
Conclusions: Vaxfectin™-formulated NP + M2 pDNA vaccines were shown to offer HA-independent protection against human influenza virus isolates. A DNA vaccine composed of H5 + NP + M2 is being advanced to mouse and ferret lethal challenge studies with H5N1 viruses. Phase 1 clinical testing is contingent upon successful proof of concept and safety studies as well as additional financial support.
J. S. Smith1 , S. C. Stevenson2 , J. Tian1 , A. P. Byrnes1 , 1CBER, FDA, Bethesda, MD, 2Department of Diabetes and Metabolism, Novartis Institutes of Biomedical Research Inc., Cambridge, MA
When adenoviral vectors are administered systemically, they are quickly taken up by phagocytes of the reticuloendothelial system, including Kupffer cells (KCs) in the liver. We have recently shown in mice that E1/E3-deleted Ad5 vectors cause rapid KC death, followed by a slower depletion of KCs from the liver (Mol. Ther. 13:108-117, 2006). KCs lost membrane integrity within 10 min after i.v. injection of Ad5 vectors, and this eventually resulted in significant depletion of KCs from the liver. In the current study, we investigated which features of the adenoviral fiber contribute to KC toxicity by administering Ad5 vectors with the fiber partially or completely replaced by the type 35 fiber. When compared to the type 5 fiber, the type 35 fiber does not bind to CAR, has a much shorter shaft, and lacks a putative heparin-binding domain (KKTK) in the shaft. We also directly examined the role of the KKTK domain in the Ad5 shaft. All vectors were provided by Cell Genesys, Inc. and were described in Hum. Gene Ther. 14:777-787, 2003.
C57Bl/6 mice were injected i.v. with vectors and F4/80+ KCs in the liver were counted at 18 h. An Ad5 vector dose of 1011 vp/kg depleted KCs by approximately 80%. KCs were depleted to a similar extent when the type 5 fiber head was replaced with the type 35 fiber head (5S35H), indicating that the type 5 and type 35 fiber heads were interchangeable and that CAR binding was not essential. However, when the type 5 shaft was replaced with the type 35 shaft, no KC depletion was seen, regardless of whether the fiber was completely replaced (35F) or only the shaft was replaced (35S5H). In addition, KC depletion was also absent with an Ad5 vector that had the shaft KKTK sequence replaced with GAGA (S*). This indicated that the shaft length did not determine toxicity, but rather that the putative heparin-binding domain was involved. Of note, all of the vectors were capable of producing KC depletion to some degree when injected at higher doses (1012 vp/kg), indicating that none of the alterations provided absolute protection against KC toxicity.
In summary, we found that the type 5 fiber shaft made a major contribution to KC toxicity. Vectors that lacked the KKTK motif or contained the type 35 fiber shaft were impaired in their ability to deplete KCs. There was no obvious contribution from the type 5 fiber head, and CAR binding played no apparent role in KC toxicity. This information is likely to prove valuable in developing less toxic adenoviral vectors for systemic administration.
R. L. Sprando1 , T. F. Collins1 , T. N. Black1 , N. Olejnik1 , M. Ramos-Valle1 , J. I. Rorie1 , D. I. Ruggles2 , 1CFSAN,FDA, Beltsville, MD, 2CFSAN,FDA, College Park,MD
Background: The ability of half and half unpasteurized cream (HH) to reduce the acute toxicity of a single oral dose of sodium arsenite (As) was assessed.
Methods: Male, non-pregnant female and pregnant rats received a single oral dose of As in HH (As-HH) at 0.41, 4.1 or 41.0 mg/kg body weight. Male and non-pregnant female rats also received a single oral dose of As-HH at 410 mg/kg body weight. Control rats received deionized water alone, HH alone or 41.0 mg/kg As-water. Male and non-pregnant female rats were observed for 14 consecutive days after dosing. Pregnant rats, dosed on gestational day (GD) 10, were observed until GD 20.
Results: All animals receiving 410 mg/kg As-HH died within hours of dosing. No effects on feed consumption, water consumption, body weight gain or organ weights were observed in control or the remaining As-HH treated male and non-pregnant female rats. Changes in feed consumption were observed across the As-HH dose levels in pregnant rats. A statistically significant reduction in gravid uterine weight and mean numbers of viable female fetuses was observed in the 41 mg/kg As-water control group. However similar effects were not observed in the other control and treated groups.
Conclusion: HH did not reduce the acute oral toxicity of As when As was administered at very high doses. Female fetuses appeared to be more sensitive to the effects of As when As was administered in water. Effects on the female fetus were not observed when As was administered in HH.
H. H. Chen, C. J. Stark, C. D. Atreya, DVP, CBER, FDA, Bethesda, MD
Rubella virus protease trans-activity requirement of the substrate is unknown. Here we analyzed the cleavability of RV P200-derived substrates varying in their N-terminal lengths (72-475 amino acids) from the cleavage site by the RV protease trans-activity. Only substrates with at least 309 amino acid residues N-terminal to the cleavage site were able to undergo cleavage. Further, rubella sequence was found necessary in the N-terminal region of the substrate, whereas a heterologous sequence C-terminal to the cleavage site was tolerated. These results demonstrated a requirement for residues located between amino acids 994-1102 of the RV P200 polyprotein, besides its cleavage site for RV protease trans-activity. This region overlaps with the starting site of the essential cis-protease activity of RV P200 polyprotein. This is a novel observation for a viral protease of Togaviridae family.
A. A. Stearns, L. L. English, D. G. White, P. F. McDermott, A. Walker, S. K. Hubert, S. L. Ayers, E. Hall-Robinson, T. Prescholdt, R. D. Walker, FDA, Laurel, MD
Background- The National Antimicrobial Resistance Monitoring System (NARMS) includes surveillance of retail meat for Campylobacter, a leading cause of foodborne illness. Methods- In 2004, FoodNet sites in CA, CO, CT, MN, NM, NY, OR, GA, MD, and TN conducted monthly sampling of chicken breast, ground turkey, ground beef, and pork chops purchased from retail stores and cultured these meats for Campylobacter.Presumptive isolates were sent to the FDA/CVM/OR for confirmation and speciation by PCR and susceptibility testing against eight antimicrobials using the CLSI broth microdilution procedure. Results- Campylobacter were recovered from 15.3% (N=4699) of retail meat samples which included 60.2% of the chicken breast samples, < 1.0% of the ground turkey and pork chop samples, and none of the ground beef samples. C. jejuni was the predominant species isolated (71.7%) followed by C. coli (28.3%). Forty-nine percent of the Campylobacter were resistant to tetracycline (MIC ≥16 µg/mL), 16% to nalidixic acid (MIC ≥32 µg/mL), 15% to ciprofloxacin (MIC ≥4 µg/mL), 4% to telithromycin (MIC ≥8 µg/mL), 3% to azithromycin (MIC ≥16 µg/mL), 3% to erythromycin (MIC ≥32 µg/mL), and 2% to clindamycin (MIC ≥8 µg/mL).All isolates were susceptible to gentamicin (MIC <2 µg/mL) and florfenicol (MIC <4 µg/mL). With the exception of tetracycline, a greater percentage of C. coli showed resistance to antimicrobials than C. jejuni. Multiple resistances were observed in 18.4% of the isolates. Conclusions- Campylobacter, including antimicrobial resistant strains, contaminate retail chicken and can serve as a reservoir of resistant strains in the food processing chain.
S. Tang1 , J. Zhao1 , J. J. Storhoff2 , A. Dhar1 , T. Patno2 , C. A. Mirkin3 , S. Wolinsky4 , I. K. Hewlett1 , 1CBER, FDA, Bethesda, MD, 2Nanosphere Inc., Northbrook, IL, 3Northwestern Uni., Evanstone, IL, 4Northwestern Uni., Chicago, IL
Background: Nanotechnology is being increasingly evaluated and applied to biomedical testing. The nanoparticle-based Bio-barcode amplification (BCA) assay offers some unique advantages for disease diagnosis.
Methods: BCA assay uses oligos or antibodies to tag disease markers: DNA/RNA or protein. Nanoparticles are functionalized with both target-specific probe and bio-bar code sequence while magnetic microparticles are labeled with another target-specific probe. The targets in the samples are then captured, separated magnetically. Finally, the bio-bar code sequence covalently bound to nanoparticles is released, detected in a chip-based scanometric method and identified to indirectly represent the disease markers.
Results: For detection of HIV-1 p24, the nanoparticle based BCA assay could detect 5 pg/ml of HIV-1 p24 antigen. At the concentration of 500 pg/ml, the ratio of positive / negative was > 10 while the ratio for ELISA methods was around 2-3.
For detection of HIV-1 RNA, a pair of oligos from HIV-1 Gag gene were chosen and functionized to magnetic beads and 30nm gold nanoparticles, separately. A synthesized HIV-1 Gag nucleotide oligo was used as template. The detection of HIV-1 DNA down to 10-50pM is achievable in the absence of PCR amplification.
Conclusion: As a rapid sensitive new testing system, BCA assay is feasible for detection of HIV-1 RNA and p24 antigen. We are currently optimizing the assay for use of human plasma or serum for detection of HIV-1, hepatitis B and C as well as WNV.
* The project was funded by Office of Science & Health Coordination, FDA
V. J. Tomazic-Jezic, T. H. Umbreit, M. E. Stratmeyer, FDA, CDRH, White Oak, MD
Background: The potential toxicity of nano-scale particles has been recognized. Evaluation of immune effects may need special attention, as changes of this complex network of cellular and humoral interactions may result in serious long-term malfunctions of immune responses. Studies of micro particles demonstrate immunotoxic effects of biomaterials that are biocompatible as solid materials. We have reported immuno-modulatory effects, with intensity inversely proportional to the particle size; polymethylmethacrylate 1µm diameter spheres altered the Th1-Th2 balance in mice, while larger size particles had less or no effect. Polystyrene (PS) particles of the same sizes had no such effect. The properties of ultrafine particles are very different from their larger analogs and their effects on immune functions are unpredictable.
Methods: This study investigates effects of nanoparticles in vivo to identify predictable biomarkers of immunotoxicity.PS particles 50 and 500 nm size, alone or antigen coated, were studied in mice. Proliferative response, cytokine production and serum antibody levels were analyzed.
Results: Both uncoated and antigen-coated particles induced strong monocyte and macrophage infiltration at the injection site.Gross histological analyses did not reveal any liver, kidney or lung toxicity. In conjunction with antigen, both type of particles had an adjuvant effect on the IgG production. However, the 50nm particles strongly enhanced the production of IgE antibody.This finding correlated with the observed changes in the regulatory cytokine production.
Conclusions: This study demonstrates that PS , known as biocompatible material, when in nano-size particle form,may affect immune functions, without demonstrating other signs of toxicity.
L. Zhu1 , D. Stewart2 , S. Ravishankar1 , M. Tortorello2 , 1National Center for Food Safety and Technology, Summit IL, 2FDA-CFSAN, Moffett Center, Summit IL
Background: Listeria monocytogenes (Lm) may become persistent in food processing plants, so environmental swabbing programs are recommended for its control. Environmental swabs are shipped from production plants to the laboratory in transport media. This study compared swab transport media for Lm recovery.
Methods: For studying effects of swab storage temperature, Lm cells were exposed to sanitizer, then transferred to sponges in transport media: Buffered Peptone Water (BPW), D/E Neutralizing Broth (DE), Neutralizing Buffer (NB), Letheen Broth (LE) or a proprietary formulation (PF). After storage at 4 or 25°C, cells were released by stomaching and plated. For studying effects of competitive microflora, media were compared for LM recovery in presence of L. innocua (Li), Escherichia coli (Ec), Pseudomonas fluorescens (Psf), Enterococcus faecalis (Ef) or Lactobacillus plantarum (Lp). For studying effects of drying, Lm cells were dried on a stainless steel surface, swabbed, and plated after storage in media at 4 or 25°C.
Results:Transport media showed similar recoveries after 4°C swab storage; at 25°C, NB showed approx 1-log decrease; BPW, DE and LE supported increases up to 3 logs; and PF allowed nearly 100% recovery. PF allowed Lm overgrowth by Psf and Ec, DE supported overgrowth of Lm by Ec and Ef, and NB allowed LM overgrowth by all five species. Recoveries of dried Lm cells by media after 25°C storage were in order: DE>or=LE >BPW>PF>NB.
Conclusion: Transport media vary in recovery efficiency and should be chosen based on specific environmental and shipping considerations.
V. H. Tournas1 , J. S. Kohn2 , E. J. Katsoudas2 , 1CFSAN, FDA, College Park, MD, 2NERL, FDA, Jamaica, NY
In an effort to identify a more suitable antibiotic for utilization in mycological media, 12 foodborne fungal species from various genera including Alternaria alternata, Aspergillus flavus, A. niger, Eurotium chevalieri, Fusarium moniliforme, Penicillium sp., Rhizopus sp., Saccharomyces cerevisiae, Candida tropicalis, Geotrichum candidum, Rhodotorula glutinis and Kluyveromyces thermotolerans along with 21 chloramphenicol-resistant bacterial isolates from fresh produce and ATCC cultures of Pseudomonas aeruginosa, P. fluorescens, Pectobacterium carotovorum, E. coli, Bacillus cereus and Staphylococcus aureus were tested for their abilities to grow on dichloran-Rose-Bengal agar containing various levels of gentamicin, chlortetracycline or chloramphenicol. Results indicated that all fungal isolates except for R. glutinis and Rhizopus sp. grew well on all media tested. R. glutinis did not grow on media containing gentamicin whereas Rhizopus sp. produced very restricted or no growth on these media. All bacterial isolates from fresh produce grew well at 100, 125 and 150 mg chloramphenicol/liter medium, but they did not grow on media containing chlortetracycline (100, 125, or 150 mg/liter) or gentamicin (15, 25, or 35 mg/liter). P. carotovorum, E. coli, B. cereus and S. aureus did not grow on any of the tested media; P. aeruginosa and P. fluorescens grew well on media containing chloramphenicol but not in the presence of chlortetracycline. P. aeruginosa (ATCC 10145) also grew very well on media containing gentamicin (15 or 25 mg/l). The best performer among the antibiotics tested was chlortetracycline.
V. H. Tournas1 , E. J. Katsoudas2 , 1CFSAN, FDA, College Park, MD, 2NERL, FDA, Jamaica, NY
Various herbal teas including German chamomile, chrysanthemum Vascuflow herb tea, hop, jasmine and orange flowers, sweet marjoram, spearmint and thyme leaves, and papaya-mint tea as well as coffee substitutes (Bambu instant Swiss, Teeccino chocolate-mint, and Teeccino Mediterranean Espresso) were analyzed for fungal contamination and the presence of aerobic mesophilic bacteria (APC). The results of this investigation showed that mould and yeast (MY) counts reached levels as high as 5.8 x 105 colony forming units (cfu) per gram. The highest MY contamination was observed in German chamomile. A variety of microfungi including members of the toxigenic genera Aspergillus, Alternaria, Eurotium, Fusarium and Penicillium were isolated from herbal teas. Aspergillus niger, Penicillium spp., eurotia (E. rubrum and E. chevalieri), A. flavus, Fusarium spp. and Alternaria alternata were the most common moulds recovered from the analyzed samples. Less common were A. carbonarius, A. ochraceus, A. versicolor, Ulocladium, Phoma and Rhizopus spp.Yeasts were found in all herbal teas (except jasmine flowers and thyme leaves). Among the coffee substitutes, only the chocolate-mint coffee was contaminated with fungi. Low numbers (<1.0 x 103 cfu/g) of Eurotium rubrum, and Ulocladium spp. were isolated from 17% and Phoma spp. were recovered from 33% of these samples.Yeasts were found in 67% of the chocolate-mint coffee substitute samples in numbers reaching as high as 6.8 x 103 cfu/g. Aerobic mesophilic bacteria were recovered from 100% of the herbal tea, chocolate-mint and Mediterranean Espresso, and from 50% of the Bambu instant Swiss coffee samples. The highestAPC counts of 1.2 x 107 cfu/g were observed in spearmint leaves and the lowest (<100 - 1.0x 103 cfu/g) were found in Bambu instant Swiss coffee substitute.
I. E. Valentin-Bon1 , K. H. Seo2 , R. E. Brackett2 , 1USPHS, DHHS, FDA, 2CFSAN, FDA
The efficacy of iron supplementation on the recovery of Salmonella Enteritidis (SE) in white large shell eggs using Ferroxiamine E (FE) was determined. It was then evaluated using a direct plating method and enrichment methods. Two bulk lots of blended, pooled eggs were thoroughly mixed manually and artificially contaminated with various low levels of SE as inoculum (0.3, 0.002 and 0.004 CFU/ml). Twenty samples containing 500 ml of the contaminated liquid eggs were withdrawn from each of the inoculated bulk lots. One set of twenty samples was supplemented with FE with a final concentration of 200 ng/g of egg and incubated for 24 h or for 4 days at room temperature. The other set of twenty samples was mixed only with sterile distilled water as a control group and incubated at the same temperature and days.For the direct plating method, each sample was cultured by direct plating onto brilliant green (BG), brilliant green with novobiocin (BGN), brilliant green with Rifampicin (BGR), bismuth sulfite (BS), xylose lysine desoxycholate (XLD), and xylose lysine agar Tergitol 4 (XLT4). For the enrichment methods, 25-g portions from each pool were enriched in modified tryptic soy broth (FeSO4, 30 mg/L).After 24 h of incubation, the preenrichment samples were subcultured to tetrathionate and Rappaport-Vassiliadis selective broths, and streaked to the agar plates. SE was confirmed biochemically and serologically. At 25°C, SE concentrations in FE-supplemented egg pools reached 105 CFU/ml by 24h.From a total of 120 pools of eggs analyzed, most of all supplemented or unsupplemented samples were positive (between 25 to 100%) when enrichment steps were combined. However, using direct plating, 15 out 20 (75%) FE-supplemented samples were positive for SE while 4 out of 20 samples (20%) were positive with an inoculum level of 0.3 CFU/ml. These results demonstrated the potential use of Ferroxiamine E to detect low number of SE in eggs without enrichment steps in less than two days.
L. G. Valerio, Jr.1 , G. F. Gonzales2 , 1Office of Food Additive Safety, CFSAN, FDA, College Park, MD, 2Department of Biological and Physiological Sciences, Universidad Peruana Cayetano Heredia, Lima, Peru
Natural products have played a significant role in drug discovery and development especially for agents against cancer and infectious disease. For example, an analysis of new and approved drugs for cancer by the United States Food and Drug Administration over the period of 1981-2002 showed that 62% of these cancer drugs were of natural origin. Natural compounds possess highly diverse and complex molecular structures compared to small molecule, synthetic drugs and often provide highly specific biological activities likely derived from their rigidity and high number of chiral centers. Botanicals as a mixture can represent a library of natural product molecular structures some of which may possess bioactivity. This source of chemical information may be useful in efforts to approach new potential therapeutic targets or alternatively, assess potential risk from toxicity. Therefore, understanding the chemical composition of botanical mixtures, is a crucial step in discerning the relationship between molecular structure and biological activity or mode of action of a medicinal compound(s), especially in cases where novel chemical entities have been identified. A number of plant-derived preparations native to the Andean and Amazonian regions of South America have a long historical record of medicinal use in ethnotraditional practices, and continue to be in used in modern times in the form of botanical preparations. There are three plant preparations known as, Uncaria tomentosa (Cat's claw), Lepidium meyenii (Maca), and Croton lechleri (Dragon's blood) which have each been discovered from the empirical knowledge of local aborigines populations, and have since been scientifically investigated for their chemical composition and biological effects potentially applicable to therapeutic uses as, anti-viral, anti-inflammatory, anti-bacterial, cicatrizant, and anti-tumor agents. Moreover, some limited toxicity testing has also been reported with these mixtures and will be summarized. Against this background an evidence-based presentation will be given regarding the known chemical constituents and biological properties that have been reported for these plant-derived natural products.
D. V. Volokhov, J. George, A. A. Neverov, H. Kong, D. Chandler, C. Anderson, V. E. Chizhikov, FDA, Rockville, MD
BACKGROUND: Mollicutes, which include the genera Mycoplasma and Acholeplasma, contamination is a common problem in research and production cell lines. Traditional systems of detecting these contaminants are resource intensive and time consuming. The use of rapid detection technologies would provide a major advantage in maintaining the purity and safety of biological products. This study describes development and evaluation of a PCR/Microarray that enables rapid and sensitive mycoplasma detection and accurate species identification of the contaminant.
METHODS: The current version of the microarray relies on the use of short oligonucleotide species-specific probes (20-30 nts in length) for identification of 25 species of Mycoplasma and Acholeplasma known to be human pathogens and common cell substrate contaminants. The assay procedure includes PCR amplification of the ITS and subsequent analysis of the amplified ssDNA or RNA using hybridization with the microarray. Two protocols, fluorescent dye and the nanogold particle staining, were developed for the preparation of the hybridization targets.
RESULTS: Broadly specific primers targeting the ITS were developed and efficiently used for amplifying more than 300 strains of Acholeplasma, Entomoplasma, Mycoplasma, Mesoplasma, Spiroplasma, and Ureaplasma species. Sequence analysis of these species revealed the presence of unique signature sequences in the ITS suitable for Mollicutes species identification using microarray technology and DNA sequencing. The use of nested PCR allowed us to increase the limit of detection to a few genomic copies of Mollicutes DNA per reaction. We demonstrated that our developed microarray was able to efficiently and unambiguously identify the species of target Mollicutes. We demonstrated that combining PCR/microarray with an initial step of in vitro enrichment, which includes 3-7 days cultivation of sample with Vero cells, we could significantly shorten the time and sensitivity of mycoplasma detection.
CONCLUSIONS: Sequence data showes that the 16S-23S rRNA ITS region is a suitable marker for Mollicutes detection and identification. Our designed PCR primers were very sensitive and highly specific. Evaluation of the microarray chips by hybridization with their appropriate target samples has demonstrated the feasibility of the PCR/Microarray method. The results demonstrated the potential of this highly sensitive and specific assay for detection and speciation of Mollicutes contamination in biological products and research applications.
W. G. Wamer, P. K. Fu, R. R. Wei, J. C. Hubinger, CFSAN, FDA, College Park, MD
Background: There are currently no in vitro or in vivo experimental tests for identifying indirect photosensitizers, i.e., chemicals that do not absorb light yet increase the sensitivity of skin to sunlight.
Methods: The effects of a known indirect photosensitizer, glycolic acid, on the ultraviolet light-induced erythemal response of the hairless guinea pig were investigated. Groups of 12 hairless guinea pigs received either no treatment, a vehicle (control) cream, or cream containing 10% glycolic acid, pH 3.5. Creams were applied once daily, 6 days a week, for 4 weeks. The skin's sensitivity to ultraviolet light was then assessed by determining the minimum dose of ultraviolet light needed to produce barely perceptible erythema, i.e., the minimal erythema dose (MED).
Results: Topical treatment with the vehicle cream resulted in a 30% reduction in the MED compared to the untreated site. Treatment with the cream containing 10% glycolic acid resulted in an additional 11% decrease in the MED, i.e., a 41% decrease in the MED compared to the untreated site. These reductions in the MED were statistically significant.
Conclusions: The effects of topically applied glycolic acid, an established indirect photosensitizer, on the ultraviolet light-induced erythemal response of the hairless guinea pig suggest that the hairless guinea pig may be a useful animal model for identifying indirect photosensitizers.
C. Wang1 , N. Sadovova2 , C. X. Zou1 , A. C. Scallet1 , T. A. Patterson1 , J. P. Hanig3 , M. G. Paule1 , W. Slikker1 , 1NCTR, FDA, Jefferson, AR, 2Toxicologic Pathology Associates, Jefferson, AR, 3CDER, FDA, Rockville, MD
Previous data suggest that exposure of neonatal rats to various anesthetics, including NMDA antagonists and GABA agonists, triggers widespread apoptotic neuro-degeneration during development. The purpose of this study was to determine: 1) if treatment of forebrain cultures with NMDA antagonists, such as ketamine, or GABA agonists, e.g. midazolam, results in a dose-related increase in neurotoxicity; and 2) if combinations of NMDA antagonists and GABA agonists will promote or prevent each other's neurotoxic effects. Forebrain cultures were treated for 12 hrs with 1, 5, 10 and 20 µM ketamine alone, 0.1, 1, 5 and 10 µM midazolam alone, or ketamine plus midazolam at various doses. After washout of ketamine and midazolam, cultures were kept in serum-containing medium (in the presence of glutamate) for 24 hrs. Ketamine (10 and 20 µM) resulted in a substantial increase in DNA fragmentation as measured by cell death ELISA, increased number of TUNEL-positive cells (Terminal dUTP Nick-End Labeling Assay), and a reduction in mitochondrial metabolism of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dye (MTT, for cell survival). No significant apoptotic effect was observed in midazolam- (0.1, 1, 5 and 10 µM) treated cultures. Co-incubation of midazolam (5 µM) with ketamine (5 µM) reduced ketamine-induced apoptosis. These results indicate that ketamine administration (5, 10 and 20 µM) causes enhanced apoptosis in rat forebrain cultures. Co-administration of midazolam mildly prevented this increase caused by ketamine, suggesting that the GABA and glutamatergic systems could be co-activated during physiological and pathological patterns of activity, and stimulation of the GABA system could mildly inhibit the activation of NMDA receptors. These data suggest that combinations of mild doses of NMDA antagonists along with GABA agonists may reduce the risks of NMDA antagonists used alone.
This work was supported by Interagency Agreement IAG 224-93-001 between the U.S. Food and Drug Administration and the National Institute for Environmental Health Sciences.
D. G. White1 , S. L. Ayers1 , A. Glenn1 , E. Hall-Robinson1 , R. D. Walker1 , T. M. Chiller2 , P. F. McDermott1 , 1CVM, FDA, Laurel, MD, 2CDC, Atlanta, GA
Background:The National Antimicrobial Resistance Monitoring System (NARMS) expanded in 2002 to include surveillance of select foodborne bacterial pathogens and commensal organisms from retail foods of animal origin.
Methods: As part of the 2004 study, E. coli were recovered from a convenience monthly sampling of 40 retail meats per month, including 10 samples each of chicken breast, ground turkey, ground beef, and pork chops from grocery stores in 4 participating FoodNet states (GA, MD, OR, TN). The goal of each FoodNet site was to collect 480 retail meats per year (40 retail meats per 12 months); however, at times not all types of retail meats were available at selected grocery stores. Minimum inhibitory concentrations of recovered E. coli isolates were determined via broth micro-dilution methods and interpreted according to CLSI standards, where available.
Results:Sixty-nine percent of 1900 retail meat samples were contaminated with E. coli. Contamination rates varied from 84% of chicken breast samples to 45% of pork chops tested. Among the 1312 E. coli isolates tested to date, resistance was most often observed to tetracycline (51%), streptomycin (38%), sulfamethoxazole (33%), ampicillin (18%), and gentamicin (18%). Ninety-eight percent of gentamicin resistant E. coli isolates were recovered from either ground turkey or chicken breast. All isolates were susceptible to ceftriaxone; however, 2% were resistant to the veterinary extended-spectrum cephalosporin, ceftiofur. Three isolates, all recovered from ground turkey, were ciprofloxacin resistant, however, 5% of isolates were resistant to nalidixic acid with 67/71 isolates recovered from either ground turkey or chicken breast.
Conclusion: Results indicate that antimicrobial-resistant E. coli isolates are present in retail meats, in particular poultry products, and could serve as reservoirs of antimicrobial resistance genes for other enteric pathogens.
S. C. Wood1 , G. S. Bushar1 , B. Tesfamariam2 , 1CDRH, Silver Spring, MD, 2CDER, Silver Spring, MD
Introduction: Arteries blocked by plaque are treated with stenting to restore flow. Unfortunately, these interventions often result in restenosis. Drug eluting stents reduce restenosis by releasing anti-proliferative drugs, rapamycin or taxol, in high, localized concentrations. Stent associated thrombosis has been reported, particularly when anticoagulation therapy is reduced or stopped. Coagulation is initiated by Tissue factor (TF) on endothelial cells and is down regulated by thrombomodulin which inactivates thrombin. Importunately, vascular cell growth factor (VEGF) is released from endothelial cells in response to injury and hypoxia.
Hypothesis: Anti-restenotic drugs and VEFGF may induce a prothrombotic state in endothelial cells.
Methods: Endothelial cell line EA was exposed to either rapamycin or taxol followed by VEGF. TF expression was followed by a plasma recalcification coagulation assay using a Molecular Devices plate reader. Expression of thrombomodulin was followed with a BD FACSCAN flow cytometer.
Results: Treatment of EA cells with either rapamycin or taxol resulted in a slight increase in coagulation. However, addition of VEGF significantly enhanced the coagulation cascade. Importantly, coagulation correlated with expression of TF in EA cells. Thrombomodulin expression was not altered by rapamycin/VEGF or taxol /VEGF.
Conclusions: These results suggest that anti-restenotic drugs have prothrombotic effects that are potentiated by VEGF. Significantly, VEGF is released in high concentrations in atherosclerotic plaque and is responsible for the extensive neovasculaization of these lesions. Current experiments are focused on the factors that may enhance VEGF release by endothelial cells.
Acknowledgment: Supported by Office of Science and Engineering Laboratories.
J. J. Yourick1 , C. T. Jung2 , R. L. Bronaugh1 , 1Office of Cosmetics and Colors, CFSAN, FDA, Laurel, MD, 2OPS/OGD/DBE, CDER
Background: Percutaneous absorption of retinol from cosmetic formulations was studied to predict systemic absorption and to understand the significance of the skin reservoir.
Methods: Viable skin from rats and humans was assembled in flow-through diffusion cells for in vitro absorption studies. In vivo rat skin absorption studies utilized glass metabolism cages to collect urine, feces, and body content. Retinol (0.3%) formulations (hydroalcoholic gel and oil/water emulsion) containing H3- retinol were applied for 24 or 72 hr. All values reported represent % of applied dose.
Results: In vitro human skin studies (gel) found 7% in skin and 0.3% in receptor fluid at 24 hr. Human skin receptor fluid values did not increase over 72 hr. In vitro rat skin studies (gel) found 23% in skin and 6% in receptor fluid at 24 hr, while 72-hr studies found 18% in skin and 13% in receptor fluid. Significant amounts remained in rat skin at 24 hr and decreased over 72 hr, with proportional increases in receptor fluid. In vivo rat studies found 4% systemic absorption of retinol after 24 hr and systemic absorption did not increase over 72 hr. Retinol remaining in skin after in vivo application was 23% and 15% after 24 and 72 hr, respectively. Retinol systemic absorption after 24 hr was comparable to in vitro results after 24 hr.
Conclusion: Retinol formed a rat skin reservoir both in vivo and in vitro; however, little additional retinol was bioavailable after 24 hr. This research was supported by FDA, Office of Women's Health.
J. Zhang1 , T. J. Miller1 , N. P. Clayton2 , J. P. Hanig1 , P. Espandiari1 , 1CDER, FDA, Silver Spring, MD, 2NIEHS, Research Triangle Park, NC
Nonclinical studies suggest that gentamicin (Gen) induces renal tubular apoptosis in rats via several pathways (caspase-3, cytochrome C, nuclear endonuclease and Bax) and that reactive oxygen species may be responsible for Gen-induced nephrotoxicity. To explore the role of nitric oxide, different age groups of SD rats (25, 40, 80 days old) were treated with saline, 50 or 100 mg/kg/daily (ip) for 6 or 14 days. Gen-induced renal injury was evaluated using a semiquantitative scale (0-5). Histopathological evaluation showed that Gen induced degeneration, necrosis, and regeneration in the proximal and distal convoluted tubules, tubular dilatation and protein casts, glomerular vacuolization, and interstitial lymphocytic inflammation and that Gen-induced renal injury is age-, time- and dose-dependent.The immunoreactivity of apoptosis, caspase-3, cytochrome C, iNOS (inducible nitric oxide synthase), and nitrotyrosine was assessed immunohistochemically in the kidneys.The expression of caspase-3, cytochrome C, iNOS, and nitrotyrosine increased with age, time and dose and the levels of the increased immunoreactivity correlated with degree of tubular apoptosis.These findings suggest that nitric oxide may play an important role in Gen-induced nephrotoxicity.
J. Zhang1 , A. D. Knapton1 , T. J. Miller1 , P. Espandiari1 , R. Anderson1 , E. H. Herman1 , R. Snyder2 , J. P. Hanig1 , J. L. Weaver1 , 1CDER, FDA, Silver Spring, MD, 2Schering-Plough Research Institute, Lafayette, NJ
Our previous rat studies showed that drug-induced vascular injury in mesenteric tissue can be accompanied by MCD. To investigate the role, if any, of MCD in the vasculitic process, we performed studies to determine if blocking MCD in rats reduced drug-induced vascular injury. MCD blockers cromolyn (20 mg/kg, ip) or ketotifen (1 mg/kg, ip) were given at -0.5, +2, +4 and +6 hrs relative to the drug treatment. Rats were treated with (1) a single dose of the dopamine receptor agonist fenoldopam (60mg/kg, sc.) +/- cromolyn or ketotifen; (2) the PDE4 inhibitor SCH 351591 (20mg/kg/day, gavage) +/- ketotifen once daily for 3 days;(3) a single sc injection of the PDE3 inhibitor SK&F 95654 (50mg/kg) +/- ketotifen; (4) a single IP injection of the mast cell degranulator Compound 48/80 (1mg/kg) +/- ketotifen. Rats were sacrificed 24 hrs after the last drug dose. Histopathological evaluation showed that, depending on timing and dosage, MCD, inflammation, and arterial medial necrosis were general features of all three drugs, consistent with previous observations. Compound 48/80 induced MCD, inflammation and eosinophil recruitment. Concomitant treatment of rats with mast cell blockers moderately to strongly reduced histopathological mesenteric lesions in animals treated with any of the four compounds. These findings suggest that activation of mesenteric mast cells may be a key component in the pathway leading to vascular injury induced by fenoldopam and phosphodiesterase inhibitors.
S. Zhao, D. G. White, S. F. Friedman, A. Glenn, S. L. Ayers, J. W. Abbott, S. K. Hubert, E. Hall-Robinson, H. C. Harbottle, R. D. Walker, P. F. McDermott, CVM, FDA, Laurel, MD
Salmonella Heidelberg is frequently associated with foodborne illness in humans and is commonly isolated from poultry and its derived meats.The objective of this study is to compare the prevalence of S. Heidelberg in four types of meat, including chicken breast, ground turkey ground beef and pork chops collected during 2002-2004 NARMS retail meat surveillance program.Isolates were analyzed for antimicrobial susceptibility and PFGE. A total of 154 S. Heidelberg isolates were recovered, representing 22.4% of all salmonella serovars from retail meats.Among the 154 isolates, 90 from ground turkey, 58 from chicken breast and 6 from pork chop; no S. Heidelberg was found in ground beef. 61% of the isolates were resistant to at least one of the 16 antimicrobial agents tested and 16.2% were resistant to >5 antimicrobials. 3.2% were resistant to >9 antimicrobials, which were all recovered from ground turkey meat. The highest rates of resistance from poultry meat isolates were observed for tetracycline (39.86%) follow by streptomycin (38.51%), sulfamethoxazole (25%), gentamicin (22.97%), and kanamycin (22.30%), ampicillin (16.89%), cephalothin (8.1%), cefoxitin (6.76%), ceftiofur (6.76%), amoxicillin-clavulanic acid (5.41%), chloramphenicol (2.03%), and nalidixic acid (0.68%).All isolates were susceptible to amikacin, ceftriaxone, and ciprofloxacin and trimethoprim/sulfa. PFGE with XbaI and BlnI generated 61 patterns for the 154 isolates. Certain clones were widely dispersed in different types of meats and meat brands from different store chains in all three years. These date indicate that S. Heidelberg is a common serovar in retail poultry meats, and includes widespread clones of multidrug-resistant strains.
S. Zhao1 , P. F. McDermott1 , D. G. White1 , S. Qaiyumi1 , S. F. Friedman1 , J. W. Abbott1 , A. Glenn1 , S. L. Ayers1 , P. K. Post2 , W. H. Fales3 , R. B. Wilson4 , C. Reggiardo5 , R. D. Walker1 , 1CVM, FDA, Laurel, MD, 2Rollins Animal Disease Diagnostic Laboratory, Raleigh, NC, 3University of Missouri, VMDA, Columbia, MO, 4CE Kord Animal Laboratory, Tennessee Department of Agriculture, Nashville, TN, 5Arizona Veterinary Diagnostic Laboratory, University of Arizona, Tucson, AZ
A total of 380 Salmonella isolates representing 47 serotypes recovered from diseased animals in year 2002-2003 were characterized using antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and examined for the presence of class-1 integrons and the blaCMY gene. Overall, 82.4% of isolates were resistant to at least one antimicrobial, 69.7% to > 3, and 15.5% to > 9 antimicrobials. Many isolates exhibited resistance to tetracycline (78.2%) follow by streptomycin (72.6%), sulfamethoxazole (67.6%), ampicillin (53.7%), chloramphenicol (37.1%), kanamycin (36.8%), cephalothin (29.2%), amoxicillin-clavulanic acid (20.3%), cefoxitin (17.6%), ceftiofur (17.4%), gentamicin (12.6%), trimethoprim-sulfmethoxazole (8.7%), nalidixic acid (3.9%), ceftriaxone (2.1%), and amikacin (0.5%). All isolates were susceptible to ciprofloxacin. Swine isolates had the highest resistance rate; 91.5% were resistant to at least one antimicrobial. The resistance rates of Salmonella isolates from turkey, cattle, chicken, and equine were 90.6%, 77%, 68%, and 20%, respectively. However, more than one third of cattle isolates (34.9%) were resistant to > 9 antimicrobials, whereas only approximately 10% equine or turkey isolates showed resistant to > 9 antimicrobials. Antimicrobial resistance of the Salmonella isolates was also related to serotype. A large percentage of isolates belonging to S. Uganda (100%), S. Agona (78.6%), and S. Newport (62%) showed resistant to > 9 antimicrobials, compared to S. Heidelberg (11.4%) and S. Typhimurium (7%). Class-1 integrons were commonly detected (43.2 %), containing aadA, aadB, dhfr, cmlA and sat alone or in combinations. PFGE showed considerable genetic diversity across all isolates, however, several multiple drug resistance clones were widely spread among animals.
A. D. Lucas, R. P. Brown, M. E. Stratmeyer, CDRH, FDA, Silver Spring, MD
Background:Patients undergoing various medical procedures can be exposed to phthalate esters, such as DEHP, released from medical device materials and to other phthalates esters from exogenous sources such as food, cosmetics, and consumer items. Since phthalate esters exert many of their toxic effects via a common toxicological mechanism, the potential exists for additive or synergistic effects to occur following simultaneous exposure to multiple phthalate esters. A key assumption in risk assessment approaches developed to account for aggregate exposures to multiple compounds with a similar mechanism of action (e.g., Hazard Index approach) is that the effects will be additive at low doses. An in vitro cytotoxicity study was conducted to determine validity of this assumption for apoptosis and cell death produced by binary and tertiary mixtures of phthalate monoesters.
Methods:The concentration-response relationship for 3 phthalate monoesters, mono(2-ethylhexyl) phthalate (MEHP), n-butyl phthalate and n-benzyl phthalate, individually and in combination, was determined for their ability to produce apoptosis and cell death (using flow cytometry) in a Jurkat human lymphoma cell line following incubation for 24, 48, or 72 hours.
Results: An additive relationship was seen between the binary mixture constituents at 48 and 72 hours and among tertiary mixture components at 24 hours for both apoptosis and cell death; however, a synergistic effect was observed among the tertiary mixture components at 48 and 72 hours.
Conclusions:The assumption of low-dose additivity is valid for binary mixtures of phthalate monoesters, but synergism is possible in more complex mixtures.
J. K. Suagee1 , L. H. Garthoff1 , P. L. Wiesenfeld1 , T. J. Sobotka1 , C. N. Barton2 , 1OARSA, FDA, Muirkirk Road Complex, Laurel, MD, 2OSAS, FDA, College Park, MD
INTRODUCTION: One approach for assessment of general toxicity of unknown chemicals is based on live/dead counts. However, a more rapid, informative, and effective assessment of toxicity is possible if the animals are monitored and evaluated for early signs of toxicity.The ability to predict impending death will allow for collection of better quality tissue/blood samples and minimize animal stress and suffering. To improve the flexibility and speed needed to assess chemical terrorist events, we devised a simple, rapid COB test that was subsequently included in a non-standard experimental design of a food defense study.
OBJECTIVE: The objectives were: 1) establish a battery of clinical observations (clinobs) that accurately identify dose related toxicity and 2) apply this battery to a compound with an unknown oral LD50 to determine how well it predicted mortality.
METHODS: Replicate groups of young adult female Sprague-Dawley rats were pretreated (ip) with low dose lipopolysaccharide (LPS) and
1 hour later dosed with colchicine (COL) (experimental design and clinobs details are found on the related poster by Wiesenfeld et al.). The animals were monitored for signs of clinical toxicity at 1, 2, 4, and 6 hr, and then 1-4 days post dose. Observers were blinded to the experimental groups.
RESULTS: Ten endpoints were grouped into 4 levels of increasing toxicity. Toxicity levels included: piloerection, red eye discharge, and ptosis (1); unresponsiveness and listless behavior (2); loss of balance, head tremors, and diarrhea (6); and morbidity (18). For data analyses, we chose the highest score of each rat at each observation time point. An exact Mantel-Haenszel trend test was performed to determine whether higher symptom levels at each time point were significantly related to animal survival.Symptom levels were significantly related (p < 0.05) to survival at 1, 2, 3, and 4 days post dose.Therefore, the toxicity level described by the score, paralleled and predicted mortality.
Time point P-value
PD1 1.479E-05
PD2 4.736E-04
PD3 5.148E-04
PD4 0.0286
CONCLUSIONS: The inclusion of COB produced a flexible, rapid, qualitative and quantitative estimate of toxicity that helped predict mortality, while reducing animal pain and suffering. It could be used in conjunction with counter-terrorism programs.
D. E. Godar1 , S. G. Coelho1 , W. B. Grant2 , M. F. Holick3 , 1CDRH, FDA, Rockville, MD, 2Sunlight, Nutrition and Health Research Center, SUNARC, San Francisco, CA 94109, 3Department of Medicine, Boston University School of Medicine, Boston, MA 02118
Background: Sunshine contains UV radiation (290-400 nm) that affects human health in both detrimental and beneficial ways. The detrimental health effects include melanoma and non-melanoma skin cancers. The beneficial health effects include vitamin D3 formation, which is not only important for bone and muscle health, but also reduces the incidence of multiple sclerosis and type 1 diabetes mellitus in children. An evaluation of the vitamin D status (measured as 25-hydroxyvitamin D in serum) of many young Americans (0-19 yr) show they have deficient (<27.5 nmol/L) or insufficient (<50 nmol/L) levels, indicating they are not getting adequate solar doses to make sufficient vitamin D3, according to the current recommendation (200 IU/day).
Methods: We decided to investigate this situation using the solar UV doses of young Americans (about 2,000) to see if they are making enough vitamin D3.
Results: We find many youths are getting insufficient incidental solar exposures to make sufficient amounts of vitamin D3 without wearing sunscreen: Fitzpatrick skin types I/II during the winter; skin types III/IV all year, except summer; skin types V/VI all year. If children and adolescents always wear SPF 15 sunscreen, they will reduce their production of vitamin D3 by up to 99.5%. Furthermore, no youth makes ample vitamin D3 by the current suggestion (1,000 IU/day), except skin types I/II during the summer.
Conclusion: Thus, according to our calculations, most American youths are not going outside enough to get UV doses that would make sufficient amounts of vitamin D3 for about half the year or more, depending on skin type.
D. E. Godar1 , W. B. Grant2 , M. F. Holick3 , 1CDRH, FDA, Rockville, MD, 2Sunlight, Nutrition and Health Research Center, SUNARC, San Francisco, CA 94109, 3Department of Medicine, Boston University School of Medicine, Boston, MA 02118
Background: Sunshine contains UV radiation (290-400 nm) that affects human health in both detrimental and beneficial ways. The major detrimental health effect is skin cancer, while the major beneficial health effect is vitamin D3 production. Vitamin D3 reduces adults' risk for getting osteoporosis, osteomalacia, hypertension, cardiovascular disease, type II diabetes, rheumatoid arthritis, tooth loss and falls that can result in hip fractures in the elderly. It also reduces the mortality from some cancers like colon, breast, prostate and melanoma. An evaluation of the serum levels of vitamin D (measured as 25-hydroxyvitamin D) of many adult Americans show they have deficient or insufficient levels, indicating they are not going outside enough to get adequate UV exposures to make ample vitamin D3.
Methods: We decided to investigate this situation using the outdoor UV doses of adult Americans (22-40, 41-59, 60+ yr) to see how much vitamin D3 they can make from incidental UV exposures.
Results: According to the current recommendation for adults (200-600 IU/day), many people are not getting adequate incidental UV exposures to make sufficient vitamin D3 without wearing sunscreen for about half the year. If people wear SPF 15 sunscreen, they will make virtually no vitamin D3 all year long. For the same incidental UV dose, the amount of vitamin D3 produced decreases with increasing age and skin type. Furthermore, hardly anyone makes ample vitamin D3 by the current suggestions (1,000 IU/day), except some skin types I/II during the summer.
Conclusion: Our results agree with the published findings of vitamin D insufficiency in America.
L. Hu, M. D. Bray, M. Osorio, D. J. Kopecko, LESTD, CBER, FDA, Bethesda, MD 20892
Campylobacter jejuni are a leading bacterial cause of human diarrheal disease in both developed and developing nations. Colonic mucosal invasion and the resulting host inflammatory responses are thought to be the key contributing factors to the dysenteric form of this disease. Dendritic cells play an important role in both the innate and adaptive immune responses to microbial infection. In this study, the interaction between human monocyte-derived dendritic cells and C. jejuni was studied. We found that C. jejuni were readily internalized by dendritic cells (DCs) over a 2 hr period. However, after a prolonged infection period (24 or 48 hrs) with C. jejuni, only a few viable bacteria remained intracellularly. Minimal cytotoxicity of C. jejuni to dendritic cells was observed. C. jejuni induced the maturation of dendritic cells over 24 hrs, as indicated by up-regulation of cell-surface marker proteins CD40, CD80 and CD86. In addition, Campylobacter-infected DCs triggered activation of NF-κB and significantly stimulated production of IL-1β, IL-6, IL-8, IL-10, IL-12, IFN-γ, and TNF-α when compared to uninfected DCs. Active bacterial invasion of DCs was not necessary for the induction of these cytokines, as heat-killed C. jejuni stimulated similar levels of cytokine production as live bacteria. Purified lipooligosaccharide of C. jejuni appears to be the major stimulant for the increased production of cytokines by DCs. In summary, these data indicate that during infection, Campylobacter triggers an innate inflammatory response through increased production of IL-1β, IL-6, IL-8, and TNF-α and initiates a Th1-polarized adaptive immune response as predicted from the high level production of IL-12.
A. Rodriguez1 , A. Margolin2 , 1WEAC, Winchester, MA, 2UNH, Durham, NH
Background: The CDC estimates that 86,731 food-borne illnesses each year are caused by Y. enterocolitica. This bacterium can grow at the low temperatures used to store vegetables. And because these are ready to eat foods, the bacteria would not be killed before the product reaches the consumer. Real time PCR is an advancement to the basic PCR technique which allows for the instantaneous quantitation of the amplification product.
Methods: Romaine lettuce samples were placed in broth and inoculated with 1 to 106 CFU of Y. enterocolitica. The samples were then incubated at 30° C for 24 h. Sample aliquots were tested by real time PCR both before and after the incubation. No DNA extraction procedure was required. Two sets of probes were used, one for the chromosomal ail gene, and another for the virulence plasmid virF gene.
Results: Before incubation, the sensitivity limit for both genes was 105 CFU. Following a 24 h incubation the sensitivity increased to 1 CFU for the chromosomal gene and to 10 CFU for the plasmid gene.
Conclusions: The assay evaluated here was sensitive and faster than conventional cultivation methods for the detection of Y. enterocolitica. It can potentially be used to detect this pathogen in green leafy vegetables.
K. Y. Song1 , K. H. Seo2 , S. Lee1 , R. E. Brackett2 , 1JIFSAN, UMD, College Park, MD, 2CFSAN, FDA, College Park, MD
Enterobacter sakazakii has subsequently been implicated worldwide in neonatal and infant cases of meningitis, septicemia, and necrotizing enterocolitis. Hence, it is critical need to detect and isolate E. sakazakii from the contaminated samples with the rapid and accurate. This study was conducted to evaluate the efficacy of quaternary ammonium compounds (QAC) as commercial disinfectant on inactivating clinical or environmental E. sakazakii isolates in water. QAC acts primarily by disruption cellular permeability of the cytoplasmic membrane. The sequence of events leading to cell death are as follows: (i) adsoption of the compound on the bacterial cell surface, (ii) diffusion through the cell wall, (iii) binding to the cytoplasmic membrane, (iv) release of potassium ions and other cytoplasmic constituents, and (vi) precipitation of cell contents and death of the cell. Commercial disinfect used was QAC consisted of both 2.25% n-alkyl dimethylbenzyl ammonium chloride and 2.25% n-alkyl ethylbenzyl ammonium chloride. QAC was prepared at concentrations ranging from 0 (pH 7.00), 5 (pH 7.4), 10 (pH 7.42), 20 (pH 7.40) and 50 ppm (pH 7.32). Total 62 strains (58 E. sakazakii and 4 non-E. sakazakii) were treated with the disinfectant solutions for the exposure 10 min, and then spreaded on TSA so as to enumerate live bacteria for 24 hours at 37 C. All of them except Salmonella spp. were decreased by more than 6 log CFU/ml at 50 ppm QAC, but only Salmonella spp. could reduced by more than 3 log CFU/ml and showed the survival of 2.89 log CFU/ml at same condition. According to EPA guideline, the maximum level permissible of QAC on food contact surfaces without rinsing was 200 ppm. The QAC concentrations used in the present study were 4 times lower then EAP guideline. Also, it is need to further study to (1) determine the efficacy of QAC on E. sakazakii that has been sublethally stressed by exposure to acid, heat, cold, and starvation conditions, and also (2) to screen the survival rate of E. sakazakii in accordance with the initial inoculation.
K. Y. Song1 , K. H. Seo2 , S. Lee1 , R. E. Brackett2 , 1JIFSAN, UMD, College Park, MD, 2CFSAN, FDA, College Park, MD
Enterobacter sakazakii is a member of the family Enterobacteriaceae. Several outbreaks or sporadic cases of either severe neonatal meningitis in premature infants or necrotizing enterocolitis have been attributed to E. sakazakii. At present time, there are no published data pertaining to the resistance to commercial disinfectants of E. sakazakii. Sodium hypochlorite is used widely as sanitizer in food industries. The objective of this study was to evaluate the antibacterial efficacy of chlorine for inactivation of E. sakazakii strains (9 clinical and 49 environmental isolates), and also to compare the biofilm formation on two different conveyor belts used for food industry. In the antimicrobial effects of sodium hypochlorite, there was no significant difference between the adjusted pH and the unadjusted pH. All of E. sakazakii tested were killed under the 10 ppm (FAC range of 3.7 to 5.1 ppm) at 10 min exposure. Also, in the removal of biofilm formed two different conveyor belts (1x2 cm), there was no difference between pH-adjusted Sodium hypochlorite and pH-unadjusted one. But pH-adjusted Sodium hypochlorite was more effective in only reducing range of bacterial population than pH-unadjusted one. The biofilm cells formed in Conveyor belt A (Buna-N, Nitrile rubber) were removed only 50 ppm (FAC range of 11.5 to 23 ppm) at 10 min exposure both pH-adjusted and pH-unadjusted sodium hypochlorite solution. But those of conveyor belt B (2-ply monofilament PVC, Style 2AR10/OW belt) could be survived at 400 ppm (FAC range of 92 to 184 ppm) at 10 min exposure but all of them were removed under 1000 ppm (FAC range of 230 to 460 ppm) at 10 min exposure. It was suggested that the removal of biofilm cells depended on the type and materials consisted of conveyor belt. Also, to compare with the resistance of biofilm cells formed TSB and (10% w/v) dried infant formula, there was showed the same pattern as described above, but it showed only difference of cell population formed in biofilm. The results showed that the vegetable cell of E. sakazakii tested could remove under the 10 ppm concentration of sodium hypochlorite. But the biofilm cells of E. sakazakii showed the more resistance than vegetable cells in sodium hypochlorite as sanitizer, and the efficacy to remove biofilm varied in accordance with the conveyor belt type, conveyor belt materials, and media used to form biofilm.
K. Y. Song1 , K. H. Seo2 , S. Lee1 , R. E. Brackett2 , 1JIFSAN, UMD, College Park, MD, 2CFSAN, FDA, College Park, MD
Among the reported outbreaks in the United Sates during 1993-1997 period for which the etiology was determined, bacterial pathogens caused the largest percentage of outbreaks (75%) and the largest percentage of cases (86%). Salmonella Enteritidis and and E. coli O157:H7 accounted for the largest number of outbreaks, cases, and deaths. So, it is need to detect it with the quick and accurate using by updated biotechnology. Recently, semiconductor quantum dot or nanocrystals have emerged as a novel and promising class of fluorophores for cellular imaging. Quantum dot can be excited by a wide spectrum of wavelengths to give great photostability, and their emission spectra, which differ according to size and material composition, are narrow, symmetrical, and tunable. The new dyes as quantum dot could provide quantitative measurement with great sensitivity in one bacterium.To detect the multiple pathogens by using quantum dot and multi-antibody-coated magnetic beads simultaneously, the procedure could be divided into two steps. The first step was quantum dot-labeled antibody conjugate. Anti-Salmonella antibody was labeled quantum dot 525 nm and anti-E. coli O157:H7 antibody did quantum dot 609 nm by using antibody conjugation kit. The second step was binding between antibody conjugated quantum dot and target bacteria. Immunomagnetic bead specific to Salmonella, or to E. coli O157:H7, quantum dot-conjugated anti-Salmonella antibody or anti-E. coli O157:H7 antibody, Salmonella or E. coli O157:H7 solution containing of 109 CFU/ml were added to micro-tubes and then followed by protocol. The mixtures were analyzed by Ocean Optics spectrometers with OOIBase32™ Spectrometer Operating Software. The total peaks of mixtures showed 3 different patterns (1) the excitation wavelength of 473 nm, (2) Salmonella Entertidis of 525 nm, and (3) E. coli O157:H7 of 605 nm.Also, it is need to further study to seek for the detection limit at the low concentration (ca.101-105 CFU/ml) of mixture culture.
K. Y. Song1 , K. H. Seo2 , S. Lee1 , R. E. Brackett2 , 1JIFSAN, UMD, College Park, MD, 2CFSAN, FDA, College Park, MD
Bacillus anthracis, a Gram positive, nonmotile, facultative anaerobic and sporeforming rod-shaped bacterium is a causal agent of anthrax, a serious and often fatal infection in both livestock and humans. Recent interests in anthrax is due to its potential use in bioterrorism and as a biowarfare agent against civilian populations. Cases of pulmonary and cutaneous anthrax occurred in postal workers and in mail handlers exposed to envelopes contaminated with B. anthracis spores in the United States. Therefore, it is critical to develop a reliable-and-rapid strategy for B. anthracis to decontaminate infected humans and wild stock from contaminated areas. This study was undertaken to evaluate the efficacy of sodium hypochlorite, calcium hypochlorite, a quaternary ammonium compound, chlorine dioxide against vegetative ecells and spores of B. anthracis. Also the removal of biofilm formed by spores of B. anthracis from stainless steel chips and two different conveyor belts was examined using by commercial disinfectants. When B. anthracis cells were in planktonic state, 50 ppm hypochlorite (pH 7), 1 ppm calcium hypochlorite, 1 ppm QAC and 100 ppm chlorine dioxide induced a more than 6.0 log CFU/ml reduction of cell numbers within 10 min exposure. Also, on the condition of 3000 ppm hypochlorite (pH 7), 300 ppm calcium hypochlorite, and 100 ppm chlorine dioxide for the exposure of 10 min, spores of B. anthracis showed a more than 8.0 log CFU/ml reduction. But it did not kill the spore on more than 50000 ppm QAC, since QAC was not sporocidal, its activity exhibiting inhibition of the outgrowth of germinating spores. In the removal of the biofilm formed by spores of B. anthracis at stainless steel (1x2 cm), a significantly higher population reduction of 7.0 log CFU/chip was noted when chips were treated with 8000 ppm hypochlorite (pH 7) for the exposure of 10 min, but in same concentration of pH 9, there was only 1.5 log CFU/chip reduction. Also, in comparison of two different conveyor belt chips (1x2 cm), the biofilm formed in conveyor belt A (Buna-N, Nitrile rubber) could be inactivated at 300 ppm chlorine dioxide for the exposure of 10 min, but that of conveyor belt B (2-ply monofilament PVC, Style 2AR10/OW belt) could be survived at more than 500 ppm chlorine dioxide. The results showed that the vegetable cells of B. anthracis were easily inactivated than spore or bioflm cells formed by spores of B. anthracis. Also, the efficacy to remove biofilm would vary in accordance with the type of conveyor belt, the materials of conveyor belt, and pH of disinfectants used. Among disinfectants tested, chlorine dioxide showed the best effect to kill or remove B. anthracis in vegetable cells, spores and biofilm cells.
K. Y. Song1 , K. H. Seo2 , S. Lee1 , R. E. Brackett2 , 1JIFSAN, UMD, College Park, MD, 2CFSAN, FDA, College Park, MD
Until 1980, Enterobacter sakazakii was referred to as yellow pigmented Enterobacter cloacae. It was then re-classified as a unique species based on differences from E. cloacae in DNA relatedness, the specific yellow pigment production, biochemical reactions, and antibiotic susceptibility. Several outbreaks or sporadic cases of either severe neonatal meningitis in premature infants or necrotizing enterocolitis have been attributed to E. sakazakii. Hence, this study was to evaluate that the performance of FDA method was compared with the state-of-art Real-Time PCR by testing powdered infant formula artificially contaminated with strains of E. sakazakii clinical isolates, so as to (a) improve the efficiency of isolation and identification of E. sakazakii, (b) to exclude the false positive as E. vulneris. FDA method as conventional cultue methods for isolation of E. sakazakii in dehydrated powdered infant formula involve steps of pre-enrichment, subculture, re-incubation, and picking and streaking the yellow colonies, which takes up to 6 to 7 days. And the confirmation method that can be routinely performed is only a biotyping assay, such as API 20E. However, the Real-Time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii with 1 and half day. This experiment was to carry out 4 different groups; (1) Group 1 (control)- no artificially inoculation to 25 g of powdered infant formula, (2) Group 2 (E. sakazakii)-only each culture (more than 107 CFU/ml of E. sakazakii) inoculation to 25g of powdered infant formula, (3) Group 3 (E. vulneris)-only each culture (more than 107 CFU/ml of E. vulneris) inoculation to 25g of powdered infant formula, and (4) Group 4 (mixture)-mixed culture (more than 107 CFU/ml of both E. sakazakii and E. vulneris) inoculation to 25 g of powdered infant formula. The results showed that an initial inoculum of group 1, group 2, group 3, and group 4 was detected in 0%(undetectable), 100%(6/6), 0%(0/6), and 90%(18/20) by FDA methods, respectively, while by Real-Time PCR, in0%(0/2), 100%(4/4), 0%(0/4), and 100%(10/10), respectively. According to this study, Real-Time PCR can be used more successfully than FDA method in order to detect and isolate E. sakazakii. Further studies to evaluate the detection limit at the low concentration (ca.101-105 CFU/ml) of mixture culture of E. sakazakii and E. vulneris.
K. Y. Song1 , K. H. Seo2 , S. Lee1 , R. E. Brackett2 , 1JIFSAN, UMD, College Park, MD, 2CFSAN, FDA, College Park, MD
Enterobacter sakazakii has subsequently been implicated worldwide in neonatal and infant cases of meningitis, septicemia, and necrotizing enterocolitis. Hence, it is critical need to detect and isolate E. sakazakii from the contaminated samples with the rapid and accurate. According to U.S. FDA method for isolation of E. sakazakii in dehydrated powdered infant formula, there involved several steps of pre-enrichment, subculture, re-incubation, and picking and streaking the yellow colonies, which will take at least 6 to 7 days, and also the confirmation method is performed by a biotyping assay, such as API 20E. This study was to evaluate the performance of 10 different pre-enrichments using by state-of-art Real-Time PCR so as to improve or upgrade the FDA method. After artificially inoculation of E. sakazakii to 25 g of powdered infant formula, it was mixed with 225 ml of 10 different pre-enrichments, prewarmed to 45 C, and gently dissolved by hand swirling until the powder was uniformly suspended, After incubating the reconstituted formula samples overnight at 37 C, 1 ml of each pre-enrichment were treated to extract the DAN to evaluate the Ct value using by Real-Time PCR. In the inoculation volume of 7.2 CFU/g E. sakazakii, the Ct value showed 22.72 (Tryptic soy broth), 25.82 (TSB-0.6% yeast extract), 23.43 (Distilled water), 28.27 (VRBG), 21.37 (Enterobacteriacease), 22.31 (TSB-0.1% sodium pyruvate), 24.95 (Nutrient broth), 23.02 (VRBL), 21.95 (Brain Heart Infusion), and 24.34 (PBS). In the inoculation volume of 1.82 CFU/g E. sakazakii, the Ct value showed 22.88 (TSB), 22.16 (TSB-0.6% YE), 23.01 (DW), 24.66 (VRBG), 20.34 (EE broth), 23.79 (TSB-0.1% SP), 23.19 (NB), 22.71(VRBL), 22.37 (BHI), and 24.70 (PBS). And in the inoculation volume of 0.182 CFU/g E. sakazakii, the Ct value showed 25.14 (TSB), 23.07 (TSB-0.6% YE), 23.31 (DW), 29.00 (VRBG), 31.55 (EE borth), 24.14(TSB-0.1% SP), Undetected (NB), 21.93 (VRBL), 23.70(BHI), and 23.47 (PBS). This results showed that there was no significant between 10 different pre-enrichment broths (p<0.05). Further studies are being evaluated the detection limit at the low concentration (ca. less than 0.1 CFU/g) and to compare the Ct value of heat-, acid- or cold-injured E. sakazakii using by 10 different pre-enrichment broths.
D. V. Volokhov1 , S. Duperrier2 , A. A. Neverov1 , J. George1 , R. E. Duvall3 , C. Buchrieser2 , A. D. Hitchins3 , 1FDA, Rockville, MD, 2Institut Pasteur, Paris, France, 3FDA, College Park, MD
Background: In a previous study the atypical hemolytic Listeria innocua strain, PRL/NW 15B95, was shown to contain a cluster of genes analogous to the L. monocytogenes pathogenicity island 1 (LIPI 1) gene cluster (Johnson J. et al., Applied and Environmental Microbiology, 2004 70(7), pp.: 4256-4266). This cluster includes the hemolysin gene coding for the hemolytic activity of PRL/NW 15B95. While some non-LIPI 1 L. monocytogenes specific genes were found to be absent the possible presence of other non-LIPI 1 L. monocytogenes specific genes still existed.
Methods: The isolate's genome was examined with a Listeria biodiversity DNA array. Regions of interest were nucleotide base sequenced and subjected to comparative genomic and phylogenetic analyses.
Results: The array results confirmed that the isolate was a L. innocua strain with LIPI 1 genes and indicated that it was serotype 6a consistent with previous serological analysis. Surprisingly, evidence for the presence of the non-LIPI 1 L. monocytogenes specific gene, inlA, previously thought to be absent, was obtained. Significantly, although inlA's expected neighbor, inlB, is absent several other neighboring L. monocytogenes serotype 4b specific genes are present.
Conclusion: The presence of more than one cluster of L. monocytogenes specific genes makes it less likely that PRL/NW 15B95 is simply a L. innocua strain that has been altered by horizontal gene transfer in recent times. Overall the evidence suggests that the atypical isolate is a living relic that tangibly represents a stage in the devolution of L. innocua from a common L. monocytogenes and L. innocua ancestor, a process that has been postulated by others.
M. O. Paul, NRL, ORA, FDA
Background: Enterobacter sakazakii is a Gram-negative rod belonging to the family Enterobacteriaceae. Listed previously as yellow-pigmented Enterobacter cloacae, Enterobacter sakazakii is a life-threatening cause of epidemic and sporadic cases of meningitis, sepsis, and necrotizing enterocolitis among neonates, with reported mortality rates of 40 to 80%.
Methods: During routine examination of a sample of sesame seeds which was Salmonella VIDAS positive (but biochemically and serologically negative) for Salmonella spp., we isolated E. sakazakii. Isolation was possible at each stage of the Vidas procedure, viz., after: pre-enrichment in lactose broth, selective enrichment in RV/TT broths, and post enrichment in M-broth. Each broth culture was plated onto HE (Hektoen Enteric) and XLD (Xylose Lysine Desoxycholate) agar plates from which typical colonies were streaked onto TSAYE (trypticase soy agar with yeast extract) plates. E. sakazakii isolation was confirmed on yellow-pigmented colonies by VITEK.
Results: Between Nov., 2004 and July, 2005, we have confirmed five isolates of E. Sakazakii from: sesame seeds (three samples, all VIDAS false positive), Haleem Mix (a mixture of grains and spices-also VIDAS false positive), and from edible pumpkin seeds (VIDAS negative).
Conclusions: Our results indicate that laboratories like ours, both for convenience and economical considerations, may not need to employ specialized media for E. sakazakii isolation, but may do so during routine bacteriological examination for Salmonella spp. in food products. Such specialized media (VRBG, DFI) could be utilized to determine other parameters, such as the comparative sensitivity of our routine methods for E. sakazakii isolation.
R. D. Beger1 , L. K. Schnackenberg1 , R. D. Holland1 , D. Li2 , Y. P. Dragan1 , 1NCTR, FDA, Jefferson, AR, 2University of Texas, MD Anderson Cancer Center, Houston, TX
Background: In this study, we hypothesized that the altered insulin and glucose levels in male pancreatic cancer patients reported in a recent JAMA article would result in an altered lipid profile in the blood of pancreatic cancer patients when compared to case controls (Stolzenberg-Solomon, et al., 2005).
Methods: Nuclear magnetic resonance (NMR) and mass-spectrometric (MS) metabolic profiling techniques were applied to lipid extracts of plasma samples from pancreatic cancer patients and matched controls obtained from another study (Li et al., 2006).
Results: The NMR data from the lipid plasma extracts were used to build partial least squares discriminant function (PLS-DF) models that classified samples as belonging to the pancreatic control group or to the pancreatic cancer group. PLS-DF models based on 4 bins classified pancreatic cancer and pancreatic controls with 91 percent accuracy in training and 90 percent accuracy in leave-25%-out cross-validation testing.PLS-DF models based on 5 bins classified pancreatic cancer and pancreatic controls with 96 percent accuracy in training and 94 percent accuracy in leave-25%-out cross-validation testing.Results from our MS-based phospholipids profiling investigations of lipids in plasma from pancreatic cancer and control patients are consistent with the NMR results presented and indicated altered levels of phosphatidylinositols and glycophospholipids.
Conclusions: The cancer models are based upon changes in lipid profiles in plasma that appear to provide more accurate diagnosis of pancreatic cancer than current methods that are based upon a single biomarker.
A. R. Boobis1 , D. Jacobson-Kram2 , J. S. MacDonald3 , N. G. Doerrer4 , 1Imperial College London, London, UK, 2CDER, FDA, Silver Spring, MD, 3Schering-Plough Research Institute, Kenilworth, NJ, 4ILSI Health and Environmental Sciences Institute, Washington, DC
Background: The standard two-year rodent bioassay is a primary source of information for assessment of potential human carcinogenic hazard posed by chemicals. A committee of government, academic, and industry scientists convened under the auspices of the ILSI Health and Environmental Sciences Institute (HESI) hypothesized that, for non-genotoxic chemicals, the signals of importance for human cancer hazard identification can be detected in shorter-term studies, thus providing critical information on mode of action for tumorigenesis.
Methods: The HESI committee is testing the hypothesis in a pilot program using existing US government databases. A query tool was developed to assess short-term signals in 90-day studies for their ability to predict tumor findings in two-year studies. Chemicals that were positive in liver, lung, kidney, or urinary bladder in two-year rat or mouse studies over the last five years were included. Signals from 90-day studies were evaluated in those tissues where tumors were seen, along with evidence for genotoxic and immunosuppressive activity.
Results: Cellular changes indicative of a proliferative process can be identified for some, but not all, of the chemicals producing tumors in two-year bioassays after 13 weeks of chemical administration. Preliminary results suggest that 90-day studies utilizing conventional endpoints may not be adequate to identify all chemicals that will eventually produce tumors after two years. Not all of these will be relevant to humans. However, it may be possible to include additional endpoints to identify those critical signals of human relevance not detected with routine evaluation.
Conclusions:These analyses are not yet complete. Conclusions and next steps are predicated on further examination of the data.
P. L. Goering1 , V. S. Vaidya2 , R. P. Brown1 , Z. Vakili1 , B. A. Rosenzweig3 , A. M. Johnson2 , K. L. Thompson3 , A. I. Steen1 , J. V. Bonventre2 , 1CDRH, FDA, Rockville, MD, 2Harvard Medical School, Boston, MA, 3CDER, FDA, Silver Spring, MD
Background: Sensitive biomarkers are needed to detect kidney injury at the earliest stages because routinely-used biomarkers, such as BUN and serum creatinine (Cr), do not indicate renal injury until a significant degree of renal function is lost. The objective of this study was to determine whether Kim-1 mRNA levels in kidney and Kim-1 protein levels in urine are more sensitive than traditional biomarkers of nephrotoxicity.
Methods: Male rats were administered either a single injection of mercuric chloride (Hg) at 0, 0.1, 0.25, 0.5, or 1 mg/kg, iv, or injected daily for 3 days with gentamicin sulfate (gent) at 0, 25, 50, 100, 150, 200 or 400 mg/kg, sc. Immediately after the injections, rats were placed in metabolism cages for 24-hr urine collection, after which blood and kidneys were removed.
Results: BUN, Cr, and U-NAG levels were elevated above control values only at the highest dose (400 mg/kg) of gent and at doses of 0.5 mg Hg/kg and higher. In contrast, both kidney Kim-1 mRNA and urinary Kim-1 protein levels were significantly increased above control values in a dose-related manner after injection of gent doses > 100 mg/kg. Kidney Kim-1 mRNA and urinary Kim-1 protein levels were elevated at Hg doses > 0.25 mg Hg/kg, except urinary Kim-1 was similar to controls after 1 mg/kg, likely due to severe renal damage.
Conclusion: Increased Kim-1 mRNA expression in kidney and Kim-1 protein in urine may serve as biomarkers of renal injury for improved detection of nephrotoxicity following exposure to medical device residues and drugs.
E. Thomas1 , L. R. Grillone2 , T. R. McNamara2 , J. A. Gow2 , A. M. Hochberg3 , R. K. Pearson3 , 1L.A. Retina, Beverly Hills, CA, 2ISTA Pharmaceuticals®, Inc., Irvine, CA, 3ProSanos Corporation, Harrisburg, PA
Background: Successful completion of panretinal photocoagulation has been used as an endpoint in phase III clinical trials of intravitreous, highly purified, preservative-free, ovine hyaluronidase (Vitrase®) treatment for severe vitreous hemorrhage (VH). However, this endpoint in clinical practice may be affected by factors including patient scheduling and insurance coverage. A predictive model for hemorrhage clearance in patients with diabetes is needed.
Methods: A training set drawn from subjects with diabetes (N=350) from the Vitrase® for Vitreous Hemorrhage phase III clinical trials was retrospectively analyzed. At baseline and at one month post-injection, hemorrhage density was scored on a 0-4 scale, in each of 12 radial segments of the fundus. The change in the sum of the measurements ("Change in Total Hemorrhage Point Score", CTHPS) was evaluated, using Prentice's criteria, as a surrogate marker for successful completion of panretinal photocoagulation by three months without vitrectomy.
Results: By the Wilcoxon test, treatment with hyaluronidase had a significant effect upon the surrogate marker. By logistic regression, the surrogate marker CTHPS had a significant effect on the laser endpoint and the entire treatment effect was accounted for by the influence of treatment on CTHPS, thus satisfying Prentice's criteria.
Conclusions: The change in the measurement of VH density, CTHPS, meets established criteria as a surrogate marker for laser treatment and allows prediction of success by one month after an intravitreous injection of ovine hyaluronidase. This surrogate marker can guide future trials and enhance the management of patients with diabetes and VH.
E. Herman1 , A. D. Knapton1 , T. Miller1 , P. Espandiari1 , R. Anderson1 , S. Moore2 , J. P. Hanig1 , 1OPS/OTR/DAPR, 2Ciphergen Biosystems, Inc
Therapy of type II diabetes with thiazolidinedione drugs such as rosiglitazone (RSG) can be limited by toxicity such as fluid retention and cardiac hypertrophy. The present study was initiated to develop an experimental model which could be used to identify biomarkers associated with the onset of RSG-induced cardiovascular toxicity. Groups of 10 week old female Sprague-Dawley (SD) rats (5-6/group) were treated orally with RSG (80 mg/kg), 1% carboxymethylcellulose (CMC) or water daily for 28 days. At necropsy (day 29) blood and tissues were collected for analysis. Animals treated with RSG gained more weight (77 vs 66 vs 62 grams) and had lower glucose levels (186 vs 214 vs 223 mg/dL) than observed in the two control groups. As noted with clinical use, values for RBC, hematocrit and hemoglobin were significantly reduced in animals treated with RSG. Serum samples from RSG and CMC-treated rats analyzed by SELDI, using weak cation exchange and immobilized metal affinity Protein Chip array surfaces, revealed 11 differentially expressed peaks in the RSG-treated group not seen in the CMC control group. Multivariant analysis demonstrated that principle analysis and heirarchial clustering could differentiate RSG-treated from CMC vehicle rats. Myocyte changes consisting of minimal to moderate degeneration and /or necrosis were noted in the hearts of RSG-treated animals. Cardiac troponin T levels were not elevated in any of these animals. RSG also induced diffuse changes in brown (perirenal) as well as multifocal changes in white (peripancreatic and/or periovarian) adipose tissues. No hepatic alterations were detected in the RSG-treated animals. These findings sugget that female SD rats develop certain alterations which have been reported in patients and thus could be an appropriate model to search for potential early biomarkers of RSG-induced toxicity.D. S. Hirsch, Y. Shen, W. J. Wu, DMA, OBP/OPS, CDER, FDA
Background: Epidermal Growth Factor Receptor (EGFR) overexpression correlates with high proliferative index, invasive capacity and poor prognosis in breast cancer. Cdc42, a member of the Ras superfamily of small GTPases, is a GTP binding protein that regulates cell proliferation and migration. Stable expression of active Cdc42 in NIH3T3 fibroblasts causes increases in EGFR protein levels and cellular transformation. Here, we used two EGFR overexpressing breast cancer cell lines, MDA-MB-231 and BT20, as models to test the hypothesis that reduction of Cdc42 protein levels would lower EGFR protein levels resulting in inhibition of processes critical to breast cancer progression.
Methods: A series of breast cancer cell lines were screened for Cdc42 protein levels by western blot. Activated Cdc42 protein levels were determined using GST-p21 binding domain assay, an affinity chromatography assay that specifically detects activated Cdc42 protein levels. Cdc42 protein levels were reduced by transfecting cells with Cdc42-targeted siRNA; non-targeting siRNA was used as a negative control. Migration was studied using boyden chamber and wound healing assays.
Results: Higher levels of activated Cdc42 were found in EGFR overexpressing MDA-MB-231 and BT20 cells as compared to MCF7 which has low EGFR protein levels. Cdc42-specific siRNA reduced Cdc42 protein levels by greater than 50%. siRNA-mediated reduction of Cdc42 protein levels lowered EGFR protein levels, leading to a marked inhibition of cell proliferation and migration.
Conclusions: Cdc42 is a critical positive regulator of EGFR protein levels, cell proliferation and cell migration. Disruption of Cdc42 signaling can reduce cell proliferation and migration in MDA-MB-231 and BT20 cells. Targeted introduction of Cdc42-specific siRNA to cancer cells may offer a novel therapeutic approach to inhibit these processes. Future studies are aimed at determining the suitability of Cdc42 as a therapeutic target.
C. S. John, Y. M. Choi, G. Q. Mills, CDER, FDA
Background: The imaging biomarkers are currently being developed and qualified for the use in drug development. The objective of this presentation is to compare three radiopharmaceuticals (tracers) that bind to central nervous system (CNS) neurotransmitters such as dopamine transporters (DAT). The pharmacokinetics and pharmacodynamics of three tracers using Single Photon Emission Spectroscopy (SPECT) imaging is described.
Methods: [I-123]β-CIT has been shown to bind (label in-vitro and in-vivo) dopamine transporters (DAT) with high affinity. SPECT imaging with [I-123] β -CIT showed that radioactivity in striatal regions in PD patients was significantly reduced (50-70%) as compared to healthy volunteers (PNAS 1993, 90, 11965-11969). These results suggest that [I-123] β -CIT can be used as a biomarker for the loss of striatal dopamine terminals, a hallmark of PD pathology. A comparison of [I-123] β -CIT is being made with a drug X (Tc-99m labeled) and Y (I-123 labeled).
Results: All three tracers bind to DAT in-vivo with a high affinity. However, the whole brain uptake of three agents differ significantly. Y showed localization in the striatum accumulated rapidly and reached a maximum at 10-15 minutes after administration. Radioactivity accumulation in striatum in patients with PD was lower compared with healthy volunteers. Patients with moderate and advanced PD showed marked bilateral reduction in uptake of Y in caudate and putamen sub-regions of the brain which is the site rich in dopamine transporters. X also crossed blood brain barrier but only 0.4% ID was taken up in the brain. X was washed off significantly within 30 min. It also exhibited two diastereomers that had slightly different in-vivo kinetics.
Conclusion: All three tracers showed a decreased uptake in caudate-putamen sub regions of brain in PD patients as compared to healthy volunteers. Thus imaging can help a neurologist or movement disorder specialist in early diagnosis of PD. The imaging biomarkers may be also useful in initial phase of drug development in PD and in diagnosing PD, but its usefulness in clinical efficacy determination has not been successfully demonstrated yet.
B. H. Joshi, P. Leland, F. Varricchio, S. R. Husain, R. K. Puri, CBER, FDA, Bethesda, MD
Background: Bladder carcinoma is a difficult public health problem requiring identification of new targets for therapy. We have reported that many murine and human carcinoma cell lines and tumors express high levels of receptors for interleukin-4 (IL-4) and interleukin-13 (IL-13) in vitro and in situ. Here we have examined whether these receptors are also overexpressed in bladder carcinoma and they can serve as a potential biomaker of disease.
Methods: Highly specific antibodies to IL-4Rα, IL-13Rα2, IL-13Rα1 and IL-2Rγc chain were used for immunohistochemistry (IHC) studies in 15 biopsy samples of bladder carcinoma and 16 normal bladder specimens. RT-PCR analysis was also performed.
Results: Our data indicate that 11 of 15 bladder carcinoma specimens are strongly positive (2+ to 3+) for IL-4Rα and 3 of 15 samples are positive (1+ to 2+) for IL-13Rα2. In contrast, normal bladder specimens did not express either IL-4Rα or IL-13Rα2 chains. The expression of IL-13Rα1was ubiquitous in most cancer and normal samples studied; while IL-2Rγc was absent in cancer as well as normal bladder specimens. There was no correlation between the receptor expression and the age of the patients or histologic subtype of disease.
Conclusions: Most human bladder carcinoma specimens over express type II IL-4R in situ more frequently than IL-13Rα2 compared to normal bladder. A much larger sample size needs to be analyzed to determine whether these receptors can serve as a potential biomarker of disease. These studies help us understand the heterogeneity of bladder carcinoma.
D. S. Kaplan1 , V. M. Hitchins1 , P. V. Phan2 , R. Y. Au2 , M. W. Hungerford3 , C. G. Frondoza3 , 1CDRH, FDA, Rockville, MD 20850, 2Nutramax Laboratories, Edgewood, MD, 3Johns Hopkins Univ., Dept. Orthopaedic Surgery, Good Samaritan Hosp., Baltimore, MD
A critical obstacle in the use of cell-based therapies is the limited number of chondrocytes available to repair cartilage defects. Chondrocytes isolated from the extracellular matrix of cartilage and propagated in monolayer culture dedifferentiate into a fibroblastic phenotype. These cells shift from producing type II to type I collagen and shift from producing high molecular weight (MW) aggrecan to low MW proteoglycans. Analysis of growth characteristics and phenotype changes at the gene level may help identify tissue culture conditions favoring proliferation and maintenance of chondrocyte cellular functions. In the present study human chondrocytes derived from three cell lines were seeded and propagated on natural polymers as scaffolds and the propagated chondrocytes were evaluated for type II collagen and aggrecan production.
Articular chondrocyteswere grown on 1) control polystyrene (PS) tissue culture flasks, 2) type I collagen, and 3) alginate gels. Cells growing on both PS and collagen gels appeared refractile with well defined morphology, and all cells remained 100% viable. In contrast, chondrocytes seeded on the alginate gel were sparsely distributed throughout the scaffold and did not appear as healthy as cells cultured on PS and collagen. Evaluation of phenotypic markers using reverse transcriptase polymerase chain reaction showed that all three lines expressed a fibroblastic phenotype, characterized by expression of type I collagen. Aggrecan expression patterns varied according to the cell line.
The present study demonstrates that these two natural scaffold materials, type I collagen and alginate gels, did not consistently enhance retention of the chondrocyte phenotype.
Acknowledgements: Supported by the FDA Office of Science and Health Coordination and the Good Samaritan Hospital.
A. D. Knapton1 , J. Zhang1 , J. L. Weaver1 , F. D. Sistare2 , J. P. Hanig1 , 1FDA, Silver Spring, MD, 2Merck, West Point, PA
Our previous study showed the number of mesenteric tissue mast cells (MC) and the percent of MC degranulating increased as early as 1-2 hr after treatment with SK&F 95654. We hypothesized that MC may serve as drug targets and that MC mediators (histamine, serotonin, etc.) may participate in early drug-induced vascular injury. This study was to determine if perfusion with MC degranulator compound 48/80 or vasodilators fenoldopam (FP) and aminophylline (AP) caused early changes in MC, and if perfusion with MC mediators histamine or serotonin caused early changes in the mesenteric vasculature. The mesentery of Sprague-Dawley rats was perfused in situ, via the superior mesenteric vein, with 0.12 or 1.2 mg/ml of compound 48/80, 2 or 4 mg/ml of AP, 0.95 mg/ml of FP, 1 or 2 mMhistamine, or 800 µM serotonin for 30 min. The mesentery was then perfused with monastral blue and Evan's blue for 15 min. Free dyes were washed out with drug-free perfusion fluid for 15 min. Mesenteries perfused in-situ without drug were used as controls. The whole mesentery was then fixed in formalin for histopathologic evaluation. On gross examination, dye deposits were found in the mesenteric vessels in rats perfused with compound 48/80 or AP. Histopathological evaluation showed that blue dye was deposited in the walls of mesenteric capillaries, venules, arterioles, and small veins in rats treated with compound 48/80, AP, histamine, and serotonin, indicating increased vascular permeability by these agents. Also, MC degranulation, eosinophil recruitment, and vascular inflammation were detected in rats perfused with compound 48/80, AP, and FP. Effects of histamine and serotonin on mesenteric vessels were characterized by vasodilatation, increased vascular permeability, increased number of eosinophils, and perivascular inflammation. This suggests that agents causing mesenteric inflammation in the rat target the MC and that MC mediators released by degranulation may play an important role in mesenteric vascular injury.S. Kumar1 , V. Majam1 , H. Zheng1 , B. Mahajan1 , M. Parise2 , M. Wilson2 , 1CBER, FDA, Rockville, MD, 2CDC, Atlanta, GA
Malaria has a significant impact on U.S. domestic and international interests. There are about 1,500 clinical cases of malaria reported in the United States each year, and approximately 50,000 US military personnel and more than 27 million American visitors are at the risk of contracting malaria annually due to their presence in malaria endemic areas. Furthermore, transfusion-transmitted malaria (TTM) remains a rare but serious risk of blood transfusion that occurs at a rate of about one case per four million blood units collected [Mungai et al. N Eng J Med 2001 344: 1973-1978]. In the absence of a licensed blood-screening test, each year approximately 150,000 donors are deferred based on their history of travel or prior residence in the malaria endemic regions. An antibody-based blood-screening test that detects all four species of human Plasmodium might confirm the absence of a prior or current malaria infection and thus allow reinstating the majority of donors after a significantly shortened deferral period. We, in partnership with the CDC, are working to develop a recombinant multiple malarial-antigen based enzyme-linked immunosorbent assay (rMELISA) as a blood-screening test to detect a current or past exposure to all species of human malaria parasites, Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale (Po), that are known to cause TTM in the U.S. in prospective donors of blood and blood-products. The availability of such a test will allow us to revise the current FDA guidance based on actual identification of donors for malaria-risk, and thereby minimize the risk of TTM, while increasing the blood supply by reinstating a substantial number of otherwise suitable donors. Currently, some European countries utilize malarial antibody-based tests to shorten the deferral period for malaria at-risk donors. However, European tests are unsuitable for U.S. needs because of limited sensitivity and detection of antibodies to only two of the four human malaria parasites. The urgent need for a blood-screening test for malaria has been recognized as a significant topic under the CBER Critical Path Initiative and is a prime example of the FDA's mission-oriented research to facilitate the development of a potentially marketable product needed by industry to improve blood safety.Results showing the progress in the development of rMELISA as a blood screening test will be presented.
J. E. Leakey1 , F. W. Lee1 , S. M. Lewis1 , G. Olson2 , J. K. Dunnick3 , W. T. Allaben1 , 1OSC, 2TPA, 3900 NCTR Rd, Jefferson, AR 72079, 3NIEHS, POB 12233, Research Triangle Park, NC 27709
AZT/3TC antiretroviral drug combinations are given during pregnancy to reduce maternal-fetal HIV transmission. AZT is genotoxic in fetal mice and monkeys and carcinogenic in mice. We assessed a new C3B6-trp53tm1Brd[N12]F1 p53 haplodeficient transgenic mouse model for use in cancer bioassays. These mice, produced by mating Taconic C57Bl6(N12)trp53(-/-) males and C3H females, possibly have similar tumor profiles to B6C3F1 mice. Haplodeficient C3B6-trp53tm1Brd[N12]F1 mice were dosed with 0, 40, 80, 160, 240 mg/kg AZT or 160 mg/kg AZT combined with100 mg/kg 3TC, by gavage in aqueous methylcellulose/polysorbate 80 (0.2/0.1%), transplacentally from GD12 to GD18 then postnatally from PND1 until6 or 9 months old. During the initial postnatal period (PND1- PND28), survival was >95% and >82% for the control and dosed groups respectively. The AZT and AZT/3TC treatment produced only small (<10%) reductions in body weight gain, but mice from the AZT/3TC dose groups showed increased hprt mutation frequency when evaluated at PND 28. Both AZT at all 4 concentrations and AZT/3TC increased blood reticulocyte micronuclei formation at PND1, 10 and 28. AZT treatment did not significantly reduce hepatic mitochondrial gene expression or increase serum lactate levels, but did increase tumor incidence at 6 months of age. In male mice, preliminary data showed overall tumor rate increased from 0/25 in controls to 6/26 (23%) in the 240 mg/kg AZT group. Tumor types included 3 malignant lymphomas and 2 brain tumors. Thus, the C3B6-trp53tm1Brd[N12]F1 mouse may be a good model for evaluating cancer risk of antiretroviral drugs.
B. D. Lyn-Cook1 , A. Haefele2 , B. Word1 , G. J. Hammons1 , 1FDA, 2University of Arkansas at Little Rock
Genetic variants of genes, such as DNA-methyl-transferases, are known to affect regulation ofother genes in cancer. DNMT 3A and 3Bgenes are up-regulated in pancreatic cancer. Recently, a novel C to T single base transition in the promoter of DNMT 3B was detected in lung cancer and was found to correlate with greater than 2-fold increase in risk of lung cancer. A higher frequency of this polymorphism was also found in female lung cancer patients. In this study, we have identified this C to T polymorphism in pancreatic tumor cells and tissues. A significant level of DNMT 3B expression was found in the female pancreatic cell line (SU 86.86)with the C to T polymorphism compared to the lack ofdetectable expression of DNMT 3B in the C to C wild-type male pancreatic tumor cell lines (Mia or Panc-1). Preliminary findings have also showed a higher frequency of the C to T genotype in female pancreatic tumor tissues. This genotype, along with the TT variant, has been associated with increased expression, activity and tumor grade. We have found that a natural agent modulates the expression of DNMT 3A and 3B differently than the chemotherapeutic drug used in a number of solid cancers, gemcitabine. Gemcitabine alone or in combination with indole-3-carbinol increased expression of DNMT 3B but decreased DNMT 3A expression in the female tumor cells. Data also demonstrated a 2-fold higher level of DNMT 3A in the female tumor cells compared to the male cells without any treatment. Indole-3-carbinol decreased expression of DNMT 3A 3-fold in the female cells but had no effect on the male cells. These data suggest gender differences in DNMT 3A and 3B expression in pancreatic tumor cells and modulation by a natural chemoprevention agent, I3C, in pancreatic cancer.
R. P. Maguire1 , W. A. Hallett2 , 1Pfizer Global R&D, Groton, USA, 2GlaxoSmithKline, London, UK
Introduction:
Recently guidelines propose standards for FDG/PET-CT acquisition and analysis for clinical trials in oncology. However, in the industrial clinical trial environment, we need a more detailed level of protocol specification and standardization that allows trial execution partners: acquisition sites, software developers, analysis labs, statisticians and study teams to confidently and independently develop their own methods and procedures. This poster discusses some of the most important issues that need to be specified and proposes a protocol that is sound practice, practical and flexible enough to be accomplished by clinical nuclear medicine centers.
Methods:
We reviewed the literature and consulted with experts in the field. Issues such as the use of contrast in image acquisition and the radiation doses associated with measurement were considered. By acknowledging the interdependence of injected activity, scan time per bed position and scanning mode 2D/3D we attempted to ensure that image counts and signal to noise are within acceptable limits. We also considered the inclusion of further parameters such as plasma glucose level, which may be important covariates in the development of more complete models of observed SUV. Image analysis was also addressed, based on measurement of the SUVmax endpoint.
Conclusions:
The protocol recognizes a need for rapid outcome classification - using the EORTC criteria and the need to generate quantitative data for PK/PD modeling. We intend to share this protocol with our partners in oncology drug development as a step toward establishing standards for FDG/PET-CT trials. Whilst image quality guidance is only 'rule of thumb' at this stage, it is a move toward establishing firm image quality norms. The protocol will undoubtedly evolve over time, but it is thought that it represents an appropriate balance between prescription and flexibility and captures the issues at a level that is appropriate for interdisciplinary clarity.
T. J. Miller, P. Espandiari, J. Zhang, A. D. Knapton, J. Weaver, E. Herman, J. P. Hanig, CDER, FDA, Silver Spring, MD
Limitations of existing biomarkers to detect liver injury in experimental animals highlights the need for additional tools to predict human toxicity. Serum levels of cytochrome c (cyt c), were evaluated in two rodent liver injury models, acetaminophen (APAP) and D-galactosamine (GalN), as a potential indicator of drug-induced liver injury. Treatment of adult male SD rats with APAP (1g/kg, i.p.) for 24 h caused a significant increase in both ALT (U/L) and AST (U/L) compared to saline control. Similarly, ELISA analysis for serum cyt c demonstrated an increase in APAP-treated rats and an undetectable level of cyt c in the control group (86.28 +/-8.2 vs 0.0 ng/mL respectively). Likewise, SD rats given GalN (300, 500, 1100mg/kg) i.p. for 24, 48, 72, and 96 hours showed dose & time dependent changes in serum ALT and AST levels, maximal at 24 h for all treatment groups. Increased serum levels of cyt c (ng/mL) detected in GalN treated rats at 24 hours (1.1 g/kg: 122.7+/-10.6; 500 mg/kg: 5.374+/-0.7; 300 mg/kg: 3.1+/-0.4; vehicle: 2.8+/-0.3), mirrored closely the dose dependent increase in serum ALT and AST detected at this timepoint. Immunohistochemistry performed on liver tissue from GalN treated rats revealed a dose-related change in the location of cyt c from the perinuclear, punctuate-stained mitochondria to the diffusely-stained cytoplasm of affected hepatocytes. In mid and high-dose treated rats, cyt c can be found within the lumen of the hepatic central artery. Histopathology (H&E) of liver tissue from GalN-treated rats correlated well with the serum markers for toxicity, revealing moderate hepatocyte necrosis and increased hepatic inflammation in the mid and high-dose GalN treatment groups, decreasing in severity at later timepoints. In addition, a dose-dependent, increase in TUNEL positive apoptotic nuclei in hepatocytes was observed at 24 h, providing additional evidence for extracellular cyt c release. These studies demonstrate cyt c to be a useful indicator of hepatic injury in these animal models, and supports its potential utility as a clinical predictor of drug-induced liver toxicity.A. Agrawal, J. Pfefer, CDRH, FDA, Rockville, MD
Background: Light-based imaging approaches such as optical coherence tomography (OCT) can facilitate diagnosis of retinal pathologies and mucosal pre-cancers.Recently, there has been increased research activity in using nanoengineered particles which provide optical contrast enhancement and enable molecular targeting.Quantitative data on the fundamental optical properties of nanoshells will be necessary to understand the performance characteristics of future drug-device combination products as well as to evaluate the potential of these particles as components in test methods for optical devices.
Methods: In this study, we investigate the fundamental optical properties of silica core, gold-coated particles called nanoshells.The extinction and backscattering properties of nanoshells can be tuned by varying core and shell size. Measurement methods included OCT and spectrophotometry, while theoretical estimates of light scattering were calculated using Mie theory. Measurement techniques for determining backscattering and extinction properties were validated using well-characterized samples of polystyrene microspheres.
Results: Experimentally determined trends in the extinction cross section of nanoshells were consistent with Mie theory calculations. Although quantitative agreement between experimental and theoretical data was excellent when extinction was low,theory tended to under-predict experimental values by up to 30% when extinction values were at their highest.As expected, the OCT signal intensity displayed a linear increase with the square root of the theoretical backscattering coefficient.
Conclusions: These results provide a solid foundation for understanding of OCT performance with nanoparticles and for evaluating their incorporation into tissue phantom-based test methods.
Y. Shen, D. S. Hirsch, W. J. Wu, OBP, DMA, CDER, FDA
In NIH3T3 fibroblasts, expression of constitutively activated Cdc42, a Ras-like GTP-binding protein, causes an aberrant accumulation of EGF receptor (EGFR) by inhibiting c-Cbl-regulated EGFR degradation.Cdc42-regulated cellular transformation is blocked by c-Cbl overexpression.Overexpression of EGFR contributes to increased proliferation and tumor cell motility in human breast cancer.However, mechanisms of EGFR overexpression are not well characterized, and often cannot be attributed to gene amplification.Here, we useEGFR overexpressing breast cancer cell line MDA-MB-231 as model to test the hypothesis that impaired EGFR degradation contributes to its overexpression. Stable expression of c-Cbl-N480, a c-Cbl mutant that no longer associates with Cdc42, but still has E3 ubiquitin ligase activity, enhances the rate of EGFR degradation resulting in reductions in EGFR protein and in ERK activity.These effects inhibit the growth of MDA-MB-231 cells.MDA-MB-231 cells expressing c-Cbl-N480 in turn have significantly decreased levels of activated Cdc42 and Rac1, resulting in dramatic inhibition ofcell migration.These findings indicate that Cdc42 and c-Cbl are critical components involved in the regulation of EGFR protein levels in MDA-MB-231 cells.Given these findings, disruption of Cdc42 inhibition of c-Cbl function could serve as a therapeutic target to restore proper EGFR degradation and thus reduce cell proliferation and cell migration in breast cancer.
T. Shimamura1 , R. Royal2 , M. Kioi1 , K. Angra1 , S. R. Husain1 , R. K. Puri1 , 1CBER, FDA, Bethesda, MD, 2National Cancer Institute, NIH
Background: We previously reported that pancreatic carcinoma cell lines express Interleukin-4 receptors (IL-4R) and a chimeric fusion protein, comprised of circularly permuted IL-4 and Pseudomonas exotoxin (IL4-PE) prolongs survival of immuodeficient animals implanted orthotopically with human pancreatic tumors. In this study, we established an advanced pancreatic cancer animal model and examined the safety and efficacy of IL4-PE.
Methods: We examined IL-4R expression in 70 pancreatic ductal adenocarcinoma samples by immunohistochemistry. The cytotoxity of IL4-PE in pancreatic cancer and normal cell lines was determined by protein synthesis inhibition assays. We developed a primary and metastatic pancreatic tumor model by orthotopic implantation of GFP-transfected Miapaca-2 pancreatic cancer cells and detected their localization by in vivo whole body fluorescence imaging.
Results: We found that 42 of 70 tumor samples from pancreatic ductal adenocarcinoma expressed moderate to high density IL-4R. IL4-PE was highly cytotoxic (IC50= >0.1-10 ng/ml) to six pancreatic cancer cell lines, while it was not cytotoxic (IC50= >1000 ng/ml) to normal cells. In vivo whole body fluorescence imaging on day 14 post-tumor implantation confirmed the metastatic lesions in lymph nodes and peritoneal locations in 15 out of 29 mice. The treatment of these mice with i.p. IL4-PE (200 ug/kg, twice a day x 5 days) inhibited the growth of both primary tumors and metastatic lesions as determined by imaging studies. IL4-PE treatment was well tolerated and it significantly (p =0.018) prolonged the survival of these mice compared with excipient treated controls.
Conclusions: IL4-PE could be a useful therapeutic agent for the management of patients with advanced pancreatic ductal adenocarcinoma.
D. R. Tavris1 , B. A. Gallauresi1 , S. Dey2 , R. Brindis3 , K. Mitchel2 , 1FDA, 2American College of Cardiology, 3San Francisco Kaiser Hospital
Purpose
To assess the reason for the relative high risk of women for local complications following cardiac catheterization by evaluating the associations between gender, sheath size, and local adverse outcomes following cardiac catheterization.
Methods
The data used in this study was obtained from a portion of the American College of Cardiology-National Cardiovascular Data RegistryTM (ACC-NCDRTM), which included 13,878 patients who underwent cardiac catheterization at one of 59 participating cardiac catheterization institutions throughout the United States during late 2003. Rates of serious local vascular adverse events were calculated by gender following cardiac catheterization, by type of vascular hemostasis used, stratified by arterial sheath size.
Results
Serious local vascular events were reported in 3.54% of patients, most commonly hematoma (2.00%). The relative risk for women of any vascular complication was 1.40 [95% CI = 1.17, 1.67, p=.0002]. A statistically significant relative risk for woman was evident when collagen plug devices or manual compression alone were used as the first method for hemostasis. The rate of vascular complications increased progressively with increasing sheath size, more so in women than in men.
Conclusions
High relative risk for women of local vascular complications following cardiac catheterization was demonstrated with use of manual compression, as well as with collagen plug devices to control femoral artery bleeding. Large sheath size is associated with both a relatively high absolute risk and a high relative risk for women. Knowledge of this information should be considered by interventional cardiologists in making decisions on how to achieve hemostasis following cardiac catheterization.
S. A. Williams1 , D. E. Slavin1 , J. A. Wagner2 , C. J. Webster3 , 1Pfizer, 2Merck, 3Millenium
Background
The current system of subjective acceptance or rejection of biomarker signals fails to optimize error rates and costs, and leads to healthcare inefficiency, delays to drug development and does not minimize harm to patients.
Methods
We applied a tolerability of risk approach to explain current behaviors around acceptance or rejection of biomarkers by industry and regulators, and evaluated the likely effects of these behaviors on public health.
We propose an alternative to the subjective perception of harm [the key driver of biomarker acceptance or rejection] namely societal monetary cost, and describe biomarker qualification in terms of cost-benefit. We generate principles that enable the practical application of this method under different circumstances.
Results
Industry behaves to minimize the risk of biomarker failures that lead to false termination decisions or delays [false negative efficacy, false positive safety]. Regulators behave to minimize the risk of biomarker failures that lead to false "proceed" decisions [false negative safety and false positive efficacy]. Errors that lead to "dread" are subjectively amplified and distort management of particular types of biomarker risk.
Conclusions
Biomarker signals, monitoring programs or surrogate endpoints regarded as acceptable by one stakeholder will likely be regarded as unacceptable by others because of subjectivity in ranking importance of different risks. This leads to stagnation, increased costs, and a failure to maximize societal good. Conversely, a cost-benefit approach based on tolerability of risk principles can define acceptable biomarker performance that maximizes benefits and reduces the unintended consequences of precautionary behaviors.
A. Ottesen1 , G. C. Ziobro2 , 1JIFSAN, University of Maryland, College Park, MD, 2CFSAN, FDA, College Park, MD
Seedpods of Illicium verum Hook f., (Star Anise), are used in the preparation of teas, herbal remedies, and culinary dishes. Shikimic acid, extracted from the seedpods, is a crucial precursor for the synthesis of Avian bird flu medication, Tamiflu®.
Adulteration by seedpods of other Illicium species poses a health risk due to elevated levels of neurotoxic compounds (veranisatins), such as anisatin and neoanisatin present in other Illicium species. Over the last few years the FDA has received reports of seizures, vomiting, jitteriness and irritability after individuals have consumed Star Anise. Suchsymptoms are consistent with adulteration by other Illicium species. The Agency issued an advisory on Star Anise "teas" on September 10th , 2003.
Similarities in seedpod morphological characters preclude their use to definitively speciate individual seedpods, whereas adulteration at low levels by other Illicium species is not detectable by biochemical methods because Star Anise contains similar compounds at trace levels.
This poster reports on use of multiple different PCR sequence sites, biocoding regions, from plastid and nuclear DNA to develop a reliable method of distinguishing between Illicium species. Upon identification of successfulbiocoding regions to distinguish between toxic and non toxic Illicium species, a protocol will be developed for the rapid identification of adulterated preparations of Star Anise.
CATEGORY D: GENOMICS, PROTEOMICS, PREDICTIVE TOXICOLOGY AND MODELINGM.C. Aguila, CVM, FDA, Rockville, MD
Background: Many synthetic chemicals have been identified as known or suspected endocrine disruptors (EDCs). The long term impact of the potential interactions in human health is unknown. These chemicals are used in foods, personal care products, prescription drugs, household cleaners, and lawn care products. They may have estrogenic or antiestrogenic actions, androgenic or anti-androgenic and /or disrupt adrenal and/or thyroid functions.
Methods: The hazard evaluation of these EDCs represents a complex challenge from the scientific and regulatory point of view.
Results: Estimation of the potential and relative potencies of EDCs, in comparison with the endogenous hormones or natural hormone-like chemicals can be accomplished by in vitro test systems. An initial screening should be followed by in vivo evaluation of the rates of absorption and metabolism of exogenous EDCs. In conjunction, the binding to sex hormone binding globulin in plasma should be considered because it may differ from exogenous EDCs in vivo. Next, it is important to determine the ability of the EDCs to activate downstream events that, ultimately may lead to changes in gene expression and function, by examining different systems and focusing on the target organs. More information is needed with respect to the identification of susceptible animal models, exposure windows, and genetic markers associated with biological responses. Lastly, the effects found in animals need to be correlated to humans.
Conclusion: An integral evaluation of EDCs using a combination of mechanism-based toxicology with physiological, cell biology and molecular biology tools might be used to predict the safety of EDCs.
F. W. Frueh1 , L. J. Lesko1 , A. Rudman1 , F. Goodsaid1 , S. Amur1 , J. Woodcock2 , 1CDER, FDA, White Oak, MD, 2FDA, Rockville, MD
Purpose. The use of pharmacogenomics has increased rapidly over the last several years as indicated in the increasing number of regulatory submissions to the FDA containing pharmacogenomic information. Initiatives such as FDA's Critical Path, the recently issued Guidance to Industry: Pharmacogenomic Data Submissions, the creation of a new voluntary genomic data submission (VGDS) process and the formation of an Agency-wide Interdisciplinary Pharmacogenomics Review Group (IPRG) not only encourage industry to use pharmacogenomics in drug development, but also provide the support and resources needed to review genomic data at the FDA. In addition to reviewing VGDS data, the IPRG also works on policy development, and, upon request, advises review divisions on interpretation and evaluation of pharmacogenomic data that is part of regular submissions.
Methods. Sponsors can submit a VGDS to the IPRG by following the procedures and decision trees outlined in the Guidance and the associated operating policies (MaPPs) that have been published. In addition there have been numerous genomic workshops with industry and academia has established a dialog on the use of pharmacogenomics in drug development.
Results. Since the publication of the guidance there have been 23 requests for VGDS meetings to date with several sponsors requesting subsequent VGDS meetings as follow-up. The development of the IPRG and the VGDS has also led to increasing communications between global regulatory agencies, and joint pharmacogenomic meetings are now held with the European Medicinal Evaluation Agency in Europe.
Conclusion. Early experience with VGDSs has been very positive.Among the benefits to sponsors are the possibility to: meet informally with FDA and receive peer review assessments of scientific data from pharmacogenomic experts at the Agency, obtain insight into the evolving regulatory decision making process as it relates to genetic and genomic information, familiarize FDA scientists with novel pharmacogenomic experiments, data analysis, and interpretation approaches at an early stage. In addition, there have been numerous lessons learned in areas including: data submissions and review, regulation and policy, education, and international collaborations.
S. Amur1 , A. Noory1 , W. Tong2 , F. Goodsaid1 , F. W. Frueh1 , 1CDER, FDA, White Oak, MD, 2NCTR, FDA, Little Rock, AK
Background: The purpose of this analysis was to study differential expression of genes in cancer using PBMCs and also to identify differentially expressed genes during treatment with drug T in PBMCs and to interpret the biological significance of the findings in the VGDS study.
Methods: Hybridization data was generated using Affymetrix microarrays. The FDA microarray database and analysis tool, ArrayTrack, was used to store and analyze the VGDS data to identify differentially expressed genes. The biological interpretation of the data was carried out with the help of Ingenuity Pathway Analysis (IPA) and SafeBase tools.
Results: 108 genes were found to be differentially expressed in cancer patients. Analysis by IPA indicated that these genes are involved in pathways related to cell death, cancer and immune response. Treatment with Drug X caused changes in B-cell receptor signaling-related pathways at 8 weeks and chemokine signaling related pathways role at 16 weeks.
Conclusions: Differentially expressed genes were identified in the PBMCs of cancer patients and a subset of these genes suggested the possible occurrence of immunological events in addition to predictable cell cycle-related and cell death-related events. Drug X alters the gene expression profiles in PBMCs of the patients undergoing treatment.
P. Mummaneni, S. Amur, F. Goodsaid, A. Rudman, F. W. Frueh, CDER, FDA, White Oak, MD
Background: Currently, some genomic biomarkers are being usedas clinical adjuncts to selection and dosing of drugs, in addition to assessing drug safety. One of our objectives aligned with the FDA's Critical Path Initiative, was to compile a comprehensive list of genomic biomarkers in drug labels from 1945-2005.
Methods: Physicians Desk Reference (PDR), Drugs@FDA and Internet resources were identified as the sources of label information. Using a well-defined battery of search terms, we identified drug labels with possible pharmacogenomic (PGx) information and manually screened labels for genomic biomarkers.
Results: Approximately 10% of the approved drug labels contained PGx information with biomarkers. Her2 (Trastuzumab), c-kit (Imatinib) and EGFR (Cetuximab) are biomarkers used for responder selection based on gene expression. Patients are screened prior to therapy for G6PD (Dapsone), DPD (Fluorouracil) and OTC (Valproic acid), since deficiency of these biomarkers leads to severe toxicity. Genetic variations in NAT (Isoazanide), TPMT (6-MP), UGT1A1 (Irinotecan), CYP2D6, CYP2C19 & CYP2C9, proven to cause increased drug exposure leading to toxicity, are used for dosage adjustment. In addition, viral genotyping is used to identify drug resistance.
Conclusions: Our search highlights the importance of genomic biomarkers in identifying responders, avoiding toxicity and in adjusting dosage of drugs, for efficacy and safety. In the post-genomic era, many more clinically relevant genomic biomarkers will be added to the repertoire of the biomarkers. Genomic biomarkers will impact the future of personalized medicine that is in its infancy.
S. Amur1 , A. Rudman1 , P. Mummaneni1 , D. Bagatto2 , J. Gut2 , F. W. Frueh1 , 1CDER, FDA, White Oak, MD, 2Theragenomics Associates
Background: Currently, 121 marketed drug labels have been identified that contain pharmacogenomic (PGx) information.To facilitate capturing PGx information, a flexible database (SafebaseTM) was identified.SafeBase allows the user to store, structure, mine, visualize and interactively work with pharmacogenomic data.
Methods: We have identified the FDA-approved drugs that have PGx information in their labels and have extracted the PGx content. We are currently working with the SafeBase team to populate the database with the PGx information.
Results: The drug labels as well as the extracted genomic information from 40 labels have been added to SafeBase. From within the program, new hyperlinks to PubMed were created to enable FDA reviewers to check updates in the literature related to genomic information for the drug in question. To illustrate the functionality of SafeBase, a case study using Camptosar will be presented at the meeting.
Conclusion: We have successfully initiated the addition of drug labels and the PGx information to SafeBase. This novel knowledgebase will provide easy access togenetic and genomic information, thereby facilitating the safety and efficacy assessment of novel drugs.In addition, this information is provided in the context of chemical, biochemical, clinical and other relevant information, providing a searchable and complete overview on the impact of this information on systems biology.
R. L. Bell, T. L. Williams, D. Andrezjewski, K. A. Lampel, S. M. Musser, CFSAN, FDA, College Park, MD
Background: It has been well-documented in the literature that the ability of Shigella ssp. to penetrate into and replicate within human colonic epithelial cells is regulated by growth temperature. Bacteria grown at 37 C are virulent and invade epithelial cells, whereas the same virulent strains are noninvasive when grown at 30 C.This loss of invasive ability is reversible upon raising the growth temperature back to 37 C and therefore reflects a phenotypic change rather than a genotypic change. While the genes (both chromosomal and plasmid-encoded) have been identified as necessary for virulence, the proteins that are up or down-regulated in response to growth temperature have not been directly observed.
Methods: We have developed a method for generating bacterial protein profiles from the liquid chromatography/mass spectrometry (LC/MS) chromatogram of intact proteins of bacterial cell lysates. This method can be used to determine proteins unique to a particular strain as well as monitor expression differences due to external stimuli. When significant changes in protein expression profiles are identified, the proteins of interest can be easily purified, sequenced, and examined for alterations in primary sequence composition and/or post-translational modifications.
Results: This work describes the LC/MS analysis of S. flexneri in which the cells were harvested at 30 C and 37 C. Protein expression profiles were generated and proteins either unique or up/down-regulated to either growth temperature were identified. Since the only variable changed was growth temperature, all proteins that are differentially expressed are directly involved in virulence or are regulators of those virulence genes.
Z. K. Binienda1 , B. T. Thorn2 , L. K. Schnackenberg1 , R. D. Beger1 , P. P. Sapienza3 , C. S. Kim3 , S. F. Ali1 , B. D. Przybyla4 , 1NCTR, FDA, Jefferson, AR, 2Z-Tech, Co., Jefferson, AR, 3CFSAN, FDA, Laurel, MD, 4UAMS, Little Rock, AR
Background: A micotoxin, 3-nitropropionic acid (3-NPA), is investigated for its irreversible inhibition of mitochondrial succinate dehydrogenase (SDH).3-NPA produces neurodegeneration in several brain regions including basal ganglia. It has been previously reported that total FFA increased from 130 to 300% of baseline in the rat caudate, hippocampus and cerebral cortex within 1-2hr following acute exposure to 3-NPA.
Methods: The present study was undertaken to clarify a role of brain FFA and other metabolites during early response to metabolic stress. A global approach was undertaken to correlate changes in gene expression across the cortex, caudate, hypothalamus, hippocampus and cerebellum in adult Sprague-Dawley male rats at 0.5hr, 2hr and 4hr following exposure to 3-NPA at 30 mg/kg. The gene expression changes were correlated with changes of FFA. Additionally, the results of the monitoring metabolite composition in the cerebral cortex at the 2hr time point are presented.
Results: Custom designed microarrays containing oligos (MWG, Inc.) representing 530 rat genes were applied in the gene expression experiments. The FFA was measured by gas chromatography. The metabonomic profile of the cortex in the same animals obtained using NMR technology showed changes in the level of succinate at 2hr after exposure to 3-NPA. Eleven gene transcripts altered in the hippocampus were found to be correlated with changes in the level of FFA. The myristic (14C) and palmitic (16C) acid occurred in most of these correlations. A decrease in myristic acid concentration was associated with an increase in mRNA levels for genes related to calcium signaling pathways and transcription factor (AP-1) activators.
Conclusion: Data indicate that posttranscriptional and translational regulation may play a major role in early response to metabolic stress.
S. Dhruvakumar, PETA
The first step of drug discovery is "target identification" in which the disease state is studied to identify a molecular target for modulation through chemical intervention (i.e., a drug). Until recently, target identification relied heavily on finding targets by studying animal models of human disease (natural or engineered). However, animal-based targets are only successful to the extent that the relevant biology is replicated in humans -- a hit-or-miss proposition due to differences between species and between the animal and human diseases. However, a new paradigm is emerging as genomics, proteomics, and other 'omics' technologies are facilitating the molecular level study of humans. Targets are increasingly found by studying human tissue (e.g., normal vs. diseased or early vs. late stage) for differential gene or protein expression to identify genes that are involved in the disease process. Cellular pathways associated with the human disease can be mapped using such techniques, leading to the identification of high-quality human-relevant disease-specific targets. Another application of 'omics' technologies comes later during drug development, when these technologies can be used to identify early biomarkers of drug efficacy or toxicity. Early stage human drug testing or 'experimental medicine' utilizing such biomarkers is increasingly popular and is replacing some animal preclinical testing. The identification of 'omics' biomarkers is also being done within animal experimentation. While this enables more humane endpoints, the fact remains that attempting to extrapolate from animal-based human drug research is inefficient compared to studying humans directly, and resources should shift more quickly towards human-based 'omics' approaches.
D. G. Ranamukhaarachchi1 , D. Nandanie1 , R. Puri2 , A. X. Yang2 , J. Han2 , J. C. Fuscoe3 , T. Han3 , W. S. Branham3 , H. F. Yancy4 , H. C. Harbottle4 , J. Mason5 , S. Morris3 , W. Liggett6 , S. J. Wang7 , D. Mendrick8 , T. Martinsky9 , Rosalie Elespuru1 , 1CDRH, Federal Labs at White Oak, Silver Spring MD, 2CBER, FDA, Bethesda MD, 3NCTR, FDA, Jefferson AR, 4CVM, FDA, Laurel MD, 5Howard U. Washington DC, 6CDRH/NIST, FDA, Rockville MD, 7CDER, Federal Labs at White Oak, Silver Spring MD, 8GeneLogic, 9Telechem
Background: Microarray-based genomic (gene expression) profiling is still quite experimental and technology is changing. To prioritize issues for guidance development and product review, we are evaluating sources of variabilities in model genomic systems (irradiated and unirradiated tissue culture cells [phase 1] and primary human lymphocytes [phase 2]). [Critical path]
Methods:Four FDA Genomics Labs (CDRH, CBER, NCTR, CVM) are participating in an inter-lab validation exercise examining the following variables: microarray surfaces and chemistries; microarray printing, organization, and feature location; microarray hybridization and processing; sample RNA isolation and labeling; microarray and RNA stability; performance of commercial high density chips (phase 2). Microarrays and sample RNA's created in each lab were swapped to the other labs, allowing independent evaluation of printing, sample isolation, and microarray processing.
Results: Four different microarray surfaces have been printed and processed in the four labs, following the printer and experimental designs created by the group. Data from phase 1 are being gathered for statistical evaluation. Multiple technical issues were resolved in each laboratory before valid comparisons could be made. Each type of microarray surface required an independent approach to optimization. Technical issues included positive and negative controls, microarray printing, processing, and scale-up. Comparative results in different labs will be presented.
T. J. Flammang, J. F. Anson, W. Slikker, NCTR, Jefferson, AR
The National Center for Toxicological Research conducts FDA mission-critical peer-reviewed research to protect and promote public health. The research is designed to provide a scientifically sound basis for regulatory decisions and reduce risks associated with FDA regulated products. The NCTR program features a multi-disciplinary toxicology research team supported by extensive AALAC accredited animal research and breeding facilities; GLP-compliant safety study designs, 10 BSL-3 safety level containment laboratories; a phototoxicology center; specialized programs for genomics, proteomics, metabolomics, and toxicoinformatics; and pathology, computer, animal care, chemistry, and microbiology support teams. NCTR scientists and collaborators published 200 scientific papers and produced six patents for innovative products in 2005. The NCTR specializes in the customized quantitative bioassessment of chemicals of FDA regulatory interest in a major partnership with the National Toxicology Program. Current bioassessment studies include the toxicity of acrylamide, a newly emerged food contaminant and known animal carcinogen; the neurotoxicity of ketamine, a commonly used pediatric anesthetic; aloe vera, a dietary supplement, the phototoxicity of modern tattoo inks and permanent cosmetics, and the toxicity of combination therapies of anti-HIV/AIDS drugs. The Division of Systems Toxicology fully integrates -omics technologies that are revolutionizing personalized medicine, drug development and safety studies into side-by-side designs with traditional biochemical toxicity studies. The Division uses custom microarray printing facilities in support of genomics; mass spectrometry and nuclear magnetic resonance facilities in support of proteomics and metabolomics; and a toxicoinformatics branch continuously develops its ArrayTrack software for storage, analysis, and integration of study results. www.fda.gov/nctr
G. Fraczkiewicz, D. Zhuang, R. Fraczkiewicz, W. S. Woltosz, M. B. Bolger, Simulations Plus, Inc., Lancaster, CA
Human ether-à-go-go-related gene (hERG) K+ channel blockage is the key mechanism responsible for QT prolongation that frequently results in torsade de pointes (polymorphous ventricular tachycardia, TdP), which may degenerate into ventricular fibrillation and sudden death. A wide range of drugs with diverse chemical structures can inhibit hERG K+ channels. In silico methods employed in pre-synthetic toxicity screening can estimate hERG channel blocking activity.
We have built 2D and 3D QSAR models for prediction of the hERG K+ channel affinity using methods of artificial neural network ensembles (ANNE) and classification support vector machine ensembles (SVME). The main focus of our work was the selection of a consistent data set for model training. A set of highly variable drug-like compounds with IC50 values for inhibition of hERG K+ channels expressed in mammalian cell lines (HEK 293, CHO, neuroblastoma cells) were selected from literature. Multiple data sources were collected for each compound and the most repetitive value of hERG IC50 was used as the final value for the model building.
Our 2D and 3D ANNE regression models can predict pIC50 values within ~0.5 log units. Models were validated with external test sets (R2 = 0.88 - 0.9, RMSE = 0.47 - 0.54 log units). SVME classification model turned out to be highly effective in separating blocking and non-blocking compounds; better than the regression model. False rates for training/verification and test sets were 4% and 1% respectively.
R. Fraczkiewicz, G. Fraczkiewicz, M. B. Bolger, W. S. Woltosz, Simulations Plus, Inc.; 42505 10th Street West, Lancaster, CA 93534
The concept of partial electric charges localized on atoms serves as an approximation of the electron density distribution in molecules. The latter, in turn, is a primal determinant of ADMET properties of molecules. A data set composed of 473 organic molecules was composed with maximum diversity of individual atomic environments in mind. Molecular geometries were optimized at the B3LYP/6-311G** level, followed by extraction of partial atomic charges with the aid of the NPA (natural population analysis)scheme. One part of the data set was used to train a new empirical model for very fast estimation of the atomic charges, the remainder was sequestered as an external validation set. Next, several predictive ADMET property models have been retrained using the new charge related descriptors. Quality of these new models was subsequently compared to their earlier versions that utilized PEOE charges of Gasteiger and Marsili.J. C. Fuscoe1 , R. D. Beger1 , W. S. Branham1 , R. R. Delongchamp2 , Y. P. Dragan1 , P. H. Duffy3 , R. D. Edmondson1 , T. Han1 , D. K. Hansen3 , R. D. Holland1 , R. C. Jones1 , C. L. Moland1 , L. K. Schnackenberg1 , J. T. Taylor1 , W. Tong1 , 1Division of Systems Toxicology, NCTR, FDA, Jefferson, AR, 2Division of Biometry and Risk Assessment, NCTR, FDA, Jefferson, AR, 3Division of Genetic and Reproductive Toxicology, NCTR, FDA, Jefferson, AR
Various gender differences in cardiovascular disease and heart failure have been noted in humans, including age of onset, initial manifestations, and drug response.This variability may be reflective of differential gene and protein expression in the two sexes.To understand these sex-associated differences, gene expression, metabolite, and protein profiles in the heart will be determined in both males and females in a rat model system.This unique systems biology approach of integrating "OMICS" data is expected to result in a more complete picture of sex differences than any one method.Hearts from both sexes of young (8 weeks), middle age (21 weeks) and old age (78 weeks) Fisher 344 rats were ground into powder under liquid nitrogen and aliquots were used for each analysis. Aqueous extracts evaluated by 1D proton NMR showed differences in creatine, taurine, lysine, lactate, glucose, glutamine, and glutamate levels.MS profiling showed sex and age-related changes in specific phosphatidylcholine and ATP/ADP levels. Proteomic and gene expression studies are in-progress.All data sets will be integrated in an NCTR-developed database/analysis system.Completion of this project will result in: (1) a compendium of differences between the sexes in terms of metabolites, gene expression, and protein expression; and (2) definition of sex differences in metabolic and regulatory pathways in the heart.This knowledge in an animal model system will help focus resources on human studies aimed at the proper treatment and management of disease in males and females.
J. Han1 , R. L. Farnsworth2 , J. L. Tiwari1 , J. Tian1 , H. Lee1 , P. Ikonomi2 , A. P. Byrnes1 , J. L. Goodman1 , R. K. Puri1 , 1CBER, FDA, Bethesda, MD, 2ATCC, Manassas, VA
Background: Changes in cell culture conditions influence the metabolism of cells, which consequently affects the quality of products they produce, e.g., viral vector, recombinant proteins, or vaccines. Currently, there is no effective technique available to monitor the overall quality of cells grown in bioreactors.
Methods: Human embryonic kidney (HEK) 293 cells, commonly used as a cell substrate to produce biological products including adenoviral vectors, were grown to different confluence states. Cells were harvested and total RNA extracted, reverse transcribed, labeled, and hybridized with 10K cDNA microarrays. Additional cells were infected with an adenoviral vector (AdGFP) and two days later the viral titers were determined. Microarray data were analyzed by phenotype scatter plot and cluster analyses for class discovery. Gene-specific real-time Q-PCR was used to verify the expression of selected genes.
Results: We demonstrate that cell confluence status directly influences the titer of the adenoviral vector produced by 293 cells. Over-confluent cells produced a lower yield of adenoviral vectors compared to 40% or 90% confluent cells. Genes that were either up-regulated or down-modulated in response to different cell confluence status were identified by DNA microarray analyses. By multivariate predictive models, a set of 37 genes were identified, which were either down-regulated or up-regulated in over confluent cells compared to 90% confluent cells and thus appeared to be predictor of cell confluence state and the quality of product.
Conclusion: These results demonstrate that gene expression profiling may be able to assess the quality of cell substrates prior to the production of a large-scale batch of biological product saving time and resources.
T. Han, C. D. Melvin, L. Shi, W. S. Branham, P. S. Pine, K. L. Thompson, J. C. Fuscoe, FDA
DNA microarrays, which have been increasingly used to monitor mRNA transcripts at a global level, can provide detailed insight into cellular processes involved in response to drugs and toxins. This is leading to new understandings of signaling networks that operate in the cell, and the molecular basis of diseases. However, the high costs of commercial oligo arrays have made them inaccessible to many laboratories. Custom printed oligo arrays have proved to be a cost effective way to facilitate the applications of DNA microarray technology. Using hybridization conditions recommended by the oligo provider, cDNA labeled from two rat mixed tissue samples (NAR 33:e187 2005), were hybridized with in-house fabricated rat oligo arrays. The expression ratios were compressed greater than expected and data analysis revealed a high degree of non-specific binding. A series of experiments was designed to optimize the hybridization and washing conditions using mixed tissue samples, rat liver RNA and universal rat reference RNA (Stratagene, Inc.) in order to minimize cross-hybridization. The results from the optimized hybridization and washing conditions greatly improved the reproducibility and accuracy of expression ratios. These experiments also demonstrated the importance of probe design using a better bioinformatics approach and the need for common reference RNA samples for platform performance evaluation in order to fulfill the potential of DNA microarray technology.
T. Andersson1 , D. A. Flockhart2 , D. B. Goldstein3 , S. M. Huang4 , D. L. Kroetz5 , P. M. Milos6 , M. J. Ratain7 , K. Thummel8 , 1AstraZeneca, 2Indiana University, 3Duke University, 4CDER, FDA, Silver Spring, MD, 5UCSF, 6Pfizer, 7University of Chicago, 8University of Washington
Pharmacogenomic data can facilitate our understanding of the sources of variability in drug response and can potentially lead to improved safety and efficacy of
drug therapy for individual patients.The type of genomic data (eg, which alleles, what genotypes) that need to be evaluated is one of the critical issues in drug development
and regulatory review.Several drug metabolizing enzymes and the clinical utility data of the pharmacogenomic tests were reviewed.A summary of recommended polymorphic alleles that need to be evaluated in various ethnic/racial groups for common, known valid metabolic biomarkers such as CYP2D6, CYP2C9, CYP2C19, and UGT1A1 are listed in the following Table. Emerging data for exploratory biomarkers (eg, CYP3A4/3A5,ABCB1, or methods involving tagging SNPs) are also discussed.
Table. Summary of recommended polymorphic alleles of specific metabolizing
biomarkers to measure in specific population groups [ref 1,2]
| Additional alleles relevant to specific population groups | ||||
|---|---|---|---|---|
| Enzymes | Basic alleles to measure in all population groups | Caucasians1 | African Americans1 | Asian Americans1 |
| CYP2C9 | *2,*3 | *5,*6 | ||
| CYP2C19 | *2,*3 | *4,*5,*6 | ||
| CYP2D6 | *3,*4,*5,*6,*2xN | *10 (*41)2 | *17 | *10 (*21)2 |
| UGT1A1 | *28 | (*60)2 | (*6)2 | |
I. R. Patel, S. A. Jackson, J. E. LeClerc, T. A. Cebula, CFSAN, FDA, Laurel, MD
Background: An important part of the public health mission is to understand factors that affect the pervasiveness of foodborne bacterial infections. Bacterial mutators, for instance, are found among natural populations of pathogens and typically contain defects in the mutS gene, part of the methyl-directed mismatch repair (MMR) system that maintains genome fidelity. Other unusual phenotypes associated with mutators, however, suggest that MutS may have additional roles that affect the infectious process.
Methods: We used DNA microarrays to analyze the global gene expression profiles of wild-type and mutator strains of Salmonella Typhimurium and Enteritidis in both exponential and stationary phase growth. The high density DNA microarrays represented each of the genes present in the reference strain Salmonella Typhimurium LT2 and have a minimum of 18 perfect match and 18 mismatch probes per gene, thus providing high sensitivity and specificity.
Results: We have found that many of the genes encoding chemotaxis and transport proteins were differentially expressed in Salmonella enterica mutators. Ribosomal genes, pathogenicity island genes, and other genes whose expression is known to change during the transition from log to stationary phase were also differentially expressed in mutator strains. Real-time PCR confirmed that many of these genes are differentially expressed.
Conclusions: Since Salmonella shows enhanced infection in early stationary phase, the mutator phenotype may augment this process by regulating the expression of specific genes. Identification of these genes by expression profiling may reveal the regulatory mechanisms involved and help to explain the persistence of mutS mutators in the environment.
S. A. Jackson, I. R. Patel, M. K. Mammel, J. E. LeClerc, T. A. Cebula, CFSAN, FDA, Laurel, MD
Background: We describe the design, application, and results from a novel DNA microarray used to interrogate genomic diversity and uniquely identify individual strains from a collection of Escherichia coli O157:H7 and other pathogenic strains of E. coli.
Methods: The high density tiling DNA microarray contains 89.4 kb (~1.6%) of the genome of E. coli O157:H7 strain EDL933 and provides information on 128 individual genomic loci, including several multi-locus sequence typing (MLST) genes and other genomic regions chosen to provide information about the diversity of individual strains. This strategy has been employed to interrogate the genomes of 35 strains of E. coli O157:H7 and 25 strains of other pathogenic E. coli representing diverse pathogroups.
Results: A majority of the O157:H7 isolates gave unique hybridization patterns, therefore providing a unique sequence signature useful for identifying individual strains of this pathogen group. Hybridization profiles generated by other pathogenic strains of E. coli demonstrated an expected increase in the level of genomic diversity measured in these pathogroups. Data acquired from hybridization signals at MLST sites were used in cladistic analyses and demonstrated that array-based data is a valid alternative to conventional sequencing for establishing MSLT relationships among pathogroups of E. coli.
Conclusion: The application of DNA microarrays is a powerful approach for identifying and discriminating among closely-related strains of E. coli O157:H7 as well as showing evolutionary relationships among distantly-related strains of different pathogroups of E. coli. This application would enhance current epidemiological studies and provide a means to help establish attribution in forensic investigations.
B. B. Goswami, M. Ayodeji, M. Kulka, T. A. Cebula, S. A. Jackson, I. R. Patel, M. Mammel, D. Ngo, CFSAN, OARSA, FDA, Laurel, MD
Background: Viruses transmitted by food or water fall into three groups, hepatovirus, enterovirus, and norovirus. Although cell-culture based detection for hepato- and enteroviruses is feasible, molecular methods based on RT-PCR are preferable due to much higher sensitivity and rapidity. However, identification of virus strains within a species by currently available techniques such as sequencing is unsuitable for rapid diagnosis.
Methods: Genomes of hepatovirus, enterovirus, and norovirus strains were grouped into clades based on sequence homology. Approximately 4kb of genomic sequence of each clade was used to design a high density oligonucleotide microarray (a "tiling" array) containing overlapping 29-mers that "walk" across the genome at a seven nucleotide resolution. This design is capable of signaling single nucleotide changes (SNPs) that occur in a "test" strain relative to a reference strain, in addition to identifying multi-nucleotide polymorphisms. Genomic RNAs of hepatitis A virus and enterovirus A were RT-PCR amplified using primers that target conserved sequence motifs within each of the viral genomes. The amplicons were labeled with Cy3-dUTP by primer extension and hybridized to the array.
Results: Samples containing either HAV or coxsackievirus B (CVB) could be uniquely identified by the hybridization signal generated from each.Moreover, the HAV and CVB strains were signaled by the probes specific for their clade, demonstrating the ability of the array to discriminate among viral pathogens at the species and strain level.
Conclusion: Array-based hybridization provides a sensitive and rapid technique to detect and identify multiple viral genomes in the same sample.
M. L. Kotewicz1 , S. A. Jackson1 , W. Jiang2 , J. Henkhaus2 , A. Briska2 , C. W. Dykes2 , J. E. LeClerc1 , T. A. Cebula1 , 1CFSAN, FDA, Laurel MD, 2OpGen Inc. Madison WI
Background: Optical mapping is a rapid method to map, analyze, and compare whole bacterial genomes.This method measures restriction fragment sizes along single molecules and assembles contiguous reads (contigs), producing an optical restriction map of the full-length bacterial chromosome.Optical maps resemble a DNA "bar code" of the entire chromosome.
Methods: Bacterial chromosomes were immobilized onto derivatized glass slides, digested with BamHI, stained, and photographed using a fluorescent microscope interfaced with a digital camera. Automated image analysis software located and sized each fragment based on flourescent dye bound and assembled multiple scans into whole chromosomes.
Results: Ten O157:H7 isolates were optically mapped.A direct comparison of the optical map of the Sakai chromosome to its sequence showed that many of the small fragments (21 to 1,500 bp) had been lost during digestion or image processing, but the overall optical map length was within 0.3% of the 5.5 Mb sequenced genome.The other O157:H7 genomes ranged from 5.326 to 5.579 Mb, a variation of 253 kb.In comparison with sequenced strains, the optical maps identified strain specific polymorphisms including chromosomal inversions, insertions, deletions, and substitutions.Many of the polymorphisms were centered about known prophage integration sites.
Conclusions: Inversions encompassing up to one-third of the chromosome in O157:H7 isolates were flanked by prophages.This suggested chromosomal structure and stability were affected by recombination at homologies among these prophages.In addition, many of the strain differences were located at these prophage sites (up to 22 sites of phage insertion in the sequenced strains), which included either different inserted phages or "scars" of prophage deletions.The optical maps thus provided chromosomal markers for individual isolates of E. coli O157:H7.
M. K. Mammel, J. E. LeClerc, T. A. Cebula, DMB, OARSA, CFSAN, FDA, Laurel, MD
Background: Salmonella enterica serovar Newport has become a human pathogen of public health importance, due to its increasing incidence and the rapid emergence of strains resistant to five or more antibiotics. From the phylogenetic analysis of antibiotic susceptible and resistant isolates, we identified two evolutionary clades of S. Newport, one of which contained the multi-drug resistant strains. To better understand the acquisition of antibiotic resistance in these pathogens, one strain from each clade has been subjected to whole genome sequencing.
Methods: Whole genome sequencing was performed by 454 Life Sciences using an ultra-high-throughput automated system. Alignment of assembled contigs onto a sequenced reference genome of S. Typhimurium was performed using MUMmer and AMOScmp software. Alignments of the genomes by BLAST were examined for base-pair changes and loss or gain of genes.
Results: Within alignments of the two Newport sequences matching S. Typhimurium genes, 28,000 single base-pair differences were found (20,000 in the third codon position), representing 0.6% of the genome. An expected sequencing error rate of one in 10,000 would contribute only about 480 of the differences. Of the 4450 annotated S. Typhimurium genes, 194 were not found in either Newport strain. At least 85 Typhimurium genes found in the drug-susceptible strain were not found in the multi-drug resistant strain.
Conclusion: A remarkable number of gene differences was identified in the closely-related strains of the serovar S. Newport. The gain and loss of genes may give insight into the ability of one type of strain to acquire multi-drug resistance. (We acknowledge Dr. J. Ravel, TIGR, for continuing collaboration in bioinformatic analysis.)
E. J. Matthews, N. L. Kruhlak, R. D. Benz, J. F. Contrera, CDER, FDA, Silver Spring, MD
A weight of evidence (WOE) reproductive and developmental toxicology (reprotox) database was constructed that is suitable for quantitative structure‑activity relationship (QSAR) modeling and hazard identification of untested chemicals. The database was derived from multiple publicly available reprotox databases and consists of more than 10,000 individual rat, mouse, or rabbit reprotox test results linked to 2134 different organic chemical structures. The reprotox data are classified into seven general reprotox categories (male reproductive toxicity, female reproductive toxicity, fetal dysmorphogenesis, mortality, growth, and functional toxicity, and newborn behavioral toxicity), and 90 specific reprotox categories as defined in the source reprotox databases. Each of the 90 specific categories is represented by over 500 chemicals, but the percentage of active chemicals is usually low, generally only 0.1 ‑ 10%. We performed statistical analyses on these 90 data sets to identify clusters of categories which are correlated, containing similar groups of active and inactive chemicals. Results revealed clusters of specific categories that contain chemicals that have been shown to be active in two or more animal species (trans‑species). Our WOE model considers trans‑species reprotoxicants as having the highest potential risk to humans. In contrast, some specific categories exhibit only single species‑specific activities and are considered a lower potential human risk. Results also showed species differences in susceptibility to chemicals causing dysmorphogenesis; the rat and mouse are more susceptible compared to rabbits (6.1‑ and 3.6‑fold, respectively).
J. Mayer, M. Cheeseman, M. L. Twaroski, CFSAN, FDA, College Park, MD
In vitro genetic toxicity assays are used to assess the carcinogenic potential of untested compounds through their ability to cause gene mutations and/or chromosomal damage. Though these tests are considerably less expensive and time consuming than 2-year bioassays, they are not inexpensive, they vary in their sensitivity and specificity, and they do not detect non-genotoxic carcinogens. Alternatively, in silico toxicology programs such as OncoLogic® and MultiCASE (MCASE) are structure activity relationship (SAR) analysis programs used to predict carcinogenicity through the use of expert rules and the identification of biophores. One of the bases of these software tools was the structural alert rules developed by Tennant and Ashby (Ashby and Tennant, 1991) to predict carcinogens. The Office of Food Additive Safety's Division of Food Contact Notifications utilizes the results of all these analyses in the review of new food contact substances. Recently, we attempted to validate and compare the accuracy of OncoLogic® (Version 4.1), MCASE (Version 3.31), Tennant-Ashby structural alerts, and genetic toxicity testing, individually and in combination at predicting compounds to be potent carcinogens. Seven-hundred and thirty compounds from the Carcinogenic Potency Database (CPDB) were analyzed, with the results of rodent bioassays applied as the standard for measuring carcinogenicity. The sensitivity of the methods for predicting carcinogens and the specificity for predicting non-carcinogens was examined. Compounds that pose a concern even below the 0.5 ppb threshold of regulation for food contact substances, i.e. those with TD50 values below 6.25 mg/kg bw/d, were then examined separately. The results of this validation exercise demonstrate that in silico SAR programs and structural alerts provide supplemental data not otherwise available that can be used to assist regulators in risk assessments and determining the need for additional data to ensure safe use of new food contact substances.
A. Y. Men, S. Amur, F. Goodsaid, F. W. Frueh, CDER, FDA, Silver Spring, MD
Purpose: To identify differentially regulated genes in tissues in response to drug X; to understand & interpret the gene expression data using ArrayTrack and to evaluate the impact of changing the parameters of data filtration such as P value and fold change on the biological interpretation of the data.
Methods: SPONSOR provided the experimental design, details and raw genomic data in a VGDS. Drug X, a MAP kinase inhibitor, or a control vehicle, was administrated orally 100mg/kg/d for 14 days to rats. Two separate tissue sections from the heart, liver, quadriceps and psoas muscles, were collected from all animals that survived until scheduled necropsy. Gene expression was determined using AffyXYZ chip and data was analyzed using Array Track Software with the data filtration parameters, P value and fold change. Biological interpretation of the gene expression was performed by Ingenuity.
Results: The number of differentially regulated genes decreased dramatically with decreasing P value and increasing fold changes in all the tissues examined. Significant differences were seen in the canonical pathways, including signaling and metabolic pathways, between the unfiltered and filtered gene sets.
Conclusions: This is an independent analysis of VGDS data submitted by SPONSOR. Our observations indicate that data Filtration plays an important role in interpretation of the gene expression data. Hybridization data cutoffs should include both p-value as well as fold-change.
A. D. Nandanie1 , S. Lababidi2 , R. K. Panguluri1 , M. Schneider1 , D. G. Ranamukhaarachchi1 , 1DB, OSEL, CDRH, FDA, 2DB, OSB, CDRH, FDA
Generating quantitative data in expression microarray devices demands robust control standards for data normalization. Previously, we showed (Proceedings of Joint Statistical Meetings, 2005) that significant data variability exists within a given commercial microarray surface using quantified mRNA from Arabidopsis thaliana. Based on these findings we argued that there is a need for positioning reference control standards that will represent the entire array surface area. Using an array format of 96 human genes and RNA from human cell lines, we were able to confirm a relationship between data variability and gene location on array surface. Fluorescence intensity signals using Cyanidine dyes (Cy-3 and Cy-5) for each corresponding gene that was hybridized onto the surface were determined at different time points post hybridization. The results derived from these experiments suggest that the data variability is independent of hybridization time and the type of sample used. This study confirms our previous findings that there is a need for positioning reference control standards representing the entire microarray surface for data normalization.
L. K. Schnackenberg1 , P. Espandiari2 , J. Zhang2 , P. S. Pine2 , R. Anderson2 , R. D. Beger1 , J. P. Hanig2 , 1NCTR, FDA, Jefferson, AR, 2CDER, FDA, Silver Spring, MD
Background: The effects of the nephrotoxin gentamicin on 25, 40, and 80 day old male, Sprague Dawley rats were investigated using a NMR-based metabonomics approach
Methods: At each age, groups of 4 rats were administered saline, 50 mg/kg or 100 mg/kg gentamicin for six days.Predose urine and urine at 8, 24, 48, and 72 hours was collected after dosing for metabonomics analysis.Tissue and serum were collected at sacrifice for clinical chemistry analysis and histopathology evaluation.
Results: Principle component analysis (PCA) of the binned NMR spectra was performed in order to investigate patterns of changes as a function of time and dose in each age group.The data clusters according to age, dose, and time of urine collection.Partial least squares-discriminant function (PLS-DF) models were produced to find correlations between NMR spectral intensities with individual kidney histopathology scores. The histopathology scores ranged from 0 (no injury) to 5 (severe toxicity). PLS-DF models were 88% accurate in predicting histopatholgy scores. Scaled-to-maximum, aligned, and reduced trajectories (SMART) analysis was also applied to compare the temporal metabolic trajectories obtained for each animal at each dose level of gentamicin.SMART analyses showed the greatest separation from saline-dosed animals occurred at 80-day rats.Clinical chemistry data showed no significant changes in BUN and creatinine levels for any age or dose group.
Conclusions: SMART and PLS-DF analyses indicated age and dose-related effects of gentamicin that correlated with histopathology findings that the order of age-dependent sensitivity to gentamicin nephrotoxicity was 80>40>25 days.
N. Sergeev1 , A. Rasooly2 , K. Herold3 , 1FDA/CDRH/OSEL/DB, Silver Spring, MD, 2NIH-National Cancer Institute, Rockville, MD, 3UMD- University of Maryland, College Park, MD
OligoDesign is a program developed to assist in design of primers and oligoprobes for PCR and microarray analysis. The program has Windows-like user interface that provides significantly enhanced utility compared to command-line operation. The program is web-enabled, and includes links to the major online sources of sequence data. The Blast and PubMed interfaces embedded in the software allow the user to efficiently search and download relevant sequences from the NCBI database. The program provides a variety of tools for short sequence analysis and manipulation. The user can select a sequence fragment in one of the dual windows and easily copy it to the secondary window for subsequent manipulations, such as computing the reverse complement, adding or removing spaces, or reciting the sequence. The melting temperature and Gibbs free energy change for denaturation are calculated using one of three nearest-neighbor models. Sequence analysis include: tiling probe design, mismatch melting temperature, LNA melting temperature, multiple sequence file analysis and display, hairpin and self-annealing analysis, automated PCR primer design, automated microarray probe design, local alignment, and dot plot. A recently added feature allows the user to evaluate a given primer set against a target sequence. The program looks for primer dimer interactions and primer pair annealing, as well as hairpin and self-annealing analysis of each of the primers, and reports a summary of all of these calculations. The poster will include experimental results from PCR primers designed using OligoDesign and Primer 3 software.
Software Download: http:\\www.enme.umd.edu\bioengineering
N. Tayebi, L. Wood, J. N. Lozier, CBER, FDA, Rockville, MD
Background: Hemophilia A (HA), the deficiency of the coagulation factor VIII (FVIII), is the most common sex-linked inherited bleeding disorder. It is cause by mutations in the large (186 kb and 26 exons) and complex FVIII gene on chromosome Xq28.The major identified mutation hot spots are in intron1 and intron 22 with 40 to 50% of severe HA cases arising from intron 22 inversions. The best characterized animal model of HA is the Chapel Hill HA dog, which has an inversion similar to the common human intron 22 FVIII gene inversion.
Methods: To understand the mechanism of HA in the Chapel Hill hemophilia A dogs better, we set out to determine the complete sequence of the normal dog FVIII gene. The RPC1-81 BAC library is derived from normal male Doberman Pinscher genomic DNA. BAC clones 291 M9, and 292 C4 from this library were shown to contain FVIII gene sequences by hybridization with dog FVIII exon probes. BAC clone 292 C4 contains exon 1-22 and BAC clone 314 O16 contains intron 21-exon 26. A third BAC clone, 291 M9 contains non FVIII sequence that is found in HA dog FVIII transcripts in the place of exons 23-26. All three BACs carry a factor 8 associated (F8A) sequence, which is the site of recombination in the common inversion mutation.
Results: High throughput sequencing established 14 contiguous fragments of sequence for BAC clone 292 C4. We have established overlaps between 10 of these fragments by PCR, subcloning and sequencing of overlap fragments from the 292 C4 BAC and dog genomic DNA. We have identified overlapping FVIII sequence in BAC clone 314 O16 beginning at intron 21.
Conclusions: Comparison of our existing Doberman FVIII genomic DNA sequence with that FVIII genomic DNA sequence derived from the canine genome project (http://ensembl.bii.a-star.edu.sg/Canis_familiaris/) that used Boxer genomic DNA showed 99% homology between exonic DNA sequence but numerous intronic polymorphisms. Complete FVIII genomic sequencing will permit development of a rapid PCR diagnostic test for the common FVIII inversion in dogs.
P. S. Pine1 , B. A. Rosenzweig1 , Y. Turpaz2 , K. L. Thompson1 , 1CDER, FDA, Silver Spring, MD 20993, 2Affymetrix Inc., Santa Clara, CA 95051
Background: The reliability and comparability of microarray data is contingent on the use of high quality RNA. The commonly used sample quality metrics provide a threshold for filtering highly degraded samples from analyses. However, the impact on data interpretation has not been well defined for the use of samples with moderate levels of RNA degradation.
Methods: To explore this issue, a dataset was created from samples with a progressive change in RNA integrity generated by thawing frozen rat liver tissue sections. The quality of RNA from these samples was measured by RNA Integrity Number (RIN) value on an Agilent Bioanalyzer. RNA samples with RIN values from 9.5 to 6 were run on Affymetrix GeneChip RAE230A arrays.
Results: Probes were identified that had the greatest increase, decrease, or invariance in signal across samples as a function of RIN and characterized by their location on the exemplar RNA sequence. The distance between the 5' or 3' end of a probe set and the 3'-end of the exemplar transcript was the most discriminative parameter of probe signal sensitivity to RNA degradation. Gene transcripts with signals most sensitive to decay as a function of RNA integrity were susceptible to false categorization in modeled analyses.
Conclusions: These results illustrate the importance of using highly discriminative metrics of RNA quality for samples analyzed with microarray technology.
B. A. Rosenzweig, K. L. Thompson, J. Zhang, J. P. Hanig, P. Espandiari, CDER, FDA, Silver Spring, MD 20993
Background: An animal model of age-dependent sensitivity to the adverse effects of gentamicin was used to explore the utility of genome-based biomarkers for the prediction and detection of nephrotoxicity.
Methods: Groups of Sprague Dawley rats that corresponded to children (25d), adolescents (40d), and adults (80d) received s.c. injections of 50 or 100 mg/kg gentamicin daily for 6 or 14 days. The relative expression levels of genes known to be induced by kidney damage were analyzed by qRT-PCR in RNA samples prepared from kidney tissue.
Results: An induction in the mRNA levels of Kim-1 and Tnfrsf12a was observed in treatment groups at time points prior to observation of renal histopathology. Induction of Lcn2, Spp1, and Clu transcript levels was highly correlative with proximal tubule epithelial cell damage. Expression levels of region-specific aquaporin genes were consistent with the site of injury observed with histopathology.
Conclusions: In this study, a panel of gene expression biomarkers supplied predictive and contemporaneous information on drug-induced renal injury, demonstrating potential utility as updated tools for preclinical toxicology assessments.
C. Alvares1 , M. A. Kuziora1 , P. S. Pine2 , K. L. Thompson2 , 1Gene Logic, Gaithersburg, MD 20879, 2CDER, FDA, Silver Spring, MD 20993
Background: A common microarray quality metric is the ratio of probe intensities mapping to the 5' and 3' ends of a transcript. Since sample amplification is usually 3' biased, the metric presumes intact mRNA is required for efficient 5' labeling, thus higher 5'/3' ratios indicate better quality data.
Methods: Rather than relying on individual probesets to define quality for an entire array, we propose an alternative method where signal intensities as a function of distance from the 3' end of thousands of probes are used to derive a global ratio. A linear regression is performed on probe intensities with known 3' distances.Since >90% of mapped probes are within 600 bases of the 3' end, we calculated a global ratio from estimated signals derived from the regression at 500 and 100 bases.
Results: Using gene expression data derived from Affymetrix GeneChip® where degradation of RNA samples was controlled, we found that global ratios correlate better to a sample's RNA Integrity Number than did GAPDH 5'/3' ratios, Β-Actin 5'/3' ratios or percent present calls.
Conclusions: The global ratio provides a robust estimate of starting RNA quality and can be used to ensure comparable samples for data analysis. It does not require the tiling of specific gene regions and can be applied to any set of genes where the 3' distance is known.The global ratio method should be applicable to multiple array platforms to determine RNA quality of the starting sample.
J. Fostel1 , K. L. Thompson2 , M. Cooper3 , S. Pettit4 , 1NCT, NIEHS ITSS, Research Triangle Park, NC, 2CDER, FDA, Silver Spring, MD, 3Biogen Idec, Cambridge, MA, 4HESI, Washington, DC
Animal housing, handling and euthanasia are all likely to impact gene expression. As toxicogenomics data are databased and subjected to meta-analysis across different studies or institutions, these factors play an increasingly confounding part in differential gene expression. To assess contributions by variables such as rat strain, diet, vehicle and route of administration, a new project area was initiated within the ILSI Health and Environmental Sciences Institute (HESI) Genomics Committee:to establish a publicly accessible database of control animal microarray data. The aim is to develop a resource for analysis of fluctuations in gene expression arising from biological or technical variables. Data has been collected from 18 institutions, representing 536 hybridizations to Affymetrix microarrays, along with 35 descriptors of experimental variables. This data set constitutes a sufficiently robust pilot database to facilitate characterization of baseline variability (most and least variant) in particular genes or gene sets. Such analysis is being carried out by members of the Committee and will be useful for identifying control genes for gene expression studies and for validating the robustness of gene expression markers of toxicity, efficacy, or mechanism of action. It could have potentially important applications as interpreting the significance or presence of a potential biomarker and, if sufficiently robust, could help inform the process of genomic biomarker identification within a regulatory context. The anonymized data will be uploaded by the Committee into the CEBS database created by the National Center for Toxicogenomics, and will be made public following the initial analysis and interpretation.
J. L. Tiwari, R. K. Puri, B. Zaslavsky, CBER, FDA, Rockville, MD
Genetic testing for evaluation of associations between genes and drug response will enter clinical trials during Phase 2 and 3 studies. Although the process of randomization produces treatment and control arms that are comparable with respect to baseline characteristics, analysis of genetic associations with drug response will utilize only patients from the drug-treated arm of the trial. Thus, the available sample size for analysis will be reduced by half.In the absence of randomization, the comparability of responders and non-responders (cases and controls) is not assured. In this setting, two important factors for producing false positive associations are population stratification and genetic admixture. In addition to matching cases and controls for usual demographic factors and ethnic background, use of genomic controls has been proposed to estimate the magnitude of bias and a correction factor for the level of significance.
The sample size for evaluating genetic association will depend on the frequency of the gene in cases and controls, pre-specified alpha level, and power. In an ethnically diverse patient population, the candidate gene frequencies may differ among the ethnic groups.
The analyses of associations can be performed using either alleles or genotypes. Data may also be analyzed by assuming a specific genetic model of inheritance (dominant, recessive, additive, and multiplicative) of the marker gene.
In general, more than one candidate gene will be tested and analyzed for associations in a single study. Thus, a critical design and analysis issue, routinely evaluated by biostatistics and clinical reviewers, is the control of error under multiple comparisons.
W. Tong1 , L. Shi1 , S. Harris1 , M. Bishop1 , H. Fang2 , H. Hong2 , Q. Xie2 , H. Sun2 , M. Cao2 , F. Ranamukhaarachchi2 , R. Perkins2 , 1NCTR, FDA, 2Z-tech
Modern toxicology has focused on understanding biological mechanisms involved in the expression of toxicity. Advanced technologies such as genomics and proteomics offer new approaches for investigating disease and toxicity at the molecular level, and for discovery of corresponding biomarkers. Data from the new experimental platforms are both huge in number and noisy in content. Extraction of useful knowledge requires bioinformatics for data management and analysis. The NCTR Center for Toxicoinformatics (CTI) provides an informatics infrastructure and data analysis capability available within FDA and to public. CTI expertise spans across computational chemistry, molecular modeling, simulation and scientific programming, bioinformatics, chemoinformatics, software engineering, scientific visualization, and Internet technology. The poster illustrates CTI capabilities by addressing several bioinformatics issues associated with the "omics" researches: (1) How to develop a validated database that will be a rich resource for data mining and experiment comparison; (2) How to make the public data readily available to in-house research; (3) How to visualize and analyze the massive information for knowledge extraction; and (4) How to integrate different types of "omics" data for answering more complex biological questions. The CTI mission is to conduct collaborative research, and we invite you to contact us.D. Zhuang, R. Fraczkiewicz, W. S. Woltosz, M. B. Bolger, Simulations Plus Inc., 42505 10th Street West, Lancaster, CA, 93534
Purpose: To compare the performance of ANN ensemble (ANNE) models and SVM ensemble (SVME) models.
Methods: We built SVME models for Blood Brain Barrier and Madin-Darby Canine Kidney (MDCK) Papp datasets by bootstrapping training data to access the difference between ensemble models and individual models. Both ANNE models and SVME models were built from a Maximum Recommended Therapeutic Dose (MRTD) dataset.
Results: The performance of the BBB classification ensemble models performance (CCR - Correct Classification Rates, 94.36%) is a little better than average performance (CCR 94.05%). The regression ensemble models built from the MDCK dataset gave an RMSE of 0.363 while the performance from a single selection of training and verification compounds was 0.374. The best ANNE models built from the MRTD dataset gave an RMSE of 0.65 for the external test set while the best SVME models was 0.68. F-test and t-test indicate that the ANNE has larger variance than SVME, but the means are more likely to be the same.
Conclusion: Using ensemble models that cover the entire training space rather than individual models that are associated with a particular split of training data always improved the average prediction accuracy, and ensemble models are more robust. Overall, there was no significant difference between the performance of ANNE and SVME. The best ANNE, however, always outperforms the best SVME at the expense of using more descriptors.
M. Chen, L. Ma, G. L. Drusano, J. S. Bertino, Jr., A. N. Nafziger, Ordway Research Institute, Albany, NY 12208
Background: Studies examining sex differences in CYP3A activity have produced conflicting results. Midazolam (MDZ) is a common biomarker for hepatic and/or intestinal basal CYP3A metabolic activity. We investigated whether sex differences exist in intravenous (IV) or oral (PO) MDZ pharmacokinetics (PK) in healthy volunteers.
Methods: Data from 13 previous studies, conducted from 1996 to 2004, were used. A single dose of IV MDZ (0.025 mg/kg) was administered to 66 healthy white adults (37W/29M, 36.3 ± 7.7 yr). A single dose of PO MDZ 0.075 mg/kg, 0.15 mg/kg (1 study) or 5mg (1 study), was administered to 72 healthy adults (71white/1Asian, 37W/35M, 38.3 ± 8.9 yr). Plasma concentrations were collected at various time points and assayed by LC-MS-MS. PK parameters were determined via population methods using a nonparametric adaptive grid program (NPAG) and a 2 compartment IV or 1 compartment oral absorption model. A Bayesian method was used to calculate each subject's PK. The unpaired t-test was used to compare IV and PO MDZ clearance by sex.
Results: MDZ clearance was significantly higher in women compared to men for both IV (0.497 ± 0.082 L/hr/kg vs. 0.443 ± 0.070 L/hr/kg) and oral administration (0.688 ± 0.279 L/hr/kg vs. 0.536 ± 0.166 L/hr/kg), respectively (p=0.006).
Conclusions: Women showed significantly higher clearance for IV and oral MDZ of 12 and 28%, respectively. Our results suggest women have greater hepatic and intestinal CYP3A isozyme activity. The clinical significance of these findings warrants further genotype and phenotype analysis by sex.
R.L. Bernstein, FDA, San Francisco District, 1431 Harbor Bay Parkway, Alameda, CA 94502-7070
Lactose metabolism and the lac operon genes and proteins of Escherichia coli comprise a major historical centerpiece of biochemical genetics and molecular biology. Lactose metabolism is a defining laboratory characteristic of E. coli, even though in nature E. coli must only rarely encounter the sugar lactose. Even in the mammalian gut, lactose is absorbed through the gut wall in the more proximal regions, while E. coli resides in the more distal regions. Further evidence that lactose catabolism is not the evolutionary purpose of lac operon genes are the largely incompatible substrate and inducer specificities and the lack of a role for thiogalactoside acetyltransferase in lactose catabolism. These observations suggest that the lac operon genes and proteins have evolved for different purpose from catabolizing lactose.
Recent genomic studies imply the ancient presence of the lacZ gene in the chromosome of Gram-negative ancestors of today's enteric coliforms. The lacZ gene appears to have been selectively lost or retained by more recent descendants (rather than introduced recently or transferred laterally through viruses or on episomes). Molecular phylogenetics analysis supports the integral role of lac operon genes and proteins among Gram-negative descendants, even those that no longer metabolize lactose. Thus the lacZ gene was probably present in the common Escherichia-Salmonella ancestor and retained in Escherichia while lost in Salmonella. (Rare present-day lactose-metabolizing Salmonella isolates may have lacZ on an episome, however.)
A possible adaptive metabolic role for the lac operon enzymes is the degradation and excretion of toxic galactosides that accumulate from catabolism of complex lipids and carbohydrates containing galactose residues in glycosidic linkage. These galactoside conjugates may originate either in endogenous gut epithelial cells or in the animal diet.
CATEGORY E: PREDICTIVE PHARMACOKINETICS AND PHARMACODYNAMICSM. Chen, A. N. Nafziger, J. S. Bertino, Jr., Ordway Research Institute Drug Development Center, Ordway Research Institute, Albany, NY 12208
Background: Because CYP3A activity exhibits wide intersubject variability, individualized dosing of substrate drugs can be challenging. CYP3A inhibitors are used to reduce the CL and potentially the variability of CYP3A substrates, allowing a more uniform dosing. Previous studies did not report the impact of CYP3A inhibition on variability of substrate metabolism. We investigated the effect of ketoconazole inhibition on CYP3A metabolic variability, measured by the CYP3A probe oral MDZ.
Methods: A single oral dose of MDZ (0.075 mg/kg) was administered to 19 healthy Caucasian adults (38.7 ± 8.8 yrs, 9M/10F) at baseline and concurrently with ketoconazole (400 mg daily for 10 days) on day 6 or 9 of ketoconazole. Plasma samples were collected over 6 and 30 hrs during both phases respectively, and assayed for MDZ by LC-MS-MS. A paired t-test and an F test were used to evaluate differences and variance in MDZ CL during both phases.
Results: Mean (95% CI) MDZ CL/F (mL/min/kg) at baseline and ketoconazole inhibition was 27.5 (22.0-32.9) and 3.0 (2.2-3.9), respectively (p<0.001). Variance in apparent oral CL decreased from 128.8 to 3.1 at baseline and inhibition, respectively. This represents a decrease in variability of almost 98% (F=41.5, p<0.001).
Conclusions: Oral MDZ CL/F decreased markedly during ketoconazole inhibition. Intersubject variability was significantly larger at baseline vs. inhibition. Use of a potent CYP3A inhibitor can reduce interindividual variability in CYP3A activity and may have applications in clinical settings for standardized dosing (e.g. cancer chemotherapy).
C. R. Bonapace1 , V. R. Jarugula1 , B. Green2 , J. V. Gobburu1 , 1CDER, FDA, Silver Spring, MD, 2School of Pharmacy, University of Queensland, Brisbane, Australia
Background: Anti-infectives represent a class of drugs in which animal infection models are commonly used to establish dosage regimens for evaluation in Phase 2/3 clinical trials. This analysis compared the exposure-response relationship for an anti-infective obtained from patients to an animal infection model.
Methods: Animal Data-Dose fractionation studies were performed with a neutropenic mouse thigh infection model to determine the unbound AUC0-24/MIC ratio associated with a static effect for S. aureus. Patient Data-A population PK/PD analysis was performed with data from three Phase 2/3 clinical studies and concentration-time profiles were simulated for patients in which microbiologic and clinical endpoints were available. The relationship between the probability of microbiologic and clinical efficacy versus unbound PK/PD parameters (e.g., AUC0-24/MIC) was assessed using logistic regression.
Results: The magnitude of the unbound AUC0-24/MIC ratio associated with a static effect in the animal infection model was approximately 160. Based on the population PK analysis in patients, the unbound AUC correlated with the probability of clinical success or failure. The unbound AUC0-24/MIC ratio associated with >90% probability of clinical success was approximately 1190.
Conclusions: Based on the population PK/PD analysis, an exposure-response (efficacy) relationship was established in patients from Phase 2/3 clinical studies and it supported the PK/PD parameter obtained from the animal infection model. However, the magnitude of the unbound AUC/MIC ratio associated with efficacy in patients was substantially greater than that obtained from the animal infection model.
K. Herold1 , R. P. Brown2 , M. E. Stratmeyer2 , 1University of Maryland, College Park, MD, 2CDRH, FDA, Silver Spring, MD
Background: Physiologically based pharmacokinetic (PBPK) models describe the pharmacokinetic behavior of a compound using a series of differential equations. These models differ from traditional pharmacokinetic models in their ability to provide a more physiologically accurate description of the absorption, distribution, metabolism and excretion of a chemical compound in the body. Until recently, PBPK models have been developed primarily using Advanced Continuous Simulation Language (ASCL) software. However, Marino (2005, Toxicol Methods Mech. 15:137-154) recently demonstrated how PBPK models originally written in ACSL can be recoded using Excel and Visual Basic (VBA). Use of either Excel or VBA has significant advantages for FDA staff that does not have access to the expensive ASCL software or are not proficient in programming.
Method: Using the approach described by Marino et al. (2005), the PBPK model of ethylene oxide written originally by Fennell and Brown (2001, Toxicol Appl Pharmacol. 173(3):161-75) in ACSL was rewritten in Excel and VBA. The model was validated for humans using pharmacokinetic data reported by Brugnone et al. (1986, Int Arch Occup Environ Health. 58(2):105-12).
Results: The results of the model written in Excel and VBA are essentially identical to those reported Fennell and Brown (2001).
Conclusions: The use of Excel and VBA to develop PBPK models will increase the utility of this approach for FDA staff. The results of the Excel PBPK model of ethylene oxide will be used to help derive the allowable limits for ethylene oxide residues on medical devices in the ISO 10993-7 standard currently under development.
M. N. Martinez1 , J. C. Kawalek2 , K. D. Howard2 , J. L. Ward2 , P. Marroum3 , W. G. Marnane1 , D. M. Bensley1 , F. R. Pelsor1 , S. W. Hoag4 , A. S. Tatavarti4 , L. Xie4 , R. M. Fahmy1 , 1CVM, FDA, Rockville, MD, 2CVM, OR, Laural, MD, 3CDER, FDA, Silver Spring, MD, 4University of Maryland
The bolus (or oblet) is a dosage form that can be used for the oral administration of pharmaceutical compounds to ruminating species.Unlike traditional tablets, oral boluses may contain quantities of drug on the order of grams rather than milligrams.Due to its size, it is only recently that USP-like in vitro dissolution methods have been developed for this dosage form.However, whether or not these dissolution tests can predict product in vivo performance has yet to be determined. The importance of this issue is apparent when the US Food and Drug Administration Center for Veterinary Medicine is faced with the decision of whether to require additional in vivo bioequivalence study data to support the approval of changes in product chemistry or manufacturing method.The current study was undertaken to determine whether an in vivo/in vitro correlation (IVIVC) can be established for bovine sulfamethazine oral boluses and to acquire insight into the magnitude of changes in in vitro product performance that can occur before corresponding changes are seen in in vivo blood level profiles.Based upon the results of this investigation, it is concluded that marked changes in in vitro sulfamethazine bolus performance can be tolerated before resulting in altered in vivo blood level profiles.However, the data also suggest that rumenal absorption may occur for some compounds.Therefore the degree to which variation in product in vitro dissolution profiles can be tolerated may be compound specific.
X. Zhang1 , J. C. Gorski2 , S. M. Huang3 , A. Lucksiri2 , J. Y. Chien4 , S. K. Quinney2 , D. R. Jones2 , S. D. Hall2 , 1Purdue University, West Lafayette, IN, 2Indiana University, Indianapolis IN, 3CDER, FDA, Rockville MD, 4Eli Lilly & Co, Indianapolis IN
BACKGROUND: The prediction of the extent of drug-drug interactions (DDIs) between the mechanism-based inhibitors, erythromycin (ERY) and diltiazem (DTZ), and the CYP3A substrate midazolam (MDZ) is confounded by the time and concentration-dependant clearance of the inhibitors. METHOD: Physiologically-based pharmacokinetic (PBPK) models were developed for individual drugs based on the reported pharmacokinetic (PK) and physiological parameters. Enzyme kinetic parameters (kinact and KI) were estimated in vitro using human liver microsomes for ERY and DTZ, and used to model the time course of changes in the amount of CYP3A. In turn, the amount of CYP3A determined the nonlinear elimination of MDZ, ERY and DTZ and the corresponding DDIs. Simulations were performed using Pharsight® Trial Simulator™. RESULTS: kinact and KI were 0.1 min-1 and 15.7 uM, respectively, for ERY, and 0.07 min-1 and 3.2 uM, respectively, for DTZ. No significant competitive inhibition was predicted. The model predicted the nonlinear disposition of ERY and DTZ following single/multiple intravenous or oral doses. The predicted fold increase in intravenous and oral MDZ AUC were 2.5 and 4.5 following pretreatment with ERY 500 mg tid for 6 days, and 1.4 and 3 following pretreatment of DTZ 60 mg for 5 doses, respectively. These results agree with actual data. The model was also used to predict the combined effect of concurrently administered ERY and DTZ on MDZ clearance. An independent model for the effect of concurrently administered DTZ and ERY on CYP3A activity was used to predict the fold increase in MDZ AUC following intravenous (4.5-fold) and oral (18.0-fold) dosing. In contrast, a mutual interaction model for the effect of DTZ and ERY on CYP3A activity predicted fold increases of 3.9 and 9.8 for MDZ AUC after intravenous and oral dosing, respectively. CONCLUSION: The PBPK models incorporating in vitro enzyme parameters predicted the nonlinear PK of ERY and DTZ, and their interactions with MDZ. Also an approach using independent model may overestimate the AUC fold change for both oral and intravenous MDZ compared to a PBPK model which incorporates both drugs into a single model.V. Lukacova, W. S. Woltosz, M. B. Bolger, Simulations Plus, Inc., Lancaster, CA
Purpose. To evaluate the accuracy of prediction of Midazolam absorption and bioavailability in a pediatric population from in-silico, in-vitro and adult in-vivo data. The significance of scaling of the clearance and gastrointestinal tract to appropriate age was assessed.
Methods. GastroPlus 5.0 with the PBPKPlus Module was used to simulate the adult human Cp-Time profiles for Midazolam in solution dosage forms. Simulated Cp-Times were compared to corresponding literature data in order to validate the non-linear dose dependence and bioavailability due to saturable CYP3A4 metabolism.The Population Estimates for Age-Related (PEAR) Physiology™, a part of GastroPlus, was used to generate tissue parameters for adult and pediatric patients. Literature data for gut CYP3A4 distribution and in-vitro Km and Vmax values were used along with rat Tissue-Plasma partition coefficients and in-silico estimation of remaining biopharmaceutical and pharmacokinetic properties.
Results. Using the default ACAT model and the observed expression levels of CYP3A4 in liver and gut, PBPK simulations accurately reproduced the non-linear dose dependence for Midazolam bioavailability and Cp-Time profiles for po administration of Midazolam in adult patients. Using a purely in-silico calculation of pediatric physiology, and scaling of the gastrointestinal tract parameters and metabolism to a pediatric population, pediatric Cmax, and Tmax were also accurately simulated.
Conclusions. In-vitro data or in-vivo Cp-Time profiles from an adult population can be successfully used to predict the Cp-Time profiles in pediatric patients if the PEAR Physiology for a given age is accompanied by scaling of the gastrointestinal tract parameters and enterocyte metabolism to the same age.R. A. Miller, R. D. Walker, R. Reimschuessel, CVM, FDA, Laurel, MD
Background: Breakpoints for antimicrobial agents may be clinical or epidemiological. The objective of this study was to develop epidemiological cut-off values for four antimicrobial agents when tested against a representative population of the major aquaculture pathogen, Aeromonas salmonicida.
Methods: Species identification of 223 submitted, geographically distributed A. salmonicida isolates was completed using polymerase chain reaction (PCR). Oxytetracycline, ormetoprim/sulfadimethoxine, florfenicol, and oxolinic acid minimum inhibitory concentrations (MICs) and zone diameters were determined for each isolate in accordance with the standardized antimicrobial susceptibility testing methods for bacterial isolates from aquatic animals, recently approved by the CLSI. Susceptibility data was tabulated in a scattergram and analyzed using error rate-bounding.
Results: Susceptibility tests on oxytetracycline, ormetoprim/sulfadimethoxine, and oxolinic acid revealed two distinct populations of bacteria. Isolates tested against florfenicol clustered in a single population. Oxolinic acid susceptibility data revealed higher MICs in the non-USA A. salmonicida isolates. Slower growing atypical isolates were generally more susceptible than typical isolates for all drugs except oxolinic acid.
Conclusions: The use of population distributions sometimes used with antimicrobial susceptibility testing of mammalian isolates to develop epidemiological cut-off values appears to be appropriate for use in aquatic medicine. This type of data, considered with pharmacokinetic and efficacy data may be useful for developing clinical breakpoints in aquaculture in the future.
R. J. Sawchuk1 , L. M. Page2 , R. L. Rauck3 , 1University of Minnesota, College of Pharmacy, Minneapolis, MN 55455, 2Medtronic Neurological, Minneapolis, MN 55432, 3Carolinas Pain Institute, Winston-Salem, NC 27103
Introduction: This clinical study investigated the pharmacokinetics of gabapentin in cerebrospinal fluid (CSF) and plasma during continuous intrathecal administration.
Methods: 20 subjects (N=4 in 5 dosing cohorts) received four consecutive, dose-escalating, 24-hour intrathecal infusions. Total daily doses ranged from 0.2 to 100 mg. Paired gabapentin concentrations in lumbar CSF and plasma were determined in each dosing phase, and in the post-infusion period. Compartmental pharmacokinetic modeling considered CSF and plasma data simultaneously and incorporated gabapentin influx from plasma to account for CSF exposure seen when dosed systemically. Noncompartmental analysis (NCA) of CSF and plasma was also conducted.
Results: CSF and plasma gabapentin concentrations showed linear increasing trends with dose. The early post-infusion time course in CSF showed a clear distributive phase; however, the post-infusion plasma level decline was monoexponential. Dose-independent efflux clearance (CLeff) from lumbar CSF was 0.0158±0.00297 L/hr. Systemic clearance (CLsys) of gabapentin was 6.05±2.23 L/hr, suggesting no gabapentin metabolism within the CNS. Volume of distribution of gabapentin was 58.8±23.9 L and harmonic mean plasma half-life was 6.3 hours. Estimates of CLeff and CLsys by compartmental and NCA agreed well. Harmonic mean terminal half-lives in CSF were 2.13 hours. CLsys was highly correlated with creatinine clearance, consistent with the extensive renal handling of gabapentin.
Conclusions: Gabapentin disposition kinetics from CSF were linear over the dosage range studied. From a calculation of the targeted delivery advantage, steady-state lumbar CSF concentrations of gabapentin can be achieved with intrathecal dosing rates 3000-fold lower than those needed to attain these levels using intravenous infusions.
W. J. Rodriguez, A. Selen, D. Avant, C. Chaurasia, T. Crescenzi, G. Gieser, J. DiGiacinto, S. M. Huang, P. Lee, L. Mathis, D. Murphy, S. Murphy, R. Roberts, H. C. Sachs, S. Suarez, V. Tandon, R. S. Uppoor, J.A. Lazor, L.J. Lesko and D. M. Chilukuri, CDRH, FDA
Introduction: Pediatric (ped) initiatives which stimulate the conduct of ped drug trials have led to the discovery of new dosing regimens and safety information reflected in the ped labeling.
Objective: To assess the impact of ped drug studies on ped dosing.
Methods: The methodology, data/results and the respective labeling changes resulting from the ped studies conducted between July 1998 and October 2005 were reviewed. We focused on differences and similarities in drug clearance and exposure.The patterns observed, coupled with labeling information illustrate the impact of new or improved dosing recommendations for ped patients. Data were collected from 244 ped studies in approximately 91,000 ped patients.
Results:104 drugs have new or revised ped labeling including new doses or dosing changes for 22 products. Based on lower drug clearance (or apparent oral clearance) dose reductions were necessary for 5 products, while higher apparent oral clearance resulted in dose increases for 4 products. Examples which illustrate lessons learned on differences and similarities in drug pharmacokinetics, pharmacodynamics, between ped and adult patients are presented.
Conclusions: The Agency's efforts and recent legislation have resulted in critical changes in drug labeling for ped patients and have shown that unique ped dosing is often necessary, reflecting growth and maturational stages. Ped dosing often can not be derived from the adult dose by applying weight-based calculations.
P. M. Sathe1 , V. A. Sayeed1 , L. X. Yu1 , M. B. Bolger2 , 1OGD, CDER, FDA, Rockville, MD, 2Simulations Plus Inc, Lancaster, CA
Purpose: To simulate an oral absorption of Cyclosporine capsule and to conduct parameters sensitivity analysis.
Methods: Mean plasma concentration vs. time profile and in vitro dissolution of oral 100 mg cyclosporine hard gelatine capsule (300 mg dose), from actual study, was used to develop an Advanced Compartmental Absorption and Transit (ACAT) model. A physiological fasted state with literature reported parameters, and a three compartment model was used to achieve a fit of the observed data. Parameter sensitivity analysis was applied to simulated bioavailability, Cmax, Tmax, and AUC. Realistic variabilities were applied to simulate mean plasma concentration vs time profiles of a virtual bioavailability trial (N=24) with 25%, 75%, and 100% probability contours.
Results: The simulation suggested 67% fraction absorbed, 42% fraction into the portal vein, and 31% fraction bioavailable. The maximum absorption was found to be from jejunum followed by ileum. Precipitation time and passive permeability were found to be most sensitive parameters for changing fraction absorbed. A precipitation time of 2.4 hours was required to match the observed Cmax and AUC. A two-fold decrease in release rate produced a 9.4% decrease in Cmax.
Conclusions: Cyclosporine capsule produced in vivo super-saturation for a significant period. Under conditions of super-saturation, slight changes in in vivo release rate do not result in significantly modified Cmax or AUC.
B. Shaikh, N. G. Rummel, C. Gieseker, R. Reimschuessel, FDA/CVM, Laurel, MD
Background: Ivermectin, a broad spectrum antiparasitic drug, is approved for use in terrestrial food animals (cattle, swine and sheep), but not in aquatic species. The marker residue (MR) to monitor for potential violative residues in both liver and muscle tissues of the above animals is the parent drug. In this study, we investigated the residue depletion of ivermectin and its potential metabolites in the muscle tissue of a fish species, rainbow trout.
Methods: 3H-ivermectin at the dose level of 0.1 mg/kg (9.25 ìCi) was administered to 42 rainbow trout in gel-capsules via stomach tube using a manual restraint. Six fish were used at each withdrawal period and necropsy was performed at 1, 3, 7, 14, 28, 35 and 42 days post dose. Prior to necropsy, fish were sedated with MS-222, euthanatized, de-scaled and muscle fillets collected. The muscles were homogenized and powdered in dry ice and used for the determination of total radioactive residue (TRR) and for liquid chromatographic (LC) analysis. Tissue Oxidizer was used to determine TRR and LC was used to determine parent drug and its potential metabolites.
Results: The results showed that TRR peaked at post dose day 3 to about 60 ppb by Tissue Oxidizer and to about 54 ppb by LC. Further LC analysis indicated that by post dose day 35, about 70% of the parent drug ivermectin was converted into an unknown metabolite.
Conclusion: Theses results of this study suggest that this metabolite could serve as a potential marker residue (MR) for ivermectin in fish, unlike in cattle, swine and sheep where parent ivermectin is known to be the MR.
I. A. Ross, P. P. Sapienza, W. D. Johnson, R. L. Sprando, K. R. O'Neill, S. C. Sahu, T. J. Flynn, T. F. Collins, P. L. Wiesenfeld, C. S. Kim, CFSAN, Laurel, MD
Background: Androstenedione is an anabolic steroid that has been used to enhance athletic performance.
Methods: Female rats were treated with androstenedione (60 mg/kg/day) from 2 weeks before mating to gestation day (GD) 19. On GD 20 the animals were sacrificed and maternal blood, brain, and livers were obtained and analyzed for steroids by a HPLC method.
Results: The plasma concentrations of estradiol were 63.3 ± 4.30 pg/ml and 94.4 ± 13.3 pg/ml in controls and in rats treated with 60 mg/kg, respectively. The treatment-related increase was borderline significant (0.05 < P < 0.10). The plasma levels of androstenedione, testosterone, and progesterone in treated rats were not significantly different from the controls. In the liver, the concentrations of androstenedione in controls and rats treated with 60 mg/kg were 0.30 ± 0.17 ng/g and 1.22 ± 0.34 ng/g, respectively, and the concentrations of estradiol were 92.5 ± 17.2 ng/g and 538.6 ± 165.2 ng/g, respectively. The concentrations of these steroids were significantly increased in the treated rats (P < 0.05). In the brain, no steroids were detectable.
Conclusions: The results indicated that androstenedione, testosterone, and progesterone are converted quickly to other steroid metabolites in the liver. The data also confirm earlier observations that steroids do not pass readily from the blood into the brain through the blood-brain barrier
CATEGORY F: MEDICAL PRODUCT DESIGN, CHARACTERIZATION, AND MANUFACTURINGE. O. Akala, O. Okunola, Howard University, Washington, DC
We have reported previously on the development of colloidal drug delivery systems (microspheres and nanospheres) for sustained delivery of bioactive agents. Naltrexone-loaded stealth hydrolyzable cross-linked PEG-MMA nanospheres were fabricated by in situ polymerization technique using emulsion polymerization method and were shown to be capable of sustaining the delivery of naltrexone for a long time.
A new polymerization method has been investigated for the preparation of naltrexone-loaded and paclitaxel-loaded stealth nanospheres: dispersion polymerization. Poly(ethylene glycol)1000 monomethyl ether mono methacrylate, methylmethacrylate, and N,O-dimethacryloylhydroxylamine were copolymerized to form nanospheres in a mixture of methanol and water using either redox-initiator system or AIBN. The nanospheres were isolated, dried by lyophilization and characterized: particles size analysis, encapsulation efficiency, surface morphology using a scanning electron microscope and in vitro availability. The stealth hydrolyzable cross-linked PEG-MMA nanospheres are capable of sustaining the availability of the drugs for more than fifteen days.
Acknowledgements: Supports from NIAAA/NIH: # 5 R21 AA13407-02 and Keck Foundation are gratefully acknowledged. This investigation was conducted in a facility supported by NCRR/NIH: #1 C06 RR14469-01
R. Duncan, C. A. Cadogan, K. Tomioka, A. Selvapandiyan, K. Stabler, J. Mellquist-Riemenschneider, D. V. Volokhov, V. E. Chizhikov, H. L. Nakhasi, CBER, FDA, Bethesda, MD
Purpose: As the public continues to demand higher standards of screening for infectious agents in transfusion products and technology continues to advance methods of nucleic acid testing (NAT), multiple independent testing of donations becomes a logistical nightmare. Multiple pathogen screening by multiplex PCR followed by hybridization to a microarray of specific DNA sequences could be one of the solutions to this challenge.
Method: A target sequence was selected in the genome of blood borne parasite, bacterial and viral pathogens as well as potential bioterror pathogens. Oligonucleotide probes were printed on glass slides that anneal specifically to the sequence amplified by unique primer pairs. The multplex microarry assay was optimized to screen for three pathogens and a human gene positive control simultaneously. The assay was tested for detection of cultured bacteria spiked in human blood and detection of Bacillus anthracis in the blood of infected mice. To expand the range of pathogens, a second array was designed to simultaneously detect the parasites Leishmania and Plasmodium. Initial testing with spiked blood will be presented.
Results: The same multiplex PCR microarray assay detected Bacillus anthracis, Francisella tularensis, and Yersinia pseudotuberculosis (a surrogate for the plague bacterium Y. pestes) at a concentration of 50 cells/ml in blood. The assay detected B. a. organisms in three of the five mouse blood samples taken 36 hrs post-infection or later. A new assay detected Leishmania cells or Plasmodium DNA spiked into blood at 50 cells/ml or 700 genome equivalents/ml respectively.
Conclusion: This study demonstrates methods to break down the technological barriers to development of a multiplex test to screen blood for pathogen contamination. Additionally, this work develops the expertise within FDA to review new technologies for blood screening devices and shows industry our willingness and ability to review new products, thereby encouraging product development.
M. Farahani1 , J. M. Antonucci2 , C. M. Guttman2 , W. E. Wallace2 , 1CDER, FDA, Rockville, MD, 2NIST, Gaithersburg, MD
Oligomeric organosilsesquioxanes, which have potential both as resins and fillers in polymeric medical and dental materials, are hybrid organic-inorganic molecules having complex structures that are difficult to characterize. Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) is a powerful tool for the characterization of monomers, oligomers and polymers, providing both molecular mass and structural information. Objectives: To determine the mass distribution and molecular structure of oligomeric methacrylic silsesquioxanes (OMPTMS) derived from the in situ hydrolysis-condensation reaction of 3 methacryloxypropyltrimethoxysilane (MPTMS) in various medical and dental monomers. Methods: The medical and dental monomers used as solvents for the oligomerization of MPTMS were: UDMA (a diurethane dimethacrylate), EBPADMA (an ethoxylated bisphenol-A dimethacrylate), TEGDMA (triethylene glycol dimethacrylate). OMPTMS was also formed from MPTMS in "wet" acetone. Conditions for OMPTMS formation involved heating at 60 ºC for 30 - 50 d in open vials. Results: OMPTMS from UDMA had a mass distribution of about 3000 - 6000 g/mol and that from EBPADMA extended from 1000 - 4000 g/mol. Compared to OMPTMS derived from "wet" acetone (5000 - 7000 g/mol), these monomer-derived oligomers had lower overall molecular masses, but had significantly greater degrees of polyhedral structures. Conclusion: MALDI-TOF-MS is a highly effective method for characterizing the complex, 3-D structures of oligomeric organosilsesquioxanesC. Guo, W. H. Doub, DPA, CDER, FDA, St. Louis, MO
Background: Impaction force and velocity are two important parameters to describe an aerosol plume. These two parameters should be good measures of local delivery equivalence for an inhalation drug, though have been overlooked for years. A new technique to measure the impaction force for nasal sprays and metered-dose inhalers (MDIs) is described here. Methods: A Stable Micro Systems TA-XT.plus Texture Analyser equipped with 750 g load cell was used to measure the impaction force. A standard 20-ml Pfeiffer nasal spray pump filled with water and a Flovent® HFA 44mg MDI were used as test products in this study. Results: The relative standard deviations (RSDs) of maximum impaction force from six repeated measurements are 2.2% and 2.6% for nasal spray and MDI, respectively. As with other inhalation drug characteristics such as spray pattern, plume geometry and droplet size distribution, the maximum impaction force varies significantly at different spray distances. Conclusion: We have developed a method for the measurement of impaction force from inhalation drug products (nasal sprays and MDIs).The low RSD from repeated measurements indicates the good repeatability of this new technique. Since impaction force is more closely related to patient sensation and aerosol deposition than other, more traditional, parameters, it may provide a better way to evaluate in vitro equivalence in support of new drug applications (NDAs) or abbreviated new drug applications (ANDAs) for orally inhaled and nasal drug products.
D. H. Kim1 , J. U. Kang1 , R. Waynant2 , I. K. Ilev2 , 1Johns Hopkins University, Baltimore, MD, 2CDRH, FDA, Rockville, MD
Background: Confocal microscopy using optical fibers has been widely used because it provides submicron spatial resolution, flexible beam delivery and 3-dimensional scanning potential. Our primary interest is to develop hollow-core photonic bandgap fiber (PBF) based high-resolution fiber confocal microscopy because solid-core fibers are not suitable for the mid-IR range (wavelengths which are attractive for various biomedical applications) and conventional hollow-core fibers are not suitable for high-resolution confocal microscopy due to technical difficulties in manufacturing small core diameter fibers.
Methods: To demonstrate the single PBF confocal microscope potential, we designed a confocal microscope arrangement using a 5-µm-diameter hollow-core PBF fiber and a 25-mW-power 532-nm continuous wave laser. For comparative experiments, we used a 700-µm-diameter gold-coated hollow core fiber.
Results: Experimental results obtained with the PBF and 10× (NA = 0.25), 20× (NA = 0.40), and 60× (NA = 0.85) focusing objectives resulted in longitudinal resolution of 14.7 µm, 6.9 µm, and 2.1 µm, respectively. In comparison, experimental results obtained with the hollow-core fiber were 55 µm and 16 µm for 40× and 60×, respectively. An additional advantage of confocal microscopy using hollow-core fiber is very low Fresnel back-reflection at the air-fiber interface. The total back-reflection reduction was 85% when hollow-core fiber vs. solid-core fiber was used.
Conclusions: We have proposed and experimentally studied a simple single-fiber confocal microscope using a hollow-core photonic bandgap fiber. Experimental results obtained demonstrate advanced properties of the hollow-core PBF for utilization in high-resolution confocal microscopy, which can extend the wavelength range to the mid-IR.
R. J. Landry, I. K. Ilev, T. J. Pfefer, R. W. Faaland, D. Calogero, CDRH, FDA, Rockville, MD
Background: It is well established that intraocular lens implants can produce spurious reflections that impede vision. A test method is needed to characterize glare from intraocular lenses (IOLs) from point light sources such as automobile headlamps.
Methods: A collimated Gaussian laser beam produced by a 15-mW-power 658-nm-wavelength continuous wave laser diode and a single-mode delivery fiber is directed to various parts of the subject IOL mounted in a water-filled model eye. Reflected images produced in the retinal plane are documented with a digital camera.
Results: Significant retinal plane images were produced only when the laser beam was incident on the haptic or edge of the IOL. The haptic produces a line glare image in a direction perpendicular to the haptic. The IOL edge produces a diffuse extended image over the field of view.
Conclusions: The test method developed can be used to characterize and pinpoint the source of extraneous glare images from intraocular lens implants from point light sources. The haptic insertion in the optic of three-piece IOL's has been identified as a source of line glare images.
D. R. Lu1 , K. Abu-Izza2 , 1ONDQA, CDER, FDA, 2University of Georgia
Background: The objective of this work was to advance the scientific understanding of the relationship between response (product performance) and the variables (critical parameters) during the development of modified-release microspheres containing Zidovudine.
Methods: Modified-release microsphere formulations of Zidovudine were developed. To understand the relationship between responses and critical parameters and to establish the formulation and process design space, a statistical approach was utilized to generate the three-dimensional response diagrams and to optimize the formulations after constructing a desirability function that combines four responses.
Results: The release rate (t85) of Zidovudine from the microspheres was found to be critically dependent on the formulation variables, including emulsifier SDS concentration (X1), drug to polymer ratio(X2), and solvent composition of ethyl acetate (X3). Based on the experimental data, the relationship was statistically calculated as:
t85 = 10.49 - 2.42X1 - 7.34X 2 - 0.094X 3 + 1.05X 1X 2 + 0.015X 1X 3 + 0.047X 2X 3 + 1.30X 22
The study further examined the optimization procedure considering multiple responses, including the t85, drug loading efficiency, product yield, and loose surface crystals. Three-dimensional response diagrams were generated as the formulation and process design space. The optimized formulations were further studied in dogs for their PK and IVIVC (Cmax=1.5 µg/mL, tmax=2.6 h, MRT=4.4 h).
D. R. Lu1 , E. C. Ware2 , 1ONDQA, CDER, FDA, 2University of Georgia
Background: The objective of this work was to implement an automated robotic approach to screen a large number of pharmaceutical salts using Biomek robotic workstation. The work represented the early attempt to use a high-throughput approach to preformulation studies.
Methods: Automation of Trazodone salt formation on Biomek workstation was performed using eight different crystallizing solvents and 13 different acids. A control program was set up to generate 104 Trazodone salts. The salts were observed under a polarized light microscope to determine crystallinity. After stepwise eliminations, the remaining salts were scaled-up for differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), hygroscopic, pH-solubility, density, surface area, and particle size analyses.
Results: Oils formed in several cases resulted in preliminary elimination of mesyl and esyl salts and four crystallizing solvents. Crystallinity was observed in 34 of 44 scaled-up Trazodone salts. PXRD, DSC, and hygroscopic analyses indicated a number of new salts that were comparable in physicochemical parameters to the reference HCl salt. Among the new Trazodone salts, the most interesting profile was from tosylate salt with a low solubility throughout the entire pH range. The solubility values range from 3 mg/mL at pH 1 to 0.2 mg/mL at pH 12.
Conclusions: The new tosylate salt gave a unique pH-solubility profile with low solubility over the entire pH range making it a potential candidate for a suspension or prolonged-action formulation. The high-throughput robotic process demonstrates its capability to increase the efficiency of pharmaceutical salt selection.D. R. Lu1 , F. Alanazi2 , Z. Fu2 , 1ONDQA, CDER, FDA, 2University of Georgia
Background: DNA vaccines have several distinct advantages over traditional vaccines. The objective of this work was to develop new artificial lipoprotein systems, as nanoscale DNA carriers, to improve the efficacy in DNA vaccination. The transfection efficiency of rabies plasmid DNA vaccine carried by our artificial lipoprotein system was evaluated in cell culture.
Methods: Phospholipid nanoemulsions (NE) resembling the lipid core of human lipoproteins were prepared. The carrier for DNA was further constructed by assembling NE-PLL-DNA complex. Gel electrophoresis, zeta potential, and mobility measurement were conducted to determine the optimal surface charge balance in various complex compositions. Transfection and transfection efficiency were examined by fluorescence microscopy and flow cytometry, respectively.
Results: The artificial lipoprotein system carrying rabies DNA vaccine was successfully constructed. The DNA vaccine was effectively transfected in the culture of SF-767 cells. The amount of PLL (palmitoyl-poly-L-lysine) incorporated into the artificial lipoprotein system represented a critical formulation parameter and had a significant effect on transfection efficiency. The new carrier system proved to be more efficient in cellular transfection of rabies DNA vaccine than commercial lipofectamine formulation. Compared to lipofectamine formulation, our carrier for DNA vaccine illustrated about a 4.2-fold increase in percentage transfection efficiency, indicating the high effectiveness of the new carrier system.
Conclusions: Effective transfection of rabies DNA vaccine in cell culture can be achieved using the new artificial lipoprotein carrier system. The amount of PLL, and subsequently the charge balance of NE-PLL-DNA complex, was a critical formulation parameter.Q. Lu, R. Malinauskas, S. F. Stewart, CDRH, FDA, Rockville, MD
Background: Thromboembolism remains a serious problem associated with blood-contacting medical devices such as heart valves, ventricular assist devices, and endovascular stents. In premarket regulatory review of these devices, hemocompatibility assays are required by FDA to evaluate their potential to damage or activate blood elements. While in vitro hemolysis testing is commonly performed and represents a valuable measurement of damage to red blood cells, it does not provide information on the critical problem of localized platelet activation and blood clot formation. As platelet activation is a major underlying mechanism in thromboembolic complications, there is a need to evaluate the interactions between platelets and medical devices. Although various assays have been proposed to evaluate platelet function, guidance documents which promote standardized testing procedures and objective pass/fail criteria have not been established.
Approach: In our effort to develop standardized procedures for platelet assays that can better predict medical-device mediated platelet activation and subsequent thrombosis, we are investigating the relationship between different flow regimes and blood cell damage by using a series of in vitro flow models. Different mechanisms of platelet activation will be assessed by a panel of test methods, including flow cytometry for platelet surface activation markers, ELISA for plasma-soluble markers, and aggregometry for platelet aggregation. Additionally, we will integrate the results from bench-top blood experiments and computational flow dynamics modeling to characterize critical variables associated with mechanical hemolysis and platelet activation in medical devices. In this poster, the flow loop model designs, experimental methods, and preliminary results will be presented.
G. Madhavan, K. J. McLeod, Clinical Science and Engineering Research Center, State University of New York, Binghamton, New York
Background: Chronic hypotension (low blood pressure) is rarely regarded as a pathological condition, yet recent evidence has emerged indicating significant adverse health effects and morbidity arising from chronic hypotension, including cerebrovascular degeneration, dementia, Alzheimer's disease, and Parkinson's disease. Despite these significant implications, the prevalence of hypotension in the adult population has undergone limited investigation as compared to hypertension. Here, we investigated the prevalence of chronic hypotension in healthy adult white women using continuous blood pressure monitoring.
Methods: Adult women in the age range of 35-80 were screened for the study. Those with no pre-existing cardiovascular conditions underwent 90 minutes of non-invasive beat-to-beat blood pressure monitoring. The protocol involved 30 minutes of supine rest, 30 minutes of upright sitting, and a 30 minute exposure to plantar-based calf muscle pump stimulation, while seated.
Results: 34 women (mean age: 53.8±1.9 years) were screened, 28 of whom had no pre-existing cardiovascular conditions. Over 65% (23/34) were found to have a diastolic blood pressure (DBP)<70mmHg after 30 minutes of quiet sitting, and over 20% (8/34) had a DBP<60 mmHg, indicative of clinical hypotension. Calf muscle pump stimulation increased DBP in all of the hypotensive subjects, and was capable of elevating DBP above the 60 mmHg threshold in 7 of 8 of the clinically hypotensive subjects, suggesting that hypotension in this group may be a result of inadequate calf muscle tone.
Conclusion: Approximately 20-25% of white adult females may be clinically hypotensive, and a dominant etiological factor in most of these individuals appears to be inadequate calf muscle tone. These results suggest that muscle training, either through exercise techniques or use of muscle stimulation devices may be sufficient to reverse this condition in many of the individuals.
This study was supported by financial grants from the New York State Office for Science, Technology, and Advanced Research (NYSTAR), Albany, New York. The plantar stimulation devices used in this study are classified as non-significant risk devices, as defined by the FDA-IDE Regulation and were supplied by Juvent Corporation, Somerset, New Jersey.
A. S. Zidan1 , M. A. Habib2 , A. NguyenPho3 , M. A. Khan4 , 1DPQR, CDER, FDA, Silver Spring, MD & School of Pharmacy, Howard University, Washington, DC, 2School of Pharmacy, Howard University, Washington, DC, 3DPQR, CDER, FDA, Silver Spring, MD, 4DPQR, CDER, FDA, Siver Spring, MD
Optimization of cyclosporine A self-nanoemulsified drug delivery systems using Box-Behnken design and desirability function
A. S. Zidana,b, M. J. Habibb,A. NguyenPhoa, M. A. Khana.aDivision of Product Quality Research, CDER, FDA, Silver Spring, MD. bSchool of Pharmacy, Howard University, Washington, DC.
Background: Quality by design (QBD) refers to the achievement of certain predictable quality with desired and predetermined specifications. A very useful component of the QBD is the understanding of factors and their interaction effects by a desired set of experiments.
Methods: The present project deals with a case study to obtain nanoemulsified particles of a model drug namely Cyclosporine A (CyA). A 3-factors, 3-level Box-Behnken optimization design was run to evaluate the main and interaction effect of several independent formulation variables namely amounts of Emulphor El-620 (X1), Capmul MCM-C8 (X2) and 20% w/w CyA in sweet orange oil (X3). The dependent variables included nanodroplets size (Y1), nanoemulsions turbidity (Y2), amounts released after 5 and 10 minutes (Y3, Y4), emulsification rate (Y5) and lag time (Y6). A desirability function was used to minimize the lag time and to maximize the other dependant variables.
Results: A mathematical relationship, Y5 = 9.09 - 0.37 X1 + 0.37 X2 - 0.45 X3 + 0.732 X1X2 - 0.62 X1X3 + 0.3 X2X3 + 0.02 X12 - 0.28 X22 + 0.471 X32 (r2 = 0.92), was obtained to explain the effect of all factors and their colinearities on the emulsification rate. The optimized nanodroplets were predicted to yield Y1, Y2, Y3, Y4, Y5 and Y6 values of 42.1 nm, 50.6 NTU, 56.7 %, 107.2 %, 9.3 %/min and 3.5 min, respectively, when X1, X2, and X3 values were 36.4, 70 and 10 mg, respectively. A new batch was prepared with these levels of the independent variables to yield Y1 to Y6 values that were remarkably close to the predicted values.
Conclusions: This investigation demonstrated the potential of QBD in understanding the effect of the formulation variables on the quality of CyA self nanoemulsified formulations.
D. M. Saylor1 , M. K. McDermott1 , B. J. Dair1 , C. S. Kim1 , J. Toy2 , D. V. Patwardhan1 , J. A. Warren3 , 1OSEL,CDRH,FDA, Rockville, MD, 2ODE,CDRH,FDA, Rockville, MD, 3NIST, Gaithersburg, MD
Background:
A popular method of controlling drug release is to incorporate the drug into a polymer matrix, which acts as a diffusion barrier and thereby slows the rate of drug release. The processing conditions during manufacture affect the composite's structure, but neither that nor the subsequent effects on release rate are well understood.
Methods:
An integrated approach has been taken in an effort to elucidate the inter-relationships between manufacturing conditions, structure, and release kinetics:
Results:
Both the theoretical predictions and experimental measurements indicate that the drug-polymer composite structures are extensively modified by variations in composition and process environment. Furthermore, these modifications have a substantial impact on the release kinetics.
Conclusion:
The rate of drug released from controlled drug delivery systems is extremely sensitive to the structure of the drug-polymer composites that evolves during manufacture. A theoretical and computational framework has been developed to predict the impact of manufacturing conditions on structure and the structure sensitivity of release in these systems. This framework is currently undergoing experimental validation.
D. M. Saylor1 , M. K. McDermott1 , B. J. Dair2 , J. Toy1 , D. V. Patwardhan1 , J. A. Warren3 , 1OSEL,CDRH,FDA, Rockville, MD, 2ODE,CDRH,FDA, Rockville, MD, 3NIST, Gaithersburg, MD
Background: The latest trend in implantable medical devices is to coat device surfaces with controlled drug delivery systems, i.e. drug-polymer composites. Recently, biodegradable polymers have replaced non-degradable polymers in order to circumvent potential clinical problems associated with the presence of residual composite after drug delivery. However, the impact of polymer selection on the composite structure of the coating and ultimately on the delivery behavior is not well understood.
Methods: To elucidate the influence of polymer material selection on the delivery characteristics of controlled drug delivery systems, we first developed a new predictive theory for the structural evolution of these composite systems. Next, we used the theory to compute expected changes in both composite structure and subsequent drug release due to variations in the type of polymer used during manufacture. Finally, we made laboratory observations to assess the validity of the theoretical predictions.
Results: For the polymers we considered, the theoretical predictions and experimental observations suggested that there is little impact of polymer selection on the composite structure under constant manufacturing conditions. However, the impact of the underlying composite structure on drug release varied significantly depending on whether (bio)degradable or non-degradable polymers are used.
Conclusion: We used the newly developed predictive theory and laboratory observations to assess the influence of polymer material selection on the delivery characteristics of controlled drug delivery systems. Although polymer selection had little impact on the initial composite structure, the relationship between structure and drug release depended strongly on the polymer type.S. Prodduturi, A. M. Wokovich, B. J. Westenberger, L. F. Buhse, W. H. Doub, DPA, OTR, CDER, FDA, St. Louis, MO
Background: Fentanyl is a potent opioid used in the palliative treatment of late-stage cancer and chronic pain. There are currently two approved Fentanyl Transdermal Systems (FTS). Innovator Duragesic FTS (D-FTS) is a reservoir system with a rate-control membrane while generic Mylan FTS (M-FTS) is a drug-in-adhesive system without a rate-control membrane.
Methods: Novel technique to study skin/membrane permeability was developed using a modified USP-5 paddle-over-disk apparatus. Four membranes with different permeabilities- two silastic (75, 125 microns) and two ethylene vinyl acetate (EVA) membranes (9%, 19% vinyl acetate content) were evaluated.
Results: The steady state output rates calculated using EVA (9% vinyl acetate) membrane for 25 µg/hr dose Mylan and Duragesic patches were 21.3 and 20.4 µg/hr, respectively. The release rate before steady state was higher from D-FTS (34.5 µg/hr) than M-FTS (24.6 µg/hr). When other membranes (silastic and EVA 19% vinyl content) having higher permeabilities were used, the steady state output rates from matrix M-FTS were very high when compared to reservoir D-FTS.
Conclusions: The higher observed initial release rate (pre-steady state) with the reservoir D-FTS might be due to its saturated drug-containing adhesive layer and/or due to alcohol incorporated as a penetration enhancer. The higher fluxes obtained from M-FTS relative to D-FTS when using high permeability membranes may indicate that matrix and reservoir FTS are not bioequivalent when the skin is compromised. EVA membrane (9% vinyl acetate) seems to be a good model to predict skin permeability. However, confirmatory experiments using cadaver skin are currently underway.
S. Salas-Vega, D. C. Richardson, CDRH, FDA, Rockville, MD
Background: Microvacuoles ("glistenings"), first reported in rigid intraocular lenses (IOLs) in 1984, are occurring in greater numbers in the new foldable hydrophobic materials. The effect of an increase in vacuolar spatial density on light scattering can impact contrast sensitivity or night vision, and necessitate surgical IOL removal. Others have developed methods to induce vacuolar growth in the lab. We are determining which of these methods best indicate phase stability in vivo and are developing a method to identify which IOLs are more likely to grow vacuoles that exceed a threshold of adverse clinical effect.
Method: To test accelerated methods for vacuologenesis, we place a lens on a temperature-controlled stage and hold at elevated temperatures for extended periods while observing via an inverted microscope. Pictures were taken at intervals and correlated time-temperature paired data were acquired and plotted.
Results: Our ability to capture time-lapse images of vacuologenesis indexed to the immediate temperature enables us to develop and justify an accelerated test method. We present data showing the time-sequenced growth of vacuoles in different, currently manufactured IOLs. The materials exhibit disparate vacuolar dimensions and densities, as well as different time courses for vacuolar stability.
Conclusion: Preliminary results show different IOL compositions have differing susceptibility to vacuologenesis, both in terms of size distribution and time course of vacuolar enlargement and shrinkage. The time-temperature protocol shows promise as a foundation for in-vitro testing of formulations, newly submitted to CDRH, to predict their propensity to grow vacuoles after IOL implantation.
E. Scott, A. Crespi, C. Schmidt, Medtronic, Inc.
Background: Rechargeable lithium-ion batteries exhibit a phenomenon called capacity fade; the amount of capacity that can be discharged on each recharge cycle tends to decrease as the battery is used. Capacity fade is often attributed to the effects of cycling, but our testing shows that the amount of time the battery is in use contributes more to capacity fade.
Methods: Medtronic's lithium-ion cells of conventional chemistry (Carbon|LiCoO2) in hermetically sealed prismatic cases were tested. Full depth-of-discharge cycling was conducted at 37°C at two different rates, approximately once per week and once per day, to quantify the effect of cycling rate and calendar time on capacity fade. Testing ran for more than three years.
Results: Although the batteries on the daily test experienced six times as many cycles as the batteries on the weekly test, the capacity retained was nearly the same (86.7% in the weekly test and 85.5% in the daily test). Furthermore, testing at two different cycling rates enabled the quantitative separation of time-dependent and cycle-dependent capacity fade parameters.
Conclusion: The passage of time had a much stronger effect on capacity fade than the number of recharge cycles in the tested batteries. Similar results have been reported on commercially available lithium-ion cells. Thus, cycling lithium-ion batteries at a single, accelerated test condition is inadequate for long-term performance projections. Proper characterization requires measurement of both time-dependent and cycle-dependent capacity fade at the application temperature.
A. S. Zidan1 , M. A. Habib2 , O. A. Khan3 , R. B. Shah4 , 1Visiting Student, DPQR, CDER, FDA, SIlver spring, MD. School of Pharmacy, Howard University, Washington DC., 2School of Pharmacy, Howard University, Washington DC, 3Summer intern, OPS, CDER, FDA, Silver spring, MD, 4DPQR, CDER, FDA, Silver spring, MD
Background. The study describes preparation and characterization of self-nanoemulsified drug delivery systems (SNEDDS) of Cyclosporine A (CyA) with Emulphor El-620. The objective was to understand the product variability due to size and other characteristics.
Methods. Ternary phase diagram was constructed to identify the efficient self-emulsification region using 20%(w/w) CyA solution in sweet orange oil (oily phase), El-620 (surfactant) and Capmul MCM-C8 (co-surfactant). The formulated SNEDDS were characterized by droplet size, turbidity, zeta potential, Fourier Transform Infra-Red (FTIR) and Differential Scanning Calorimetry (DSC) analysis. The dissolution studies were in conjunction with turbidimetry. Permeability studies were performed in Franz diffusion cells. Also near infrared (NIR) to relate both droplets size and turbidity was evaluated.
Results. The results showed an optimum surfactant to co-surfactant ratio of 2:1, above which, nanoemulsions with particle size of 10 nm and turbidity of 10 NTU were obtained. All the prepared systems were positively charged. Lack of any incompatibility among ingredients was shown by FTIR and DSC. Turbidity time profiles revealed three distinctive regions: lag, plateau, and pseudolinear phase. Emulsification rate was obtained from the corrected slope of the pseudolinear phase. The nanodroplet size was inversely proportional to the release rate. Chemometric analysis using NIR predicted particle size and turbidity over a range of oily phase percentages.
Conclusion. The study demonstrated ability of NIR to evaluate the impact of droplet size on the SNEDDS variability. The increased variability in drug permeability from the SNEDDS was due to lower droplet size (< 10 nm).
K. Vorvolakos, H. D. Luu, D. V. Patwardhan, I. S. Isayeva, S. K. Pollack, FDA-CDRH-OSEL-DCMS
Introduction
Soft polymeric materials are the key components of many medical devices such as ophthalmic fillers, lubricants, lavages, drug delivery media, and abdominal adhesion barriers. This work characterizes several adhesion barriers which function by separating and lubricating internal organs. We also describe in-house capabilities for broader exploration into soft materials and physical properties crucial to various device applications.
Results
Different physical properties are seen among adhesion barriers and peritoneal fluid. These in vitro differences, which depend on the type and molecular weight of polymer involved and the formulation conditions, could be responsible for performance differences in vivo. They could also indicate different mechanisms of action, or robustness thereof with respect to properties.
Summary and Future Work
The aim of this work is to understand wetting, lubrication, and mechanical properties on a molecular and formulation level, and improve FDA's ability to regulate all soft material devices where spreading and mechanical properties are important.
*See poster on Fe-HA by Isayeva et al.
S. Weininger1 , A. B. Shang2 , 1CDRH, 2Duke Univerisity
Introduction: As part of the validation process required for developing a testing method for assessing pulse oximeter performance in the presence of patient motion artifact, several physiologic background effects were studied. It has long been observed that the position of the probe with respect to the heart influences the resulting measurements, but this effect has not been specifically studied. As part of the American Society for the Testing of Materials commissioned project undergone jointly by Duke University and the US Food and Drug Administration1, we examined the relationship between the O2 saturation, perfusion index, and infrared plethysmogram (IR pleth) as a function of probe position with respect to the heart.
Materials & Methods: The IR pleth from 15 subjects was recorded under an IRB approved protocol1. IR pleths, saturation, pulse rate, and perfusion index (PI) were recorded as a function of hand position with respect to the heart. Four positions were studied, "Down" or hand hanging dependent at the subject's side, "Heart" or hand resting at the level of the heart, "Head" or the hand raised to the level of the head, and "Up" or the hand extended up above the level of the head. The hand was motionless during the data collection periods. Oximeters from GE and Phillips were used to collect data. Before starting, and between each elevation change, the subject underwent a rest period at the heart level.
Results and Discussion: After examination of data from 15 subjects, there were several trends noted. The PI generally increased from the down or dependent position to the raised positions. In some subjects, the PI increased consistently with increasing altitude as compared to the heart. In other subjects, the PI increased through the first three positions, and decreased as the probe was elevated above the head. The rate of the PI increase also differed among subjects. We believe these observations are consistent with the effects of physics combined with variations in subject physiology. The PI is essentially a ratio of the pulsatile arterial signal (AC) as compared to the non-pulsatile venous and tissue (DC) signal. Placing the measured extremity dependent will increase venous pooling, causing the DC signal to decrease. Elevating the extremity will cause increased venous return, increasing the DC signal. Given the known drop in blood pressure measured in the fingers with relative altitude, the PI would be expected to decrease with extremity elevation. However, the reverse is seen. We believe this is due to a proportionally larger increase in AC signal compared to the increase in DC signal. It is thought that arterial dilation occurs as a response to the decrease in blood pressure due to elevation, perhaps mediated via the endothelial release of nitric oxide2. In some individuals as the altitude of the extremity increases, the ability of the heart to maintain forward flow to that extremity may reach a maximum, and then decrease, or there may be a contribution from arterial outflow obstruction.
The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.
E. Marszal, R. A. Boykins, A. Shrake, CBER, FDA, Bethesda, MD 20892
Background: α1-proteinase inhibitor (α1-PI) deficiency is a genetic disorder that can lead to chronic lung and liver disease. The disease-associated Z-variant of α1-PI polymerizes in the liver, the major site of α1-PI synthesis. Formation of the polymers is associated with increased risk of developing cirrhosis and liver failure. Understanding polymer structure may lead to the development of new therapeutic modalities to prevent liver disease.
Methods: proteolytic digestion, PAGE
Results: We studied proteolytic degradation of α1-PI polymers formed in vitro at elevated temperature. When analyzed by electron microscopy, such polymers resemble the Z-variant polymers purified from liver. The polymers formed in vitro and exposed to proteolytic enzymes such as trypsin, chymotrypsin, Asp-N, and Glu-C were nicked on the surface but stayed structurally intact in solution. During boiling in SDS, the polymers dissociated and SDS-PAGE gels yielded patterns of polypeptides which were distinct for each protease. Polymers formed from normal and iodoacetamide-blocked monomers, thus, with and without disulfide bonds, respectively, gave similar digestion patterns.
Conclusion: Similar proteolytic digestion patterns suggest that the structures of polymers with and without disulfide bonds are similar, as we proposed recently**. To verify this conclusion, the work will be repeated with polymers that contain higher levels of disulfide bonding such as polymers formed in the presence of guanidine or at low pH.
* The research was supported with a grant from the Alpha-1 Foundation
** Marszal et al. JBC 2003
Synthesis and Characterization of an Iron-Crosslinked Hyaluronic Acid-Based (FeHa) Adhesion Barrier
I. Isayeva1, H.D. Luu1, J. DeFoe2, D. Patwardhan1, A. Chen3, K. Vorvolakos1, and S.S. Das1, 1Division of Chemistry and Materials Science, Office of Science and Engineering Laboratories, CDRH/FDA, Rockville, MD 20852, 2Department of Chemistry, Marquette University, Milwaukee, WI 53233, 3Thomas S. Wootton High School Rockville, MD 20850
Introduction: Hyaluronic acid (HA) has been used in a variety of medical applications, including post-operative adhesion barrier devices. High water solubility and enzymatic susceptibility of HA lead to a short half-life of this polymer in vivo (1-3 days), which is not always acceptable for effective adhesion prevention. To increase the HA half-life and improve the device effectiveness, HA has been ionically-crosslinked in a (FeHA)-based adhesion barrier that was approved by FDA in 2002. The post-market adverse events reported with this product prompted us to explore the chemistry and the properties of these gels.
Methods: The ionic crosslinking of HA is achieved by the reaction of sodium hyaluronate with ferric chloride. Synthesized gels were analyzed by a number of characterization methods, including a novel titration method, which is being developed to quantify the actual degree of iron crosslinking.
Results: This poster describes the effect of the crosslinking conditions, including the effect of ionic strength, pH, mixing time, and crosslink density on the homogeneity and physical properties of the FeHA gels. Data suggest, that the small variations in the pH of the starting HA solution can produce FeHA gels containing small and large size insoluble "particulates". The hypothetical cause for gel inhomogeneities and their effect on the gel properties is discussed.
Conclusions: Our ability to synthesize and characterize FeHA has enabled us to measure the effects of manufacturing deviations on properties of the final product. These capabilities may contribute to the analysis and review of other CDRH regulated soft polymer devices.
P. E. Bonangelino, E. Russek-Cohen, CDRH, FDA, Rockville, MD
We are considering the case of two treatment groups and a single binary covariate. It is well known that the coding of indicator variables as 0,1 or -1,1 does not affect the model fit, with the exception of changing the interpretation of the individual parameters. However, when there is an interaction term between two indicator variables, the significance of the main effect terms can mistakenly appear to depend on the coding of the indicator variables. This is because a test of main effect for treatment is testing a different hypothesis depending on how the indicator variables are coded. We show through an example that this confusion can occur even when the interaction term is not significant or even close to significant. There is less ambiguity in what is meant by a main effect in the presence of an interaction by considering the -1, 1 coding to be the reference coding when there is an interaction term, as is done by SAS in Proc Logistic and Proc GLM using the default of Type 3 sums of squares. Using the -1, 1 coding is particularly important for procedures such as SAS Proc Phreg, which do not have automatic indicator variable coding (i.e. a "Class" statement).
P. Chakraborty, S. Nagaraja, Cognizant Technology Solution
Title: Emerging Trends in Clinical Trial Data Integration and Management
Name of Authors: Partha Chakraborty, Srivatsan Nagaraja
Company Name: Cognizant Technology Solution.
Postal Address: 350 Parsippany Rd, Apt 88 Parsippany, NJ 07054 USA
Phone: (1) 9734620929
eMailAddress: Partha.chakraborty@cognizant.com
As the pharmaceutical industry moves towards complete implementation of EDC, a new business scenario is emerging in Clinical Data Management and Statistical Programming. Cognizant envisions that in future Pharmaceutical industry would show a shift from the existing clinical data management set up & infrastructure. A most likely alternative infrastructure would include a CDISC SDTM complaint data repository and a statistical programming tool. This would enable bio-statistician and the programmers to focus on statistical analysis rather than SAS programming. To realize this vision on Data integration and management, Cognizant has a concept of building Center for Excellence under its perusal.
The data repository would include clinical trial data from all the studies (ongoing and legacy). The data repository needs to follow CDISC ODM and SDTM structure to facilitate pooled analysis and regulatory submission. A mapping tool would enable to map the data from EDC & Legacy CRO data through a simple & intuitive User Interface. A set of generic SAS macro would convert the data to ODM and SDTM. The data repository can conceptually be divided into two parts: a) Staging (following ODM) and b) Final (following SDTM). The staging area would allow users to perform data reviews and take remedial measures on the data. The statistical programming tool would facilitate creation of Report Shell through generic SAS Macro and creation of Tables (Summary), Listing & Graphs. The reporting tool would create all the listings through a single generic macro; in stead of current ('old') way of creating a single SAS Program for a single Listing. The tool would allow building of Analytical Macro Library. The reporting tool would get the data from SDTM Data Repository.
Such an infrastructure can be easily built on current technologies available. Data repository can be built using Relational Database (Oracle) or SAS 9.1.3. An intuitive user interface would need Microsoft ASP.Net or J2EE based implementation. The foundation of data integration and statistical reporting can be built on using SAS 9.1.3 foundation server.
L. Chen, Y. Tsong, CDER, FDA, White Oak, MD
A drug abuse potential clinical trial is required by the FDA for those drugs having possible abuse potential in drug abuse community. The results from drug abuse potential studies provide important information for scheduling a drug. Due to large between subject variability in the endpoint measurements based on self-evaluated responses and the difficulty in recruiting appropriate study subjects; the designs for such studies are thus typically crossover, with self-control. In this poster, the advantages and disadvantages of currently used study designs are discussed. Design issues are presented. Some new ideas for improving existing designs are proposed.
B. D. Gallas1 , G. A. Pennello2 , 1OSEL/CDRH/FDA Rockville MD, 2OSB/CDRH/FDA Rockville MD
Background
When comparing the performances of two diagnostic devices that require interpretation by a doctor, as for imaging devices, there are at least two sources of variability: the doctors and the patients. These sources of variability generate variances and covariances in the data that can be addressed with multireader, multicase (MRMC) variance analysis, where "reader" refers to the doctor and "case" refers to the patient. MRMC analysis of the area under the receiver operating characteristic curve has supported several FDA submissions. Frequently, a fully-crossed study design is used, where every reader reads every case using both devices. For imaging devices used during in vivo procedures, a fully-crossed design is infeasible.Instead, each patient is diagnosed by only one doctor using both devices.Here we investigate sensitivity under this doctor-patient, paired-reader, paired-case study design.
Methods
From a probabilistic foundation, we derive the bias and variance of two statistics: pooled sensitivity and reader-averaged sensitivity. We also construct variance estimates of these statistics and compare them to naïve estimates. Finally, we run simulations to assess the statistics and the variance estimates.
Results
The two sensitivity statistics have the same means but different variances. The variances depend on how patients are distributed among the readers and the amount of reader variability. Regarding the variance estimates, the MRMC estimates are unbiased, whereas the naïve estimates are biased low.
Conclusions
When doctors interpret output from a diagnostic device, they contribute to the variability of the performance metric, just as cases do. Both sources of variability need to be counted.
Y. Gao, L. Thompson, FDA
Intention-to-treat (ITT) principle and per protocol (PP) approach are two common approaches for analyzing data in randomized controlled trials (RCT). Each approach has its own advantages and pitfalls under different RCTs. The two approaches can differ in the handling of patients who cross over from one treatment to another or in handling patients who drop out of the trial.In some circumstances, ITT approach is conservative (or anti-conservative), whereas in others, PP approach is conservative (or anti-conservative) . This poster will give Monte Carlo estimates of the sizes and powers of these two approaches in different simulated RCT trials so that we can have some idea of how conservative or anti-conservative ITT and PP can be under particular scenarios.
P. T. Liu, Q. F. Graves, CFSAN
We consider the situation where an identical number of samples are drawn from a population with known disease-exposure status by the following sampling schemes:
(1)cross-sectional sampling (i.e., simple random sampling) (2) exposure-stratified sampling (i.e., a prospective study) and (3) disease-stratified sampling (i.e.,a retrospectivestudy). Each scheme could conceivably yield a remarkably different sample distribution. Nevertheless, we have found for the cases simulated, that the odds ratios estimated from those schemes turn out to be close to one another and asymptotically approach an unbiased estimate. However, their corresponding variances are tremendously different. The objectives of this article are: (1) to examine the efficiency of the odds ratio estimates through the bootstrap approach in order to determine the optimal sampling strategy; (2) to examine the functional relation between the odds ratio and relative risk and to derive the relative risk distributions and their confidence intervals from a retrospective study; and (3) to evaluate the feasibility of using the odds ratio and relative risk as "signals" in analyzing the data collected from the CFSAN Adverse Events Reporting System (CAERS).
S. Hirschfeld, CBER FDA, Rockville, MD
Current analytic methods tend to examine only a subset of the available data collected during a clinical study. Outcome is typically reported for a primary and a limited number of secondary endpoints without formal integration of the results.
We propose a graphical approach assigning each variable of interest plus time a dimension in an n-dimensional space, calculating the coordinates of each patient as time progresses in that space to describe a path, and describing the sum of all paths for a patient population as bounded by a space. For any patient an event free course would be a vector along the time axis. Any events that were risk or benefit would shift the spatial coordinates away from the time axis. The coordinates at the end of the observation period and the duration spent in particular regions of the space would describe the integrated clinical course. The number of dimensions can be restricted or expanded as required and can be collapsed into composite risk and benefit indices to describe an integrated risk-benefit assessment.
Examples will be provided to illustrate the general method.
G. F. Kapke, J. Leroux, Covance Central Laboratory Services, Indianapolis, IN
Evaluation of data frequently identifies significant events as multiples of the reference interval. Due to biological variability, higher information content could be provided by reviewing events based on the Z value calculated from the analytical precision (CVA), the intra-individual biological variability (CVI), and the relative change value (RCV = baseline value - visit value) defined as RCV = 2.5*Z*[CVA2 +CVI2] or Z = RCV/[2.5 *[CVA2 +CVI2]] where Z is the probability (Z=2.58 for 99% probability). In reviewing a phase I data set of 48 patients and 483 values, no value was greater than 3 times the upper limit of normal. Thirteen values (2.69%) were greater than 2 times the upper limit of normal (86 U/L). When the data was translated into Z values, 74 values had a Z greater than 3 (15.32%), 47 values had a Z value of greater than 4 (9.73%), and 36 values had a Z value of greater than 5 (7.45%). The highest Z value was 20.7. Review of a data set by Z values would identify that more than 15% of the values were significantly elevated compared to 2.69% based on 2 times the upper limit of normal. Since Z value calculations are method independent, historical data can be reviewed and perhaps a Z value can be defined for a healthy phase 1 population that translates into inappropriate risk in the general population.
I. S. Kyprianou1 , L. Thompson2 , D. Banh1 , W. Pritchard3 , J. Karanian4 , L. Rosen5 , K. J. Myers1 , 1NIBIB/CDRH Joint Laboratory for the Assessment of Medical Imaging Systems, US Food and Drug Administration, Rockville, MD, USA, 2OSB/DBS/CODB, Rockville, MD, USA, 3OSEL/DSFM, Rockville, MD, USA, 4OSEL/DB, Rockville, MD, USA, 5NIH/CSR, Bethesda MD
Cardiovascular disease is considered the leading cause of death in the US, accounting for 38% of all deaths.There are gender differences in the size of coronary arteries and in the character and location of atherosclerotic lesions that affect the detection of coronary artery disease with the medical imaging modalities currently used (e.g. angiography, computed tomography). These differences also affect the safety and effectiveness of image-guided interventions using therapeutic devices. For the optimization of the medical imaging modalities used for this specific task we require the generation of clinically-realistic, gender-specific images of healthy and pathological coronary angiograms. For this purpose we have created a gender-specific statistical model of a pathological coronary artery tree. Starting from "healthy" heart-phantoms created from high resolution CT scans of cadaver hearts of both genders, the model uses prevalence data obtained from clinical studies of patients with significant (>50% stenosis) coronary artery disease (CAD). The model determines the plaque deposit locations and character (length, percent stenosis) for each case, based on a flow model. These data are then used to generate artificially diseased artery trees, embedded in a gender-specific torso model. Using an x-ray and optical photon Monte-Carlo simulation program, we then generate simulated angiograms exhibiting realistic disease patterns. The severity of each angiogram is determined from a set of rules that combines the geometrically increasing severity of lesions, the cumulative effects of multiple obstructions, the significance of their locations, the modifying influence of the collaterals, and the size and quality of the distal vessels. The simulated angiograms will consequently be read by model and human observers. The probability of detection derived in combination with the severity score will be used as a figure of merit for the patient- and gender-specific optimization of the imaging modality under investigation.
Q.H. Li, CDER, FDA, Silver Spring, MD
Summary: Often treatment benefit is assessed by multiple endpoints so that a comprehensive picture of treatment effect can be obtained. Multiple endpoints can be different medical assessments angled at different aspects of a disease, or the same assessment made at multiple time points to monitor treatment progress over time.An important statistical topic is how to evaluate multiple end points collectively, by properly controlling the family wise type I error rate (FWER) and endpoint specific error rates without imposing overly stringent criteria. In this presentation, an approach of evaluating multiple endpoints collectively is explored by using test results from individual endpoints. Decision rules are developed to reject null hypotheses by properly controlling the FWER as well as the endpoint point specific error rates. Applications in clinical trials are presented.
T. Ng1 , P. He2 , J. Kim1 , P. Hshieh1 , 1CBER, FDA, Rockville, MD, 2CBER, FDA, Bethesda, MD
In-vivo and in-vitro studies are conducted in the evaluation of red blood cell (RBC) products. The acceptance criteria for these studies have been evolved over the past 15 years. Evolution of these acceptance criteria will be elaborated in this presentation. In particular, the current acceptance criteria will be presented. The related issues will be discussed. Sample size calculation and statistical power consideration will be given.
T. Ng1 , B. J. Poindexter2 , J. G. Vostal2 , P. He2 , 1CBER, FDA, Rockville, MD, 2CBER, FDA, Bethesda, MD
Leukocyte-reduced Whole Blood (WB) or Red Blood Cell (RBC) products are manufactured by filtration of the Whole Blood or RBC products using FDA approved/cleared filters. The current recommendation is that leukocyte-reduced WB or RBC products contain less than 5 x 106 leukocytes after filtration, and the post-filtration RBC mass be at least 85% of pre-filtration mass to ensure clinical efficacy. The post-filtration RBC in vitro recovery is defined as the ratio of the RBC mass after filtration over the RBC mass before filtration. Therefore, the latter recommendation is equivalent to the RBC recovery being at least 85%. Since hematocrits measures the percentage of RBC mass by weight, the RBC mass before and after filtration is typically calculated by multiplying the weight and hematocrits before and after filtration, respectively. Recently, the filter manufacturers and blood centers have proposed to change the method of calculating RBC post-filtration in vitro recovery to "weight only" method for quality control (QC) validation of leukocyte reduction filters. The arguments for and against such proposal will be presented.
T. Ng, CBER, FDA, Rockville, MD
The frequentist describes the data (or outcomes) conditioned on the unknown parameters while the Bayesian describes the parameters conditioned on the observed data. The two approaches are shown to be complementing each other in two applications, namely, (i) diagnostic test kit, and (ii) hypothesis testing.
The performance of a diagnostics test kit is evaluated based on the sensitivity and specificity, which describe the test results given the disease status of an individual. So, it is the frequentist approach. On the other hand, the positive predictive value and negative predictive value answers very practical questions given that the test result is positive and negative, respectively. So, it is the Bayesian approach.
In hypothesis testing, the frequentist approach controls the error rate of rejecting the null hypothesis given the null hypothesis is true, that is the traditional type I error rate. The Bayesian counterpart of the type I error rate is the false discovery rate, that is, the expected proportion of falsely rejected hypotheses as defined by Benjamini and Hochberg (1995). Recently, Ng (2006) argues that simultaneous testing for noninferiority and superiority increases the false discovery rate for superiority although the type I error rate is controlled.
T. Ng, CBER, FDA, Rockville, MD
It is well recognized that multiplicity adjustment is not necessary in simultaneous testing for noninferiority and superiority. However, Ng (2003; Journal of Biopharmaceutical Statistics, 13, 629-662) argued that there will be more experimental treatments that are expected to have the same effect as the active control tested for superiority in simultaneous testing than would occur if only one null hypothesis is tested, thereby increasing erroneous claims of superiority. The proportion of erroneous claims of superiority is analogous to false discovery rate. Therefore, simultaneous testing of noninferiority and superiority increases the false discovery rate for superiority.
F. W. Samuelson, N. Petrick, CDRH, FDA, Rockville, MD
The ability to statistically compare the performance of two computer detection (CD) or computer-aided detection (CAD) algorithms is fundamental for the development and evaluation of medical image analysis tools. Automated detection tools for medical imaging are commonly characterized using free-response receiver operating characteristic (FROC) methods. However, few statistical tools are currently available to estimate statistical significance when comparing two FROC performance curves. In this presentation, we propose a permutation and a bootstrap resampling method for the nonparametric estimation of statistical significance of performance metrics when comparing two FROC curves. We then provide an initial validation of the proposed methods using an area under the FROC performance metric and a realistic simulation model for creating CD algorithm scoring. Validation is based on a comparison of the Type I error rate produced by two statistically identical CD algorithms. The results of Monte Carlo trials show that both the permutation and bootstrap methods produced excellent estimates of the expected Type I error rate.
L. A. Sirota1 , B. G. Zaslavsky1 , P. M. Parreiras2 , L. D. Wagner1 , S. L. Menzies1 , J. L. Arciniega1 , 1CBER, FDA, Rockville MD 20852, 2NIQCH, Rio de Janeiro, RJ 21.040-900 - Brazil
Correlation analysis, analysis of covariance, and the Altman-Bland empirical method have been used to elucidate the relationship between antigen-specific ELISA and a lethal toxin neutralization assay (TNA) of anthrax vaccine potency measurements. Both assays measure the antibody response to Protective Antigen (PA) in mice immunized once with either Anthrax Vaccine Adsorbed (AVA) or an in-house recombinant PA vaccine (rPAV) at various doses.
Coefficients of correlation and linear regression equations for the two assays were found to be immunization dose dependent. ELISA and TNA results may not be strictly interchangeable for this animal model.Further experiments are needed to study the biological mechanisms underlying the relationships found between the assays and to understand the meaning and relevance of these results for vaccine-testing purposes.
Y. Tsong, CDER, FDA, Silver Spring, MD
Walter and Irwig (2001, SIM) proposed a definition of the number needed to treat (NNT) based on normally distributed response variables without dichotomizing the data and proposed an estimate using method of moment. The NNT index is defined by inverting the proportion of benefit of the test treatment adjusted for the reference treatment. However, the definition is defined in an asymmetric way. In this presentation, the symmetric definition is proposed. In addition, the moment method proposed by Walter and Irwig underestimates the variance of the estimate. It is illustrated with a comparison with the results derived from the bootstrap method.
J. Zhang, Y. Tsong, CDER, FDA, Silver Spring, MD
The designs and analyses for non-inferiority testing have been categorized into two types of approaches: the generalized historical control (e.g. non-inferiority margin) approach and the cross-trial comparison (e.g. synthetic) approach. Both approaches need to utilize historical data.For the traditional generalized historical control approach, information of the historical data is embedded in the predetermined non-inferiority margin. For the cross-trial comparison approach, on the other hand, the hypothesis is set up to test the mean difference or the ratio between the current test product and the active control is no larger than a certain portion of the mean difference or the ratio of the active control and placebo obtained in the historical data.The regulatory agency usually requires two independent trials to provide confirmatory evidence of the efficacy of the test product.However, when either one of the above two approaches is applied in non-inferiority studies, requirement of the independence is violated when the two clinical trials share the same historical information.In this research, we evaluate the extent of the dependency of the trials designed with the generalized historical control approach and cross-trial comparison approach. A comparison between the two approaches will be made withselected powers.
Y. Tsong, CDER, FDA, Silver Spring, MD
Sequential adaptive sample size re-estimation has been proposed for clinical trials in order to accommodate random reduction due to post randomization patient exclusion and imprecision of the parameter estimate used in sample size determination. In the case of analysis with pair-matched data with binary responses, the sample size requirement is a function of the target odds ratio and the probability of the discordant pairs with unexposed case and exposed control when using a conditional formula (Schlesselman, 1982). When using a unconditional formula, the sample size requirement is a function of the target odds ratio and the quadratic polynomial of the probability of the discordant pairs with unexposed case and exposed control (Connett, Smith and McHugh, SIM, 1987). With either formula, the sample size determination is sensitive to the precision of the estimate of the joint probability of exposure in both case and control group. In this presentation, we proposed the application of the adaptive sample size re-estimation approach with a group sequential design to improve the sample size determination.
Y. Tsong, M. Shen, CDER, FDA, Silver Spring, MD
In order to assess the equivalence of two treatments, clinical trials are designed to test the null hypothesis that the difference (or ratio) of two means (proportions) is either smaller than a pre-specified lower equivalence limit or larger than a pre-specified upper equivalence limit. For example, in generic drug evaluation, this approach is used for assessing average bioequivalence. However, the average equivalence type test is often criticized as lack of the ability to assess the exchangeability of the two treatments. In this presentation, we restate the statistical hypotheses in the form of stochastic inequalities. The stochastic statement can then be generalized to define the probability of exchangeability (i.e. coverage percentage) of the two treatments. The approach will be illustrated with a numeric example.
Z. Zhang, CDRH, FDA, Rockville, MD
There has been growing interest, when comparing an experimental treatment with an active control with respect to a binary outcome, in allowing the non-inferiority margin to depend on the unknown success rate in the control group. It does not seem universally recognized, however, that the statistical test should appropriately adjust for the uncertainty surrounding the non-inferiority margin. In this paper, we inspect a naive procedure that treats an "observed margin" as if it were fixed a priori, and point out why it might not be valid. We then derive a Wald test and a likelihood ratio test, both based on asymptotic approximations, for a smooth margin, and discuss their asymptotic behavior when applied to a piecewise smooth margin. Simulation experiments are conducted to evaluate the finite-sample performance of the proposed tests as compared to a naive test, for a smooth margin and for a piecewise linear margin.
B.A. Zhen, CBER, FDA, Rockville, MD
To release a biologics product into the market, sampling inspection is required in order to assure the quality of the product. Many organizations adopted the Military Standard Tables for the inspection. Military Standard 105D is a sampling scheme and combines several individual sampling plans in a system designed to employ economic, psychological, and operational means to motivate the producer to sustain acceptable quality levels. This presentation introduces the use of Military Standard and shows its limited use, especially when there is a higher defective rate than expected in lots to be released. A new procedure using the operating characteristic curves and predicting values is developed as an alternative approach to deal with the limited use of the Military Standard. Considerations of clinical impacts are also taken into accounts when using the new procedure. A real example is presented.
J. Zhang, CDER, FDA, White Oak
QT/QTc prolongation may cause ventricular arrhythmias including ventricular fibrillation and Torsade de Pointes, which can be fatal; therefore, the current ICH E14 Guidance requests all sponsors submitting new drug applications to conduct at least one thorough QT/QTc study, normally early in clinical development after some knowledge of pharmacokinetics of the drug. It is recommended that a double blinded, randomized, crossover or parallel QT/QTc study should be considered in healthy volunteers, in general, using the investigation drug, placebo, and a positive control.
The goal of a 'thorough QT/QTc study' is to test if the mean QT/QTc difference of the drug and placebo is no greater than 10 ms (non-inferiority margin). If yes, we might conclude that the drug does not have the QT prolongation effect. It has been demonstrated that the False Negative Probability (FNP, or the type I error rate) can be controlled under α (0.05) level if using the Intersection Union Test. It has been speculated that the FPP increases with the number of time points where QT/QTc has been examined. This speculation is not always true through simulation studies. The design and design related issues for a thorough QT/QTc study will also be discussed.
CATEGORY H: PROCESS ANALYTICAL TECHNOLOGY (PAT) AND PHARMACEUTICAL TECHNOLOGYM. Ashraf1 , R. Chatpalli1 , M. A. Mahmud1 , A. Nagi1 , L. Sivieri2 , 1Wyeth Research, Pearl River, NY, 2Wyeth Pharma, Collegeville, PA
Background: This study was designed to determine the blending end point for a low dose API when mixed in a tumble mixer.The API was present at 6.25% w/w in a directly compressible formulation.Two technologies were considered for this application (light induced fluorescence (LIF) and near infrared (NIR) spectroscopy). NIR was selected for further online evaluation based on preliminary laboratory studies. Methods: The blend samples of API were prepared at 0.4 - 6.25% w/w in a placebo matrix. The feasibility studies were performed with a Foss near-IR bench top spectrometer and a LIF spectrometer (Honeywell).The on-line evaluation of the blend homogeneity of API at 6.25% w/w in a tumble mixer was performed using a NIR process analyzer equipped with an Acousto-Optical Tunable Filter, a radio frequency transmitter, and a proximity sensor. The NIR monitor was installed on the lid of the mixer and rotated with the bin. NIR spectra were obtained through a sapphire window at each revolution when the bin was in the inverted position. The proximity sensor provided a trigger signal for collecting spectra at the end of each revolution. The NIR spectra were generated from 1100-2300 nm.
Results: The results from bench top spectrometers demonstrated that API does not produce sufficient fluorescence while NIR absorbance spectrum demonstrated adequate response. The first derivative spectra show only small variations. The spectra resulting from subtraction of the spectra obtained from placebo, elicited signals arising from API. This proves that the NIR analyzer could detect the active during the blending of the product. The small deviations in the spectra between successive revolutions showed that the blends were quite uniform by the time they were monitored by NIR.
Conclusion: NIR can be used to detect the API. The online NIR analyzer is sensitive to monitor the 6.625 % active during blending using chemometrics analysis.
S. Bhansali, J. Liang, S. S. Deshmukh, Wyeth Research
Process Analytical technology (PAT) can be used to assist with determining process robustness. Specifically, in-line analytical tools provide the advantage of non-perturbational analysis of a process regardless of the scale of operation. We show in-line monitoring of an Active Pharmaceutical Ingredient (API) crystallization process to be useful in identifying key process parameters responsible for variations in the particle morphology and particle size distribution that occurred during scale-up. Focused Beam Reflectance Measurement (FBRM) was used to study the effects of process variables such as seeding and temperature cycling on the particle morphology and crystallization kinetics. The presentation will focus on potential improvements in processing that resulted from improved understanding derived from PAT data.
W. Chew1 , W. McMahon2 , C. C. Shaw1 , 1Preclinical Development, Wyeth Research, Montreal, Canada, 2Preclinical Development, Wyeth Research, Pearl River, NY
Reaction monitoring is of primary interest to chemists working in chemical process development. Information about a reaction system can be achieved in real time, allowing optimization and QbD development of chemical processes. On-line analytics is useful for determination of reaction mechanisms, reaction engineering, and process monitoring. The online ReactIR™ has been shown to be a useful spectroscopic method for the monitoring the stability and formation of unstable anhydride involved in the synthesis of the temsirolimus boronate intermediate. The mixed anhydride was found to be stable for up to 48 hr at - 5oC. The mixed anhydride formation is > 90-95% complete when it is aged for 8-10 hr at - 8 oC. This presentation highlights our efforts at using Process Analytical Technology (PAT) for optimization of chemical processes and process control towards the commercial manufacture of CCI-779 (temsirolimus).
S. S. Deshmukh1 , M. Mirmehrabi2 , 1Wyeth Research, 2Wyeth Canada
The use of in-line and at-line analysis technologies affords benefits for Active Pharmaceutical Ingredient (API) process development. The ability to analyze the process stream using spectroscopic, laser and optical technologies may provide an advantage for understanding the complex transformations occurring in a unit operation compared to traditional techniques such as HPLC. This process understanding can be used to identify critical variables and improve the design/throughput to obtain better quality and yield. In this poster presentation the use of Laser Light Scattering (LLS) for determination of crystallization induction and the use of Differential Scanning Calorometry (DSC) to quantitate crystallinity will be discussed. The utility of these techniques for the API manufacturing process and quality testing will also be discussed.B. J. Ennis1 , C. D. Ellison2 , M. L. Hamad2 , A. S. Carlin2 , E. H. Jefferson2 , R. C. Lyon2 , 1E&G Associates, Nashville, TN 37215, 2DPQR, CDER, FDA, Silver Spring, MD, 20993
Background
Near-infrared (NIR) spectroscopy is becoming a common technique to assess blend uniformity. However, impact of segregation on such measurements is less understood. Segregation interferes with representative sampling by sensor technologies, favoring constituents that settle closest to the detector. This study examines the impact of segregation on NIR measurements and the application of NIR and particle sizing measurements to process segregation.
Method
Poppy seed-salt mixtures were chosen as model blends. Blend ratios were scanned using NIR chemical imaging. Both thin and mono-layers were imaged in the blended and unblended state. These studies were used to determine a reliable method to analyze hopper samples. Discharge samples versus time were analyzed by transmission NIR, laser diffraction, and particle imaging.
Results
Good agreement for blend ratio was achieved between unmixed and mixed monolayer samples using a PCR model based on pure components. However, measurements on thin layers were biased to poppy seeds and exhibited large spatial variations. NIR transmission was chosen to minimize the effect of spatial variations in measuring hopper discharge samples. These hopper discharge results compared favorably with independent measurements by laser diffraction and particle imaging. The optimum angle calculated to minimize blend segregation (based on shear cell measurements) compared favorably with discharge profiles.
Conclusions
NIR and other spectroscopic techniques are prone to bias in the presence of sample segregation, and this can be a vital sampling consideration. In this case, analyzing a complete discharge sample with repetitive mixing by NIR transmission improves the likelihood of exposure of all blend components.
B. J. Ennis1 , C. D. Ellison2 , M. L. Hamad2 , E. H. Jefferson2 , R. C. Lyon2 , 1E&G Associates, Nashville, TN 37215, 2DPQR, CDER, FDA, Silver Spring, MD
Background
In pharmaceutical processing, magnesium stearate (MgS) affects powder flow properties and compaction efficiency based on blend time and amount of MgS used. The current work models effects of MgS on product quality by relating powder and wall frictional properties to tablet stress transmission ratio. Such process understanding is critical to pharmaceutical development.
Methods
Lactose monohydrate was blended with 0.25% or 1.0% MgS for 30 sec or 30 min. Powder and wall friction for the blends were measured by rotational shear cell. Compacts were prepared of each blend, with compaction forces monitored by load cells. Powder and wall friction determine the wall shear stress acting against the applied pressure. The resulting stress transmission ratio was calculated from Janssen's relation, and experimentally from punch forces. Chemical images were collected for each tablet to assess density variations.
Results
Compact uniformity correlated with wall friction measurements of the blends, as well as calculated and experimental stress transmission ratio. Average NIR absorption correlated reasonably well with tablet density. Uniformity of intra-tablet density was dependent upon MgS concentration: tablets with less MgS were less uniform. Intra-tablet variations in NIR absorption, reflective of density variations, correlated with stress transmission.
Conclusions
Compact density may influence tablet performance. Improved stress transmission results in lower required tablet pressure in practice. Thus consistent tablet density profiles, as controlled by stress transmission, are important for predictable performance. Stress transmission can be predicted from wall and powder friction measurements. Chemical imaging techniques can be used to nondestructively assess density profiles of tablets.
M. Farris1 , A. McDaniel2 , 1Wyeth Research, 2Wyeth Biopharma
New microbiological techniques are constantly being developed to improve process efficiency and drive down the time and cost of drug development. One such example is the Chemscan (ScanRDI™) from Chemunex. The ScanRDI™ uses fluorescent labels and rapid laser scanning techniques to detect viable cells in liquid samples without the need to be incubated to derive visible colonies.
The ScanRDI™ has been validated for monitoring water samples for parenteral drug manufacturing. Rapid laser scanning of the membrane gives results in minutes, which can then be verified by microscopy, and a result delivered in less than 2 hours from start to finish, thereby giving day of use results during drug manufacturing. Concurrent testing with the ScanRDI™ showed greater sensitivity of the system compared to the traditional water testing methods.
TheScanRDI™ is also being validated for the rapid detection ofbacterial contamination in mammalian cell culture systems. Validation of this assay has included several phases, including a period of concurrent testing in which the ScanRDI™ assay was performed side by side with traditional testing. Results from the concurrent testing were that 93% of the samples matched (ScanRDI™ and Membrane Filtration results agreed), 6% of the samples did not match (ScanRDI™ results were "false" positives, while Membrane Filtration was negative - addressed by raising the baseline of the assay), and for 1% of the samples the ScanRDI™ was unable to obtain a result.
F. R. Doucet1 , L. St-Onge1 , M. Tourigny2 , M. Sabsabi1 , R. C. Lyon3 , P. J. Faustino3 , 1National Research Council of Canada, Boucherville, Canada, 2Pharma Laser, Boucherville, Canada, 3Division of Product Quality Research, Office of Pharmaceutical Science, CDER, FDA
Background: LIBS, an atomic spectroscopy technique, analyses the elemental content of gaseous plasma discharged from the sample as a result of laser ablation. The study purpose is to evaluate the potential of LIBS to monitor the influence of manufacturing changes and formulation excipients on solid dosage forms of furosemide and assess the utility of chemometrics to determine API and excipient concentrations.
Methods: A LIBS spectrometer equipped with a pulsed Nd:YAG laser was utilized to produce the gaseous plasma at the tablet surface, without sample preparation. Calibration sets prepared under cGMP conditions differing in their avicel/lactose (0-100%) and magnesium stearate excipient compositions were used to monitor the LIBS furosemide signal intensity (e.g. Cl or S). Test sets (50/50 avicel/lactose) were prepared to monitor excipient changes during manufacturing. A chemometrics training set from broadband spectrum (e.g. CN, CH, C2) was developed. Chemometrics was used to evaluate multivariate spectra of the formulation excipients.
Results: The avicel/lactose excipient ratio significantly affected the furosemide LIBS signal. The calibration set containing lactose, had 70% of the furosemide signal intensity of a calibration set containing only avicel. Test formulation sets of 50/50 avicel/lactose were accurately predicted. In addition, chemometrics accurately determined furosemide or magnesium stearate content.
Conclusions: The impact of excipient changes on the LIBS signal can provide a novel approach to monitoring major or minor compositional or manufacturing changes. Chemometrics may assist with the accurate prediction of API and excipient content in drug product formulations demonstrating the potential of LIBS for PAT monitoring of drug products.
M. K. Ghorab, R. Chatpalli, S. Hasan, A. Nagi, Solids-Pharmaceutical R&D, Chemical and Pharmaceutical Development, Wyeth Research, Pearl River, NY 10965
Background: Thermal effusivity of a material is a physical property that is dependent on the material thermal conductivity, heat capacity and density.
Methods: Applicability of using thermal effusivity in monitoring roller compaction was evaluated by examining the relationships between effusivity and physical properties of ribbons of Avicel PH 101, anhydrous lactose, and Placebo (PBO) formulations, containing various ratios of Avicel PH 101 to anhydrous and compacted at different pressures.
Results: Correlation was established between thermal effusivity and compaction pressure of ribbons of Avicel and PBO formulation, which was best described by a 2nd order polynomial function. However, for lactose the relationship was linear.Square root of solid fraction-compaction pressure profile was similar to that of effusivity-compaction pressure and therefore, a correlation was established between effusivity and square root of solid fraction. Effusivity vs. tensile strength profiles for Avicel and all the PBO formulations ribbons were best described by 2nd order polynomial functions.However, for lactose, the profile was linear but only to a particular tensile strength corresponding to a compaction pressure of 65 bars beyond which no increase in tensile strength occurred even though effusivity continued to increase.A log-linear profile existed between the Young's Modulus and effusivity of Avicel and PBO ribbons.
Conclusions:Correlations between ribbons' effusivity and physical properties could be established but are formulation dependent.However, development of models that describe these relationships could be achieved and utilized to provide a real-time feedback about ribbons' characteristics during roller compaction.
W. Chew, G. Glasier, E. Fournier, L. Karim, Wyeth Research, Montreal, Canada
Process Analytical Technology is an FDA supported initiative that will help pharmaceutical manufacturers develop and implement new efficient tools and technologies for use in development, manufacturing, and quality assurance while maintaining or improving the current level of product quality assurance. This poster highlights our efforts in the development and implementation of process mass spectrometry to determine residual solvent drying endpoint. During the development of a synthetic process for making active pharmaceutical ingredients, many solvents are utilized. Vacuum drying is routinely used in pilot and manufacturing plants for drying of pharmaceutical intermediates and active pharmaceutical ingredients. Typically, the endpoint is determined by estimation, lowering the dryer temperature, bringing the dryer to atmospheric pressure, removing a representative sample, and performing an analysis in the laboratory to test for dryness. Literature precedence has demonstrated mass spectrometry as a simple, selective and sensitive tool for multi-component gas phase analysis of volatile compounds. In this study on-line process mass spectrometry was compared to off-line Karl Fischer residual moisture analysis and solvent analysis by gas chromatography. Process mass spectrometry has been illustrated as a fast and effective tool for establishing dryer endpoint detection on laboratory scale batches without the need to perform lengthy operations. The system saves time, reduces the analyst's exposure to API and improves process control.
C. Guo, W. H. Doub, DPA, CDER, FDA, St. Louis, MO
Background: Raman microscopic imaging is a powerful tool for differentiating formulation components and, potentially, chemical quantitation. It is a possible candidate to replace cascade impaction as a tool to determine chemical identity and particle size distribution of inhalation drugs. Methods: A commercially available combination therapy MDI, Combivent®, was used as a test product in this study. Raman images were obtained using a global scan (wide-field) Raman imaging system, Falcon II (ChemImage Corp., Pittsburg, PA), with 532 nm laser excitation. The spectral region from 960 to 1040 cm-1 is used for Raman imaging study, and the peaks at 985 and 1014 cm-1 are used to monitor albuterol sulfate and ipratropium bromide, respectively.Results: The Combivent® MDI challenges both the spatial and spectral resolution limits of Raman imaging as both APIs are weak Raman scatterers and the particle size approaches the spatial resolution (about 250 nm). Raman chemical imaging is capable of determining the chemical identities of the API particles as small as 1 µm. However, it is difficult to obtain accurate size and shape information for particles smaller than 5 µm using the Raman image alone. Conclusions: This study provides evidence that the Raman imaging technique is useful for API particle size determination for a combination ingredient MDI. Future studies will incorporate Raman image / bright-field image fusion plus additional image processing techniques as needed to facilitate the accurate particle size and shape determination.
M. L. Hamad, C. D. Ellison, A. S. Carlin, E. H. Jefferson, M. A. Khan, R. C. Lyon, DPQR, CDER, FDA, Silver Spring, MD
Background: Spectroscopic and imaging techniques for chemical and physical analysis of pharmaceutical products and processes offer significant advantages over traditional analytical methods. However, representative sampling of heterogeneous pharmaceutical materials and products during process monitoring is challenging. It is necessary to determine the appropriate spot size to measure a representative sample. Intuitively, an image is homogeneous at a given resolution if most pixels are representative of the whole. This study explores algorithms that produce quantitative descriptions of heterogeneity yielding better sampling.
Methods:Near-infrared chemical images were acquired of acetaminophen tablets of varying strengths ranging from 60-120 mg. Chemometrics was applied to each NIR image to create a concentration image for acetaminophen.Images were 256x320 pixels. "Macropixels" were constructed by binning together neighboring pixels, representing sub-samples of the original image.Histograms were used to express the distributions of pixel and macropixel intensity values. Different metrics were evaluated for their ability to characterize image homogeneity.
Results: Digital images were analyzed using histogram statistics and co-occurrence matrix analysis. In all cases, homogeneity improved as the macropixel size increased. The minimum appropriate spot size for representative sampling was predicted.
Conclusions: Algorithms for quantitatively evaluating the level of heterogeneity in chemical images were evaluated. These algorithms will serve as useful tools when examining the spatial characteristics of drug products and pharmaceutical blends with chemical imaging. This method of analysis can also be used to determine the sampling requirements of heterogeneous materials undergoing analysis with non-spectroscopic techniques.
M. Durette1 , S. Kislalioglu2 , M. L. Hamad3 , A. NguyenPho3 , M. A. Khan3 , 1DPQR, CDER, FDA, Silver Spring, MD & Department of Pharmacy, University of Rhode Island, Kingston, RI, 2Department of Pharmacy, University of Rhode Island, Kingston, RI, 3DPQR, CDER, FDA, Silver Spring, MD
Background: The purpose of this study was to develop an innovative method for the quantitative assessment of gabapentin in aqueous solution using partial least square regression (PLS) as a multivariate tool for data analysis. Near-infrared spectroscopy (NIRS) was employed as an analytical tool because it is fast, non-destructive, reproducible, and provides chemical and physical information for virtually any sample matrix.
Methods: A calibration sample set composed of aqueous solutions of gabapentin yielding concentrations of 1.333%, 2.667%, 4.0%, 5.333%, and 6.667% (w/v) was prepared. Data collection was carried out using an AOTF (acousto-optic tunable filter) NIR spectrometer equipped with a transflectance handheld dip probe. The quantitative models were computed using partial least squares (PLS) regression.
Results: Correlation coefficients of 0.9946 and 0.9925 were obtained for the calibration and prediction of gabapentin concentration in aqueous solution, respectively. For estimating the precision of the method to predict the gabapentin concentration, SEC and SEP values of 0.194 and 0.235 were obtained, respectively. To compare the efficiency of the PLS model with traditional data analysis techniques, univariate linear regression was used at wavelengths of 1396, 1726, and 1892nm. Correlation coefficients of 0.9891, 0.9905, and 0.9817 were obtained for the regression at 1396, 1726, and 1892nm, respectively.
Conclusions: In conclusion, the current method proved the feasibility of using a NIR transflectance probe for the accurate quantitation of drug in aqueous solution. Moreover, this method can be applied to complex sample matrices, such as slurries and liquid dosage forms.
S. Hassannejad Tabasi1 , R. M. Fahmy2 , D. M. Bensley2 , W. G. Marnane2 , S. W. Hoag1 , 1School of Pharmacy, University of Maryland, Baltimore, MD 21201, 2Office of New Animal Drug Evaluation, FDA, Rockville, MD 20855
Background: NIR spectroscopy is an important toolbox for quality-by-design, which can be utilized to assess physical and chemical properties of similar materials. This study examines the monitoring of sustained release coating process.
Methods: Sustained release formulations for orbifloxacin were developed using varying ratios of Eudragit RL and RS 30-D. Tablets were coated in pan coater to a target thickness of 70-80ìm, and the coat thickness was monitored using NIR spectroscopy. A total of 939 tablets from 10 batches were used to build best-fit calibrations to predict thickness and release profile. Different PLS calibrations were developed and their statistical parameters such as standard error of calibration (SEC), cross validation (SECV), prediction (SEP) and coefficient of correlation were compared.
Results: Three PLS models for coating thickness of Eudragit RL, RL:RS (2:3) and RL:RS (1:4) had SECs of 3.19, 2.97, 2.84µm and SEPs of 2.92, 2.58, 3.03µm, respectively. The combined calibration yielded a SEC and SEP of 2.91 and 3.05µm. To assess the release, seven different ratios of RL/RS were made and the release after 2hrs in acetate buffer (0.1 M, pH 3.95 ±0.05) was used to build a six factor PLS regression. The PLS regression provided SEC, SEP and correlation coefficient of 2.66%, 2.47%, and 0.9948, respectively.
Conclusion: NIR spectroscopy combined with multivariate calibration is a powerful tool for predicting drug release from blends of Eudragit. In addition, due to the similarities in the structure of Eudragit RL/RS, one properly developed model would be able to predict coat thickness from the blend with satisfactory SEP.
E. H. Jefferson, M. L. Hamad, Y. S. Yang, R. B. Shah, A. S. Carlin, R. C. Lyon, P. J. Faustino, DPQR, CDER, FDA, Silver Spring, MD
Background: Prussian blue (PB) is the active pharmaceutical ingredient (API) of an FDA approved drug product (DP) for the treatment of internal radioactive contamination of cesium or thallium metal isotopes. An experimental design was created to establish two scientific endpoints, a spectroscopic link between water content and stability and second, a spectroscopic link between water content, molecular changes and binding (capacity) efficacy.
Methods: Near-Infrared Reflectance Spectroscopy (NIRS) was developed as a tool for screening the physical and chemical properties of Prussian Blue (PB) active pharmaceutical ingredient (API) and drug product (DP). The activity of PB, based on cesium binding capacity, is dependent on moisture content. Hypothetically, PB activity can be predicted by noninvasive monitoring of moisture content by NIRS. Several API and DP lots of PB were analyzed by NIRS. Cesium binding capacity was determined by Atomic Emission Spectroscopy (AES) and moisture content was determined by Thermo Gravimetric Analysis (TGA). Partial Least Squares (PLS) methods were used to develop chemometric regression models to correlate NIRS data with Cesium binding capacity and moisture content.
Results: PLS analyses yielded correlation coefficients for NIR spectra and TGA moisture content (calibration R2= 0.99 and validation R2=0.98) and for NIR spectra and cesium binding capacity (calibration R2= 0.98 and validation R2=0.97).
Conclusions: This study demonstrates that NIR spectral data can be used to predict the activity and moisture level of PB drug substance and drug product.
S. Singh, M. Krishnan, A. Nagi, Wyeth Research, Pearl River, NY
Background. Monitoring on-line particle size distribution from roller compacted ribbons is essential to ensure manufacture of granules with optimum compressibility.
Methods. Initial powder blends were roller compacted and the ribbons milled through a two-stage process using the coarse and fine granulators within the roller compactor. A Malvern Insitec T device was installed on-line post milling to monitor real-time particle size distribution during the roller compaction process.
Results. Formulations where particle size may not have an impact on drug dissolution, a controlled granular particle size distribution is desirable to overcome processing issues such as flow and segregation. In an effort to enhance process understanding and inter batch consistency, granular particle size distribution was closely monitored post roller compaction.Roller compaction was performed in an automated roll-gap control mode at constant milling speed.Particle size distribution obtained correlated well at various roll gaps (3.0-3.8 mm). Increasing roll gap resulted in decreased particle size and this relationship was linear. The particle size (D10) ranged from ~10-28µ in diameter while D50 and D90 ranged from ~100-300µ and ~600-900µ, respectively.Ribbon porosity ranged from ~15% at a roll gap of 3.0 mm to ~19% at 3.8 mm.
Conclusions. The on-line particle size data obtained correlated well with the porosity of the roller compacted ribbons as well as the granulation flow properties. Transient states including process start-up, changeover, and shutdowns were successfully mapped and monitored. Also, the online tool was able to identify utility upset conditions such as granulator clogs and material plugging/bridging phenomena.
R. C. Lyon1 , S. Hammond2 , 1DPQR, CDER, FDA, Silver Spring, MD, 2Pfizer, Inc,, Peapack, NJ
Background: The goal of this joint FDA-Pfizer project was to develop a Quality by Design case study using a pilot-scale pharmaceutical manufacturing approach. This included the development of a design space and process monitoring by process analytical technology (PAT) tools. These tools included vibrational spectroscopy and chemical imaging techniques to predict product performance and provide quality assurance.
Methods: Extensive blending of phenytoin sodium with excipients was monitored by on-line near-infrared (NIR) spectroscopy and at-line chemical imaging techniques. The initial protocol was designed to test the instrument measurement capabilities and to correlate spectral data to product performance (capsule dissolution). Process variables include blending time, API particle size and I-bar speed.
Results: Performance results indicate that capsule dissolution increases with API milling and decreases with I-bar speed and blending time. A design space related to these process variables was established using response surface methodology. Monitoring the process by NIR spectroscopy detected multiple blending processes: rapid (chemical mixing) and slow (physical changes). Determination of a proper blending endpoint in real time was related to the spectral data.
Conclusions: Since pilot-scale manufacturing facilities are not available at the FDA, this collaboration provides the Agency with an opportunity to conduct on-line research related to the implementation and validation of PAT tools. It was possible to establish predictive relationships between processing and the final product quality, which could be used for real-time process control.R. B. Shah1 , M. A. Tawakkul1 , H. R. Prasanna1 , M. White2 , M. A. Khan1 , 1DPQR, CDER, FDA, Silver spring, MD, 2Sherwood High school, Sandy spring, MD
Purpose. The purpose of the present study was to develop a correlation between measured amounts, hardness, and porosity with prediction models employed using the rapid non-destructive near infrared (NIR) and Raman spectroscopic methods.
Methods. Tablets containing metoprolol tartrate (50, 100, 200, and 300 mg) with Eudragit-L100 (50 mg), talc and magnesium stearate were prepared on semiautomatic tablet press. The tablets containing 200 mg of the API were also compressed at different compression pressures. NIR, NIR chemical imaging, and Raman spectra were taken for all the tablets. Hardness was measured using destructive diametric crushing test. True density was measured using helium pycnometer and the porosity values were calculated. The dissolution studies were performed in USP II apparatus.
Results. Principal component analysis was used to generate a partial least square model. The model correlated NIR data with the amount, hardness, and porosity. Various preprocessing methods were evaluated for the data set to generate the best fit model. The model showed the correlation coefficient (r) of 0.974, 0.97, and 0.90 for the predicted versus measured amount, hardness, and porosity at different compression pressures. The dissolution at 10 minutes was also correlated with NIR data for tablets. It showed the correlation coefficient of about 0.89 and 0.70 for various calibration and validation set, respectively. Raman spectra correlated porosity of the tablets (r= 0.93).
Conclusions. NIR is potentially a useful PAT tool to predict hardness, porosity and amount of metoprolol tartarate tablets. However prediction of dissolution warrants the accountability of variability and mechanism of dissolution.
J. A. Spencer, R. E. Kolinski, Div. Pharmaceutical Analysis
Background: A science-based classification of various topical preparations (lotions, creams, ointments, etc.) has been developed [International Journal of Pharmaceutics 295 (2005) 101-112]. The viscosity of these materials is an important property for distinguishing liquids from semi-solids. Depending on the nature of the fluid, traditionally viscosities have been measured with mechanical or flow-through devices. Most viscometers use large quantities of material, are slow and can be difficult to clean. Further, some materials of high viscosity require specialized equipment. This work was undertaken to determine whether high frequency acoustic attenuation can be used with topicals exhibiting a wide range of viscosities.
Method: A solid state device was used to measure acoustic viscosities (AV) at 160 MHz. High frequency produces a high-shear environment where non-Newtonian liquids exhibit shear dependent viscosity. Samples as small as 100µl are placed directly on the sensor surface (~4cm2). AV and temperature were measured simultaneously in real-time. The active surface was monitored for cleanliness between samples. Reference glycerol-water mixtures were used for performance verification.
Results: Replicate AV values were measured for 63 topical preparations for which conventional viscosity had been previously determined. The AV values achieved steady state in 0.5-5 minutes depending on the sample. Numerical values for AVs ranged from 50 - 850 (AV Units) for topicals having low frequency viscosities in the range 2700 - 730000cP. %RSD values average less than 3%. Measurements of the reference mixtures showed daily RSD values of less than 2%. >
Conclusion: Many AVs correlated with the conventional viscosity measurements (low frequency - low shear) but some showed large non-ideal variations. The acoustic viscometer was found to be fast, easy to clean and required no special procedures on going from low to high viscosity samples. Since these devices are equally usable at all stages of scale-up they are particularly suitable for PAT applications.
H. Wu1 , E. J. Heilweil2 , A. S. Hussain3 , M. A. Khan1 , 1Division of Product Quality Research, OTR/OPS/CDER, FDA, Silver Spring, MD, 2Optical Technology Division, Physics Laboratory, NIST, Gaithersburg, MD, 3Global Biopharmaceutical Development, Sandoz, Princeton, NJ
Background: Identifying measurement process variability and optimizing measurement protocol are critical for Terahertz analysis of pharmaceuticals.
Methods: For THz spectral analysis, either tablet components (drug and excipients) or crushed theophylline tablets were mixed with polyethylene (PE) powder (~10 wt%), then compressed into THz transparent disks of thickness 2 mm in a 13 mm die at approximately 200 psi pressure. Prior to acquiring THz spectra, each disk was dried in the air-purged FTIR instrument (Nicolet Magna 550 with THz Si-coated beamsplitter and DTGS detector) for various times to remove moisture from the instrument. THz spectra were recorded at 4 cm-1 spectral resolution and 64 spectral averages. THz absorption spectra were corrected for PE matrix by ratioing against a standard pure PE pellet spectrum.
Results: Lower PE loading yields spectra of smoother baselines. Characteristic THz absorption peaks exist for crystalline materials, but not for amorphous components. When disc drying time increased from 1 min to 1hr, the wave-number shifts at 301 cm-1 are 30, 6, 50, 17, 29, and 58 cm-1for PE, theophylline, lactose, MCC, starch, and MgS, respectively; significant changes were observed for both the tablet THz measurement raw data and the tablet optical density data obtained from ratioing method.
Conclusions: Screening experiments yield optimal THz measurement process conditions: ca. 10 wt% PE loading and 1 hr disc drying time based on considerations from THz spectrum baseline shift, sample absorption, and PE scattering.H. Wu1 , E. J. Heilweil2 , A. S. Hussain3 , M. A. Khan1 , 1Division of Product Quality Research, OTR/OPS/CDER, FDA, Silver Spring, MD, 2Optical Technology Division, Physics Laboratory, NIST, Gaithersburg, MD, 3Global Biopharmaceutical Development, Sandoz, Princeton, NJ
Background: Quantification study using THz spectroscopy for determining pharmaceutical tablet compositions is novel for THz pharmaceutical PAT applications.
Methods:Crushed theophylline tablets were mixed with polyethylene (PE) powder (~10 wt%), then compressed into THz transparent disks of thickness 2 mm in a 13 mm die at approximately 200 psi pressure. Prior to acquiring THz spectra, each disk was dried in the air-purged FTIR instrument (Nicolet Magna 550 with THz Si-coated beamsplitter and DTGS detector) for 1hr to remove moisture from the instrument. THz spectra were recorded at 4 cm-1 spectral resolution and 64 spectral averages. THz absorption spectra were corrected for PE matrix by ratioing against a standard pure PE pellet spectrum. Characteristic peak method, linear superposition method, and multivariate data analysis method were employed to correlate the THz spectra to the compositions of tablet constituents.
Results: Characteristic peak method works better for crystalline components than for amorphous ones. The best R2 values obtained for theophylline, lactose, MCC, MgS, and starch are 0.55, 0.73, 0.86, 0.72, and 0.45, respectively. Linear superposition method provides indication that there are possible interactions between tablet components. Both PCR and PLS1 generate excellent correlations: the R2 values are 0.96, 0.96, 0.97, and 0.23 for theophylline, lactose, MCC, and starch, respectively. MgS concentration can not be extracted via THz spectra directly due to low amounts present.
Conclusions: Theophylline tablet THz spectra were analyzed using characteristic peak method, linear superposition method, and multivariate data analysis methods. The best quantification was achieved using PCR and PLS1 except starch.H. Wu, M. A. Khan, R. C. Lyon, Division of Product Quality Research, OTR/OPS/CDER, FDA, Silver Spring, MD
Background: Multivariate modeling is a critical tool which enables us to link pharmaceutical formulation parameters, manufacturing processing variables, and product performance attributes for pharmaceutical PAT applications. However, there remain regulatory and engineering challenges in the area of multivariate modeling.
Methods: Theophylline tablets were manufactured to produce a range of dissolution characteristics as per predefined testing protocol (both direct compression and wet granulation). Dissolution studies were conducted using USP II apparatus. Near-IR reflectance spectra were collected on FOSS/NIRSystems 5000 prior to the dissolution testing. Analyses by principal component regression (PCR) and partial least-squares regression (PLS) were used to correlate the NIR spectra with the dissolution data.
Results: For all of the tablets, good correlation between the tablet NIR spectra and tablet dissolution data were established by both PCR and PLS, with reasonable prediction deviation (not exceeding 10% in most cases), over the entire course of tablet dissolution process. Generally, principal component selection for multivariate modeling is based on statistical criteria. However, the stability, accuracy, and quality of the prediction model must be balanced with reliability considerations and process control practice.
Conclusions: Chemometric regression analysis was able to correlate tablet spectra data with product performance. The number of principal components used for the prediction model should be selected from a combination of statistical criteria and process consideration.
L. Xie1 , H. Wu2 , L. L. Augsburger1 , M. A. Khan2 , A. S. Hussain3 , S. W. Hoag1 , 1School of Pharmacy, University of Maryland, 20 N. Pine Street, Baltimore, MD 21201, 2Division of Product Quality Research (DPQR, HFD-940), Office of Testing and Research, Office of Pharmaceutical Science, CDER, FDA, Silver Spring, MD 20993, 3Global Biopharmaceutical Development, Sandoz, Princeton, NJ 08540
Objectives: This study investigates the relationship between formulation parameters, process parameters and their effects on capsule weight variation.
Methods: Using a fractional factorial design, 24 capsule batches were made incorporating selected formulation and process parameters in the design. Capsule formulations consisted of 4% aspirin (fine or coarse particle size) and 96% microcrystalline cellulose (PH-301 or PH-200). The batches were filled using either a Zanasi LZ-64 (dosator) machine or a Harro-Hofliger KFM/3 (dosing disc) machine. Segregation propensity was measured using the ASTM D 6940 segregation test method. Particle size, flowability, and bulk and tapped densities were measured follow standard methods.Coefficient of variation (CV) for weight was determined for (1) variation within different intervals sampled (CVi), (2) the variation between intervals (CVj), and (3) all the intervals combined along the manufacturing time (CVt). Statistical models of these weight variation indices were established.
Results: For average fill weight, the best multiple linear regression model was
Wavg = 141.3768281+54.17663621*LL*100-51.2275*CS + 2.946800732*CI (R2 = 0.83).
Where LL is lubricant level, CS is capsule size and CI is Carr's index of formulation. Machine type significantly affects all weight variation indices; whereas, angle of friction and drug-to-excipient particle size ratio significantly affect CVi.
Conclusions: Based on the regression analysis, machine type seems to be the most important parameter for filling weight variation among the parameters studied. This model approach can be used to predict weight variation problems prior to capsule filling and provides guidance to formulators for reducing variation by adjusting certain filling or formulation parameters.
J. Liang, Y. Xu, Y. Chen, J. Huang, W. Dulin, M. Tesconi, S. Ku, Wyeth Research
This presentation provided examples where conventional and environmental scanning electron microscopy (SEM/ESEM) contributes significantly in the drug discovery, and drug development process. The advent of Environmental Scanning Electron Microscopy (ESEM) has brought about new opportunities to utilize this technique to characterize APIs. It also presents a general overview of how the ESEM, in conjunction with other various analytical techniques, functions in Pharmaceutical Development. The examples show that the SEM, especially ESEM is a valuable tool for studying APIs, excipient, and formulations during pharmaceutical development. Water was used as the ionizing gas in these experiments, and changes in crystal morphology were correlated with relative humidity (RH). The instrument can easily image non-conductive powers without the use of conductive coatings, more importantly, the ESEM can be used to study the microscopic behavior of hydrated APIs, excipient, and formulations in response to wide range of wet (25-100%RH), or high temperature conditions (up to1000°C), and potentially, to identify unstable crystal compounds. Particles were examined and photographed during hydration and dehydration steps. Recently, the SEM/ESEM, EDX and microanalysis have been widely used as an indispensable instrument for the identification and classification of the pharmaceutical materials and for the observation and measurement of their microstructures. When SEM/ESEM/EDX is used as a PAT in a multi disciplinary approach, it becomes a powerful tool for the process development of pharmaceutical materials.
CATEGORY I: RISK MANAGEMENT, RISK ASSESSMENT, AND RISK COMMUNICATION FOR MEDICAL PRODUCTS AND FOODS
K. B. Arvidson1 , R. D. Benz2 , E. J. Matthews2 , J. Mayer1 , E. Lee1 , M. L. Twaroski1 , C. Yang3 , 1CFSAN/OFAS, 2CDER/OPS/ICSAS, 3Leadscope, Inc.
The ability to accurately predict toxicity using quantitative structure activity relationships (QSAR) is becoming more important as regulators worldwide incorporate QSAR-based evaluations for hazard identification and risk assessment in safety assessments. Several practical and fundamental challenges must be overcome in building reliable QSAR models, one being access to high-quality data from which accurate predictive models can be derived. In this regard, FDA has been actively collaborating with the public toxicity database standardization effort, ToxML, to create a set of controlled vocabulary to represent toxicity data. Through a cooperative research and development agreement involving the Center for Food Safety and Applied Nutrition and the Center for Drug Evaluation and Research, FDA scientists now have access to data in consolidated databases which can be more easily incorporated into the review process. These databases will greatly expand safety analysis by utilizing analog information and improve efficiency by electronic retrieval. Currently, studies are added to these databases in a separate process following the safety evaluation of the submission. This method is problematic in that it duplicates effort and creates a lag-time between completion of the review and addition of the data to the QSAR training set. In an effort to resolve this issue, FDA scientists are exploring reviewing submissions with real-time data entry in an effort to expand these databases. This poster describes the newly consolidated databases and a vision for real-time data entry. In addition to improving safety evaluations at the FDA, these databases will increase the global knowledge base and expand the scope of predictive toxicology as non-proprietary portions of these databases become available to stakeholders.S. A. Assimon, P. M. Bolger, CFSAN, FDA, College Park , MD
Egg allergy is the most common food allergy in the US, particularly in infants and young children. However, this sensitivity can emerge at any age. For those afflicted with egg allergy, the hazards of exposure to egg can be serious. The allergic response involves symptoms associated with a number of physiological systems and can include anaphylaxis. Reactions develop within minutes to several hours or more after exposure. Avoidance of egg is the principal tool for management of adverse effects. Thus, the determination of the tolerable intake level of egg protein in sensitized individuals is relevant. A health hazard assessment of the allergenicity of egg was performed. The critical effect was derived from a published food challenge study with dose-response information on the egg intake levels of adverse effects. The no (NOAEL) and low (LOAEL) observable adverse effect level for objective symptoms were identified. Utilizing an uncertainty factor of 10 for intra-individual differences and the NOAEL for objective adverse symptoms, an estimation of the tolerable acute intake of egg was determined. The tolerable intake for objective symptoms is 2.4 µg egg protein/person. The nature of individual sensitivities and responses suggests additional considerations and factors in addressing "uncertainty" may be warranted. The possibility exists that the tolerable acute intakes levels as derived above may very well provide a lower margin of safety than is normally considered appropriate based on established severe effects in humans. Thus, in this case, additional adjustments in deriving the margin of exposure may be a reasonable consideration.
R. A. Bright, J. C. Shen, CDRH, FDA, Rockville, MD
A major category of therapeutic products that has been relatively understudied in relation to adverse events is medical devices. We estimated the national hospital-related frequency of adverse medical device events (AMDE), as indicated on discharge claims.
We used a research tool on the public website of the US Agency for Healthcare Research and Quality, called HCUPnet, which provides query tools for the Healthcare Cost and Utilization Project Nationwide Inpatient Sample, for the years 1997 - 2003. The 9th International Classification of Diseases complication codes (996) or abnormal reaction (E codes) for medical devices were queried as a group for each available year. Standard errors were not available for groups of codes. We used as patient-day denominators the mean lengths of stay and total number of discharges for each year. We used as the population burden denominator the US Census estimate for the 50 states, provided for July 1 of each year.
In 2003, an estimated 1.13 million AMDE were recorded on hospital discharge summaries. The rate was 6.44/1000 patient days and the population burden was 3.89/1000 people. Of these AMDE, 486,000 were recorded as principal diagnoses, at a rate of 3.68/1000 patient days and 3.89/1000 people. In 1997, the estimates were 858,000 total AMDE (5.15/1000 patient days and 3.20/1000 people) and 385,000 principal diagnosis AMDE (2.31/1000 patient days and 1.44/1000 people).
AMDE are an increasingly significant public health burden. HCUPnet is a tool that could be easily used by hospitals, healthcare networks, and states to compare their medical device safety performance to the national experience.K. Gelperin, A. D. Brinker, M. I. Avigan, FDA
Background: Clinical studies have documented that stimulants used in the treatment of Attention Deficit Hyperactivity Disorder (ADHD) can increase blood pressure. MedWatch reports have been received by FDA which describe sudden unexplained death, particularly in children with structural heart abnormalities. We examined the rate of domestic, spontaneous reporting for these and similar adverse cardiovascular events in association with common stimulants adjusted by utilization.
Methods: Comprehensive searches of the AERS database of spontaneous (MedWatch) reports were performed to identify deaths, sudden deaths, and nonfatal cardiovascular and cerebrovascular serious adverse events reported in association with four common stimulant products, including all branded and generic forms. Reporting rates were calculated using drug utilization data for the five-year period between January 1, 1999 and December 31, 2003. Separate analyses were done for the pediatric (1-18 years) and adult (19+ years) age groups.
Results: In each of the categories studied, none of the reporting rates for amphetamine or methylphenidate exceeded one case per million prescriptions dispensed, with one exception: for nonfatal cardiovascular and cerebrovascular serious adverse events associated with amphetamine therapy in adults, the calculated reporting rate was 1.79 cases per million prescriptions dispensed. Calculated reporting rate ratios for amphetamine versus methylphenidate were greater than one in all categories, ranging from 2.3 to 7.6, with numerically higher rates noted for amphetamine.
Conclusions: Inference of risk from these data is limited. The absolute number of cases produce rates which are insufficient to suggest statistical robustness. Additional study of these events in association with these products is prudent.
R.P. Brown, CDRH, FDA, Silver Spring, MD
Background: Although rare, iatrogenic transmission of Creutzfeldt-Jakob disease (CJD), a form of transmissible spongiform encephalopathy (TSE), has been reported following the use of medical devices such as contaminated depth EEG electrodes and neurosurgical instruments. As a result, there is concern about the potential for iatrogenic transmission of CJD to occur when reprocessed neurosurgical instruments are used for neurosurgical procedures.
Methods: A risk assessment was performed to estimate the annual risk in the U.S. of iatrogenic CJD in patients undergoing neurosurgery with reprocessed neurosurgical instruments. Two approaches were used for this assessment: a deterministic model that used point estimates for default parameter values and a probabilistic approach using Monte Carlo analysis.
Results: The results using either approach were the same, specifically, that less than one case of iatrogenic CJD is expected in the U.S. each year from the use of reprocessed neurosurgical instruments, based on the assumptions used in the risk assessment. Although there is some uncertainty associated with the model-derived estimates of risk, the range of risks provided by the probabilistic model suggests that it is unlikely for even one case of iatrogenic CJD to occur in the U.S. population each year from the use of reprocessed neurosurgical instruments (95th percentile risk from the probabilistic model was about 0.5 cases/year).
Conclusions: From a regulatory perspective, these risk estimates can be used to evaluate the magnitude of the risk, help to define a risk-benefit ratio for cleaning and sterilization procedures, and assess the need for additional risk reduction measures.
R. P. Brown1 , R. Ito2 , M. E. Stratmeyer1 , 1CDRH, FDA, Silver Spring, MD, 2Hoshi University, Tokyo, Japan
Background: Di-(2-ethylhexyl) phthalate (DEHP), a plasticizer commonly used in PVC medical devices, can be converted to its monoester, MEHP, exogenously by lipases in stored plasma or blood, by hydrolysis in stored and heated IV fluid, or by gamma irradiation of PVC. As a result, some of the DEHP that is released into stored blood, plasma, or IV fluids will be converted to MEHP before reaching the patient. Previous safety assessments have only explicitly accounted for patient exposure to DEHP; however, it is also important to account for co-exposure to MEHP since MEHP is typically more potent than DEHP in producing adverse toxicological effects.
Method: A relative potency approach was used to define the potency of MEHP relative to DEHP. The total dose of DEHP equivalents following co-exposure of patients to DEHP and MEHP was then derived for various medical procedures.
Result: The weight of evidence suggests that MEHP is at least an order of magnitude more potent than DEHP in various toxicity assays; therefore, it is reasonable to assign a provisional relative potency value of 10 to MEHP. Accounting for co-exposure to both DEHP and MEHP changes the margin of exposure between the Tolerable Intake (TI) for DEHP and the dose of these phthalates that are received by patients undergoing various medical procedures.
Conclusions: The relative potency factor approach provides a means to account for co-exposure of patients to both DEHP and MEHP when conducting an aggregate safety assessment of these compounds released from medical device materials.R. Ito, N. Miura, M. Kawaguchi, K. Saito, H. Nakazawa, Hoshi University, Tokyo, Japan
Background: The plasticizer, DEHP, is released from some PVC medical devices. FDA/CDRH has recently shown that relatively large amounts of DEHP can be received by patients undergoing some medical procedures and the potential exists for large doses of DEHP to produce adverse effects in exposed patients. The DEHP metabolite, MEHP, is thought to be responsible for many of the adverse effects of DEHP. Since PVC devices typically undergo sterilization before use, this study was conducted to determine the effect of various sterilization processes on the formation of MEHP in DEHP-plasticized PVC materials.
Method: PVC sheets were sterilized with either gamma radiation, autoclaving or ethylene oxide. Control materials were unsterilized. DEHP and MEHP were extracted from the sterilized PVC sheets and unsterilized controls using either 5% glucose solution or polyoxyethylated hydrogenated castor oil (HCO-60). Analysis of phthalate levels in the extracts was conducted using LC-MS/MS.
Results: Little DEHP migrated from gamma radiation-sterilized PVC sheets into glucose solution and HCO-60, compared to the unsterilized sheets. In contrast, the level of MEHP release from the gamma radiation-sterilized PVC sheets into the glucose solution and HCO-60 was high. MEHP levels were also high in the gamma radiation-sterilized PVC sheets
Conclusion: Gamma radiation facilitates the breakdown of DEHP to MEHP in PVC material. As a result, patients undergoing treatment with gamma radiation-sterilized devices may be exposed to both DEHP and MEHP. From a risk assessment perspective, the need exists to account for co-exposure to both phthalate compounds in patients treated with gamma radiation-sterilized PVC devices.
D. D. Wagner, P. J. Carter, OR, CVM, Laurel, MD
Background: Risk to human health from contamination of animal derived food with antibiotic resistant Salmonella is continually assessed by the U.S. Food and Drug Administration (FDA). Surveys of animal feed commodities, production environments and retail meats are used to assess the distribution of salmonellae in the food production system. One hypothesis supporting feed surveys is that feed may serve as a source of phenotypes that ultimately end up on retail cuts of meat.This report summarizes serotype and antibiotic susceptibility profile distributions of Salmonella isolates derived from feed commodities and retail meat samples collected during 2002 and 2003. Methods: Feed samples were collected by FDA in national sampling projects.Samples of retail meats were collected as part of the National Antimicrobial Resistance Monitoring System (NARMS), an interagency survey program conducted by FDA, CDC, and USDA.All isolates were serotyped and tested for antibiotic susceptibility using standard testing methods. Results: Combined results across the two years showed that 20% of feeds tested were positive for Salmonella while 6 % of the retail meats (Chicken, Turkey, Ground Beef, & Pork Chops) were positive.Prevalence of Salmonella serotypes varied markedly for isolates derived from feed vs. those from retail meats.Most striking were the results from antibiotic susceptibility testing. 74 isolates of Salmonella derived from feed were uniformly susceptible to all (16) antimicrobials against which they were tested, isolates derived from the meat samples were resistant to several antibiotics. Of 365 isolates tested from retail meats, 46% were resistant to 2 or more antibiotics. Conclusions: These data suggest that antibiotic resistant phenotypes seen in retail meats are not derived from feed consumed by the animals and that resistant phenotypes are not widely disseminated in the environment.In addition, serotypes commonly found on meat products and in cases of human illness occur at very low frequency in feed suggesting a strong selective effect of the animal environment for certain Salmonella serotypes.
H. A. Clark, S. M. Snedeker, Sprecher Institute for Comparative Cancer Research, Cornell University
The Cornell Program on Breast Cancer and Environmental Risk Factors (BCERF) critically reviewed the available data on the cancer risk posed by the mycotoxin and food contaminant, ochratoxin-A (OTA). Compared to the well-known aflatoxins, OTA has received relatively little attention. In long-term animal cancer bioassays performed by the National Toxicology Program (NTP, 1989), female rats exposed orally to OTA had significantly increased incidence of mammary tumors (fibroadenomas) and both male and female rats developed tumors of the kidney. OTA has been found in animals and humans, including in tissues, blood and human breast milk. Accumulation of OTA in the mammary gland of rabbits has also been observed. Few studies to date have evaluated the cancer risk of OTA in human populations. Formed by storage fungi in both temperate (Penicillium) and tropical (Aspergillus) climates, OTA makes its way into a variety of foods and beverages, in particular, cereal grains. It is also found in wine, coffee, juices, cocoa, spices, dried fruit, pork, poultry and dairy products. Most available data on OTA occurrence are from Europe and Canada. Large data gaps exist for OTA levels in commodities from other parts of the world. The US Food and Drug Administration (FDA) is currently conducting a multi-year monitoring program in order to determine if there is a need for regulatory limits for OTA in food in the US. Current and proposed regulatory limits, as well as best farm management practices are discussed as relevant to mitigating OTA's public health risk.H. G. Claycamp, C. W. Gray, FDA, Rockville, MD
Background: FDA has sought more consistent risk-based decisions as part of its initiative, "Pharmaceutical cGMPs for the 21st Century - A Risk-Based Approach." Good Manufacturing Practices (GMP) compliance inspections are mandated on a biennial basis and are routinely performed during the approval process for the chemistry, manufacturing, and controls (CMC) technical section of new animal drug applications (NADAs) or supplemental submissions to already approved NADAs that include process- or facility-related changes. The objective of the present work is to develop a risk-informed, decision support system for pre-approval inspection decisions.
Methods: An expert elicitation process was used to define a common set of questions and elements (attributes) of the CMC reviews that are used in decision making. Weights were assigned to attributes to reflect the experts' judgments of the relative importance of each attribute in risk-informed decision making. The decision model is based on general multi-attribute utility theory (MAUT) but includes uncertainty-adjustments reflecting the quality of the evidence for each attribute. The results of the guided review of evidence for the attributes are reported as an overall score and an "uncertainty-adjusted" score. The results are used in an advisory capacity for the review team's decisions whether or not to inspect manufacturing facilities when a sponsor proposes a pharmaceutical manufacturing process. For convenience and user acceptance, the decision support tool is implemented as an Excel™ Add-In.The decision support tool is presently undergoing pilot-scale testing.
Results to Date: The decision support system has received general acceptance for user-friendliness and as providing helpful information for pre-approval inspection decisions.The addition of uncertainty adjustments to the basic MAUT models is believed to be beneficial in both decision making and in helping to understand key risk drivers in review decisions.Model refinements and simulations are presented.
V. Wiles, E. A. Grove, K. B. Ekelman, CVM, FDA, Rockville, MD
Background: The Center for Veterinary Medicine (CVM) is developing a risk-based inspection system to help ensure that regulatory resources are focused on the animal feed and drug products, manufacturing processes and facilities that pose the greatest risks to animal and human health. To help identify risk factors for animal feed and drug products, manufacturing processes and facilities, we analyzed 203 firm-initiated recalls of animal drug and feed products from fiscal years 2000 through 2005. A recall is a firm's removal or correction of a marketed product that FDA considers to be in violation of the laws it administers.
Methods: We reviewed records for 203 firm-initiated recalls of animal drug and feed products from fiscal years 2000 to 2005 to identify the types of errors associated with recalled animal drug and feed products and to determine which recalled products and errors were associated with the highest levels of health hazards. The relative level of health hazard attributed to each recalled product by FDA is reflected in an assigned recall classification number (i.e., I, II, or III).
Results: Of the 203 firm-initiated recalls of animal drug and feed products from fiscal years 2000 through 2005, 103 (approximately 51%) were for non-medicated feeds, 33 (approximately 16%) were for medicated feeds, and 64 (approximately 32%) were for animal drugs. For recalls of non-medicated feeds, 18% were classified as posing a high level of health hazard (recall classification I) and 76% were classified as posing a moderate health hazard (recall classification II). For recalls of medicated feeds, 13% were classified as posing a high level of health hazard and 53% were classified as posing a moderate level of health hazard. For recalls of animal drugs, 5% were classified as posing a high level of health hazard and 42% were classified as posing a moderate level of health hazard. The most common errors identified for recalls of non-medicated feeds were those related to the BSE rule. The most common errors identified for recalls of medicated feeds were incorrect levels of drugs in feeds or feeding medicated feeds to species or ages for which the drugs in the feeds were not approved. Other errors associated with recalls of non-medicated and medicated feeds included chemical or microbiological contamination, labeling errors, and general manufacturing errors. The most common errors associated with recalls of animal drugs were due to concerns about the drugs' stability, sterility and labeling.
Conclusions: CVM is using information from the analysis of the animal feed and drug products associated with 203 firm-initiated recalls from fiscal years 2000 through 2005 to help rank the relative risks from the products, manufacturing processes and facilities.
K. C. Worthy, L. A. Governale, DSRCS, ODS, CDER, Silver Spring, MD
Background: Trends in outpatient prescription drug usage was examined for years 2002 through 2005 with a specific focus on year 2005.
Methods: Pharmaceutical research databases from Verispan, LLC and IMS Health were analyzed to estimate national outpatient prescription drug usage patterns. The Verispan Vector One® National database provided information on prescription dispensing activity by therapeutic classes, drug product, patient demographics and prescriber specialty. The IMS Health, National Disease and Therapeutic Index™ provided information on diagnoses most frequently encountered in office-based medical practices.
Results: The total number of dispensed prescriptions in the outpatient setting increased from ~3.0 billion in year 2002 to ~3.2 billion in year 2005. The top 5 most frequently dispensed drug classes in year 2005 were narcotic analgesics, cholesterol lowering agents, beta-blockers, anti-depressants and ACE inhibitors. The top 5 dispensed drug products in year 2005 were HCD/APAP, Lipitor®, amoxicillin, lisinopril and HCTZ. General Practitioners (28%) were the top prescribers followed by Internal Medicine (22%), OB/GYN (4%) and Pediatrics (4%) specialties. Adults aged 45-64 years received the most prescriptions (37%) followed by adults aged 65 years and older (31%) in year 2005. Diseases of the respiratory and cardiovascular systems were the most commonly mentioned diagnoses during office-based physician patient contacts. Essential hypertension (ICD-9 401.9) was the single most mentioned diagnosis during all time periods.
Conclusions: There was little change in the overall patterns of prescription dispensing in retail settings from 2002 to 2005. There were slight changes in rankings and proportions for drug classes, products, and patient age groups over this time period.
A. B. Ciavarella1 , S. Jenney1 , A. Gupta1 , P. J. Faustino1 , B. Rothman2 , M. A. Khan1 , 1DPQR, CDER, FDA, Silver Spring, MD, 2OC, FDA, Rockville, MD
Background: A number of drug products are being repackaged from their original FDA approved container closures.The practice of repackaging may lead to serious stability issues with capsules due to high inherent moisture content of the capsule shells (13-15% w/w). This study, conducted in affiliation with the Office of Compliance, looks at the impact of this practice on the stability of gabapentin capsules.
Methods: 300 mg gabapentin capsules were purchased in their original HDPE containers and in repackaged USP Type A unit dose blister packs. Stability of both products was studied at 25°C/60% RH and 40°>C/75% RH over 13 weeks. Dissolution, potency and sample weight, were used to assess the products. Samples were also analyzed by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), near-infrared (NIR) spectroscopy, NIR chemical imaging and Raman spectroscopy.
Results: Potency experiments and sample weights detected no change within, and between, the original and repackaged products. The amount of gabapentin degradation product was below allowable limits in all samples. Storage up to 13 weeks under 40°C/75% RH and 25°C/60% RH did not alter the dissolution of the original or the repackaged capsules.The TGA, DSC, NIR and Raman profiles of all samples showed no difference over the storage period.
Conclusions: There were no significant differences in weight, potency, dissolution, TGA or DSC experiments for the original and the repackaged gabapentin capsules under both test conditions.Product quality attributes of gabapentin capsules are not affected by repackaging. These results should not be extrapolated to other drug products
K. Hollinger1 , E. Pinnow1 , A. Oliva2 , P. Hepp3 , G. Gensinger4 , H. Chazin2 , L. Pauls5 , D. Goldman6 , J. Ware7 , K. Uhl1 , 1OWH/OSHC, 2OND, 3OCPB, 4OBPS/RRSS, 5OEP/QAS, 6Global Net Services, Inc., 7OEP
Background:
Differences between men and women in the safety and efficacy of drugs and biologics have been of concern to the FDA since the early 1980's.Scientific studies have brought greater understanding to the biological basis for sex-based differences to medical products.[1] In 2002, Congress mandated FDA track the inclusion of women in clinical trials and gender analysis. In response the OWH has pioneered the development of SMART Templates, based upon Center policies allowing reviewers to more quickly access resources required for a review and enable the tracking of sex assessments.
Methods:
Responding to comments collected from reviewers on review templates, Microsoft Word2003 was customized creating a Task Pane with Command, Navigation and Viewing areas. The content of SMART Templates was tagged using eXtensible Markup Language (XML) to enable more sophisticated storage solutions for the data contained in reviews.
Results:
In this poster we present the SMART approach to Review Templates as piloted by CDER reviewers in the Office of New Drugs and Office of Clinical Pharmacology and Biopharmaceutics. This poster demonstrates features including: protection of the outline headers, hyperlinks to referenced resources, cross-referencing review links, integration of instructions into the Template outline, a customized Task Pane that includes a Navigation Pane facilitating movement within a review and a Command Pane from which instructions may be viewed or hidden.
Conclusions:
SMART Templates allow more effective use of reviewer resources giving them fingertip access to many resources required for a review and enhances consistency of reviews.
M. Kakhi1 , B. M. Ayyub2 , A. M. Wokovich3 , S. Prodduturi3 , W. H. Doub3 , L. F. Buhse3 , A. S. Hussain4 , N. Sadrieh1 , 1CDER, FDA, Silver Spring, MD, 2University of Maryland, College Park, MD, 3CDER, FDA, St. Louis, MO, 4Formerly CDER, FDA, Rockville, MD
Background
Transdermal Drug Delivery Systems have been in the US market place for only about 20 years. Due to the variety of external factors that can lead to dosage failure (adverse reactions), it is necessary for the Agency to have a firm understanding of the possible risks associated with TDDSs. Formalized risk analysis has long been used successfully in engineering fields. In collaboration with the University of Maryland, we have applied those techniques to the environmental (usage), chemical and mechanical aspects of these novel dosage forms.
Methods
Risk analysis focuses on the following questions: What are the modes, likelihoods and consequences of failure? The 'modes' of failure define the scenarios whose consequences are classified from 'lack of therapeutic effect' through to 'death'. Using an influence diagram, which defines the interrelationship of key variables, and an event tree, whose branches constitute various potential scenarios, the likelihood of failure can be estimated.
Results
A general influence diagram for TDDSs was constructed. Focusing on adhesion as one aspect that can lead to failure, the formalism of a success tree has been applied to quantify, for example, the probability of acceptable adhesion.
Conclusions
A framework for the risk assessment of TDDSs is proposed. The aim is to promote a systematic approach for characterizing the various factors affecting adhesion of a TDDS and assessing the consequences of the possible scenarios. Such a framework can contribute to the Agency's ability to evaluate complex and novel dosage forms.
D.L. Kallgren, OWH, FDA, Rockville, MD
Background: Following a Congressional mandate, in May 2003, the Food and Drug Administration launched the "Menopause and Hormones: What Can You Believe?" outreach campaign. The campaign provided peri-menopausal and menopausal women with reliable, easy to understand information about the benefits and risks of hormone therapy for relief of symptoms. Women were encouraged to consult with their doctors, and if hormones were needed, to use them at the lowest effective dose, for the shortest period of time possible.
Methods: Part I of the campaign enlisted both Federal and non-Federal partners to collaboratively develop outreach materials which included a fact sheet and purse guide (available in English and Spanish translations). Part II involved national and local print, radio, and Internet advertisements to complement direct community and e-mail outreach.
Results: Campaign materials were publicized to the target audience through multiple venues. Broadcasts ran nationwide on more than 30 radio stations and public service announcements on 222 radio stations. News releases ran in 1223 news outlets and press releases in 156 publications in 15 states. Print advertising ran in newspapers/publications. Internet advertising ran on the Yahoo search engine. Nearly 1100 community based organizations distributed campaign information, and nearly four million women received direct e-mail.
Conclusions: By November 2005, Web traffic to the primary campaign website, the HHS Office of Women's Health, increased by almost 200,000/month in a little over a year. Additionally, more than 221,000 free information kits have been distributed through an HHS call center and the Federal Citizen Information Center at Pueblo.
S. Kaplan, J. Staffa, G. Dal Pan, CDER, FDA, Silver Spring, MD
Context: Metoclopramide-induced tardive dyskinesia (TD) is associated with cumulative drug exposure, which can result from prolonged use of the drug. The product label recommends that duration of therapy should not exceed 12 weeks.
Objective: To estimate duration of therapy with metoclopramide and to determine the extent of adherence to the label recommendations.
Methods: Prescription claims for metoclopramide products from January 1, 2002-December 31, 2004 were extracted for participants aged one year or older, residing throughout the U.S. and contained within the Caremark database. An episode of therapy was defined as one or a series of consecutive claims with no more than a 30-day lag between the dispensing date of a new claim and the ending date of the preceding claim. Episode duration was calculated by subtracting the start date from the end date for each episode.
Results: During the study period, almost 80% of participants (total=200,907) had only one episode of therapy. The length of the longest episode of therapy for most patients (89%) varied from 1-90 days, yet 11% of patients appeared to have received prescriptions for metoclopramide for a period longer than 90 days. Cumulative therapy for longer than 6 months was found for over 10% of patients.
Conclusions: These results suggest that despite the known risk of TD and the label recommendations on duration of metoclopramide use, physicians and patients did not follow these recommendations. Physicians should carefully consider the risk-benefit profile of the drug and, if possible, avoid increased risk of TD due to prolonged exposure.
T. Khoie, K. A. O'Connell, R. L. Pierce, A. Shrake, C. E. Zinderman, R. P. Wise, CBER, FDA, Rockville, MD
BACKGROUND: FDA licensed three Alpha-1 Proteinase Inhibitors (A1PI) for emphysema secondary to alpha-1 proteinase inhibitor deficiency. Recently discovered structural heterogeneity, of unclear clinical significance, among these three A1PI products prompted our review of adverse events (AE) reported after licensure.
OBJECTIVE: Describe spontaneous post-licensure reports of suspected AEs for A1PI products.
METHODS: We reviewed FDA Adverse Event Reporting System (AERS) reports with A1PI as a suspect product. Except for required reporting of certain study AEs, AERS is a passive surveillance system. It relies directly or indirectly (through manufacturers) on spontaneous reporting by the public. Non-serious, non-electronic reports submitted by manufacturers, which have not been entered routinely into AERS since November 1997, were also reviewed if received between January 2003 and July 2005.
RESULTS: Among 849 AE reports received for all A1PI products, 805 identified Prolastin, 38 identified Aralast, and 6 identified Zemaira as suspect products. There were 113 (13%) serious reports and 765 (90%) domestic reports. Among 408 domestic reports with age information, 68% involved patients 40-59 years of age. Among 577 domestic reports with gender information, 53.7% involved females, and 46.3% involved males. The most frequently cited AEs included dyspnea, headache, and fever.
CONCLUSIONS: Most A1PI AE reports are non-serious and describe events identified as possible risks in product package inserts. Limitations in passive surveillance and lack of exposure data preclude any conclusions regarding comparative safety of A1PI products. A systematic surveillance system with consistent AE ascertainment and corresponding exposure data could provide more precise information about A1PI safety.
D. Kumar, A. S. Khan, CBER, FDA, Bethesda, MD
Background: Simian foamy virus (SFV) is widespread among all species of non-human primates (NHPs).The most prevalent mode of transmission between NHPs is thought to be via saliva.Accidental exposure to infected animals, involving wound injury, has resulted in SFV infection in zookeepers and animal handlers. The stable persistence of SFV in potential blood donors has raised safety concerns regarding SFV transmission by blood transfusion.
Methods: To investigate SFV transmission by blood, SFV negative rhesus macaques were transfused with blood from two naturally-infected rhesus macaques.The SFVs from the two donor animals were biologically and genetically distinct viruses.Recipient animals were monitored for virus infection by PCR, western blot, and coculture of PBMCs with a permissive cell line.
Results: Two monkeys that received blood from one donor animal became SFV infected: they were positive at week 8 by PCR; antibodies were detected in both animals by dot blot immunoassay and Western blot by 22 weeks; virus was isolated from the PBMCs of both animals at week 11 by coculture with Mus dunni cells. Persistent infection was seen at one year post transfusion, the last time point tested.SFV transmission was not seen in the two rhesus macaques which received blood transfusion from the second donor monkey.
Conclusions: The results indicate that SFV infection can occur by blood transfusion; however, virus transmission may depend upon various viral or host factors.This and future studies can provide useful guidance on the issue of safeguarding the human blood supply.
D. D. Levy, L. S. Pellicore, S. J. Walker, CFSAN, FDA, College Park, MD
Among the new dietary ingredients that have been submitted to FDA for safety assessment in the New Dietary Ingredient (NDI) notification program are many ingredients that are live bacteria or yeast. In the first 10 years following passage of the Dietary Supplement Health and Education Act of 1994, 5% (14/314) NDI notifications described live microbial ingredients. Since January 2004 that fraction has jumped to 15% (10/67). Most of the pre-2004 NDI notifications described ingredients that are lactic acid bacteria (e.g. lactobacilli) already widely consumed in fermented foods such as yogurts. More recent NDI notifications describe a wider variety of organisms. Evaluation of these ingredients requires safety information specific to live microbial ingredients, different from information supporting the safety of pure chemicals or plants. FDA responses to notifications describing these more diverse live microbial ingredients have addressed the lack of information about the number of organisms in a serving of the product, the relationship of the organism that was the subject of the notification to the organisms that were described in the safety information within the notification, and the source and identity of the organisms that were proposed as ingredients in dietary supplement products. Evaluation of the safety of products containing live microbial ingredients thus requires consideration of factors not considered in safety paradigms constructed for the evaluation of more traditional ingredients such as pure chemical substances or plant extracts.W. R. Long, M. D. Miliotis, R. L. Buchanan, CFSAN, FDA, College Park, MD
Better communication and coordination of risk assessment activities is essential for improved food safety decision making. The interagency Risk Assessment Consortium (RAC) was formed in response to the 1997 Food Safety Initiative to improve food safety by improving coordination among member agencies, and has steadily built recognition as an essential element of effective interagency coordination. Its membership consists of federal agencies with food safety responsibilities and/or interest. Since its inception, through the efforts of workgroups, the RAC has involved itself in many topical issues facing the risk analysis community, and serves as a resource for the member agencies. Examples include creating a data repository through the establishment of a risk analysis clearinghouse, bounding data quality, examining approaches to data utility, analyzing risk assessment data gaps and posting these for researchers, equating human and animal dose-response, relating microbiological criteria to public health goals, evaluating risk assessment frameworks, clarifying the role of peer review, and encouraging dialogue around risk ranking. Annual open meetings, symposia, and/or conference sessions encourage public input.
M. Mease, T. Nearing-Crowley, L. Zwanziger, S. Cummins, CDER, FDA, Rockville, MD
On February 15th, 2005 HHS Secretary Mike Leavitt announced drug safety reforms to improve internal oversight of the management of drug safety issues within CDER and to provide health care professionals and the public with early information and recommendations about important emerging drug safety risks. Oversight occurs through the newly established Drug Safety Oversight Board (DSB), which is a standing committee of 18 federal employees, including 14 senior scientific leaders from within CDER, two senior managers from other FDA Centers, and two practicing physician scientists from other federal agencies. Early risk communication occurs through issuance of public health advisories and information sheets targeted to the general public, health care professionals and patients.
The DSB advises the Center Director, CDER on the management of important drug safety issues, the adjudication of internal organizational disputes, the development of drug safety management policies, and on communicating about emerging drug risks. Since its inception the DSB has met six times.
By December 31st, 2005, CDER had communicated emerging drug safety risk information about 44 drugs. These communications can be found on the CDER Drug Specific Information Site (http://www.fda.gov/cder/drug/DrugSafety/DrugIndex.htm). These communications addressed six product class issues, three market suspensions, and two product withdrawals. They also involved 37 drugs that eventually had new warnings added to the product label.
The DSB is an important component of CDER's initiative to improve the management of drug safety and inform the public about important emerging medication risks. This poster will summarize the first year of DSB operations and its risk communication efforts.
M. Menis, D. R. Burwen, K. A. O'Connell, S. A. Anderson, CBER, FDA, Rockville, MD
Background: The purpose of our study was to characterize blood use patterns among the U.S. elderly population during 2001. As the U.S. population ages the demand for blood is expected to grow. However, there have been no comprehensive studies detailing blood use by U.S. elderly.
Methods: A descriptive cross-sectional study of blood use was conducted using the 5% Medicare Provider Analysis and Review (MedPAR) data file obtained from the Centers for Medicare & Medicaid Services (CMS). Each record of the file represented a Medicare patient's stay in a hospital or a skilled nursing facility (SNF). Blood use was assessed using the blood pints furnished variable, which represents the quantity of blood transfused during the patient's stay.
Results: Analysis of the CMS data showed that blood (whole blood or packed red cells) was used by 2.5% of sampled stays. Blood use prevalence was approximately 10.5 times higher for stays with at least one procedure compared to stays involving no procedures (p<0.0001). Top 20 principal procedures using the most blood accounted for about 56% of total blood pints furnished; however, these procedures represented only about 19% of all stays.
Conclusion: Our study shows a strong association between medical procedures and blood use among stays for elderly. Some of the highest blood use occurred with surgical procedures. More precise information on blood use may serve as the basis for estimating risks associated with blood transfusion and helping to balance blood supply with a need for blood.
M. Moore1 , B. Allen2 , M. Dourson3 , L. Haber3 , R. Heflich1 , R. Kodell1 , A. Shipp4 , 1NCTR, FDA, Jefferson, AR, 2Bruce Allen Consulting, 3Toxicology Excellence for Risk Assessment, 4ENVIRON International, Ruston, LA
Background: Quantitative cancer risk assessments typically utilize high-dose rodent tumor data.The quantitative model chosen for cancer risk assessment is based on the mode of action (MOA) of the chemical under consideration.In particular, it is based on whether the chemical is thought to cause tumors through a mutagenic or a nonmutagenic mechanism.
Methods: We are evaluating the potential use of benchmark dose (BMD) analysis of in vivo mutation data obtained from the tumor target tissue as a part of assessing the MOA for chemical carcinogens. Mutagenic carcinogens are expected to have BMD estimates that are lower than the corresponding BMDs for tumors. To investigate the utility of this approach, we have conducted 2 case studies using published mutation and tumor data sets for riddelliine and dichloroacetic acid and calculated and compared BMD estimates for mutant frequency and tumor incidence.
Results: The BMD analysis indicated that riddelliine is a mutagenic carcinogen and that dichloroacetic acid is a nonmutagenic carcinogen.
Conclusions: The BMD results were consistent with other available MOA information for riddelliine and dichloroacetic acid.This preliminary analysis supports the utility of in vivo dose-response mutation analysis for informing cancer MOA.
P. Nourjah, R. A. Bonnel, A. D. Brinker, M. I. Avigan, CDER, FDA, Rockville, MD
Background: Warfarin, a commonly used anticoagulant, is associated with a significant risk for bleeding, approaching 5% in some studies.We conducted an analysis of National Hospital Ambulatory Care Survey (NHMACS) to characterize patients who were admitted to emergency departments (EDs) with a diagnosis of bleeding while receiving warfarin therapy.
Methods: Publicly available NHMACS data for calendar years 1999 to 2003 were obtained from the NCHS website. International Classification of Diseases, 9th Revision, Clinical Modification (ICD -9CM) codes for bleeding (430-432, 455-578, 599.7, 623.8, 626.2, 784.7, 786.3, 459, 719.1-719.2 and 423) as well as internal NHAMCS codes were used to identify the cases related to warfarin. Results were projected to the national level.
Results: Each year on average, 112,179 visits were made to EDs by patients who had bleeding associated with warfarin. 56% of visits were by females; the median age was 72 years. 47% of visits resulted in hospitalization and an additional 2% resulted in admission for 23-hour observation. 13% of the hospital admissions were to intensive care units.
A small fraction (0.2%) of the cases were dead on arrival to the ED or associated with death while being treated in the ED. 84% of all visits were associated with a diagnosis consistent with gastrointestinal (GI) bleeding.
Conclusions: Complications of warfarin treatment continue to be linked to significant morbidity and mortality. The most common complication associated with warfarin use in this study was GI bleeding.
M. G. Paule, J. Garey, S. A. Ferguson, NCTR, FDA, Jefferson, AR
W. Qin, J. L. Brown, Department of Food Science, The Pennsylvania State University
Background: An important criterion for an effective public issue communication strategy about genetic engineering is to improve audiences' understanding of the issue and help them form informed, well reasoned opinions that are not easily swayed by inflammatory material. The heuristic-systematic model predicts that opinions produced by systematic processing would be "more persistent, predictive of behavior and more resistant to counterpropaganda" than those produced by heuristic processing. This study examined the effect of two information formats (consequence vs. perspective) developed according to public issue education principles on the public understanding of, interest in and attitude towards genetically engineered salmon, as well as the information formats effect on mode of information processing.
Methods: A survey was administered to the local residents of a small college town. The sample was stratified by age and gender, and half of the sample received the information in consequence format, half received the information in perspective format.
Results: We found that participants who read the consequence information (n=102) learned more, expressed more interest, and indicated higher level of actual confidence in judgment than those who read the perspective information (n=103). Both information formats were equally likely to promote systematic processing, but the perspective information produced lower level of heuristic processing among the readers than the consequence information (2.65±0.77 vs. 2.92±1.01, respectively).
Conclusions: We suggest use the consequence format for further communication campaigns because it is more likely to improve readers' knowledge and interest than the perspective information, and it is equally likely to promote systematic processing.
R. B. Shah1 , H. R. Prasanna1 , M. A. Tawakkul1 , B. Rothman2 , M. A. Khan1 , 1DPQR, CDER, FDA, Silver spring, MD, 2OC, FDA, Rockville, MD
Purpose. The purpose of the present study was to demonstrate whether the stability will be compromised in a repackaged unit dose container for ranitidine syrup drug product.
Methods.The original and repackaged syrup containers were exposed to accelerated (40oC/25%RH) and long-term (25oC/40%RH) stability conditions for 3 and 12 months, respectively. At predetermined time intervals, the samples were evaluated for pH, weight loss, potency, appearance, identification and quantization of impurities.
Results. The weight loss results under both conditions and packages showed no significant changes. The pH remained between 6.9 - 7.2 within the specification range for all the containers under both storage conditions. The potency of the drug substance remained between 90-110%, as specified by the USP for the two conditions and types of packaging. Additionally, the major impurity, related compound C increased from 0.21 to 0.31% w/w at 3 and 6 months for both types of packaging under long term conditions. Under accelerated conditions the total impurity profile was 0.4, 1.22, and 1.56% for original containers and 0.87, 1.4, and 1.75% for repackaged containers at the end of 1, 2, and 3 months respectively. Despite these differences, the total impurity in all cases were well below the allowable impurity level (4%) as specified in USP.
Conclusion. The results of the study indicate that the original and repackaged ranitidine syrup samples are stable up to 3 and 6 months under accelerated and long-term storage conditions, respectively.
M. Shih, J. F. Saviola, CDRH, FDA, Rockville, MD
Perfluoropropane intraocular gas is a medical device introduced into the eye to place pressure on a detached retina (ocular endotamponade). It belongs to a unique category of ocular endotamponades with variable expansile capabilities and intraocular longevities. Expansion of the intraocular gas bubble is expected during the course of treatment. Excessive increase in intraocular pressure due to gas bubble expansion can be sight-threatening. The parameters that influence gas expansion are diffusion rate of the gas, intraocular gas injection volume, concentrations of the high and the low molecular-weight gas impurities, changes in atmospheric pressure while the gas bubble is in the eye, and administration of nitrous oxide anesthesia. To mitigate the risk of excessive gas expansion, these parameters are managed through the collective efforts of stakeholders at various stages of the device life cycle.
In 1984, the Orphan Products Development Office of FDA issued a Federal Register Notice to invite submission of paper PMAs for intraocular gas devices and establish a critical pathway for marketing this type of orphan product. In 1993, FDA approved perfluoropropane intraocular gas for marketing. It is the only long-lasting ocular endotamponading gas available on the market. Since the original approval, this device has gone through challenging manufacturing changes and a recall for high intraocular pressure due to nitrous oxide interaction. Due to the collective risk management efforts of all the stakeholders, this device has remained on the market to meet a critical public health demand - a remarkable achievement for a significant- risk, single- source orphan product.
A. Shoaibi, D. R. Tavris, CDRH, FDA, Rockville, MD
PURPOSE
To assess 1) the accuracy and positive and negative predictive values of the troponin assay in the diagnosis of myocardial infarction (MI); 2) how this accuracy varies by gender; and 3) how the diagnosis of MI varies by gender.
METHODS
The data used in this study was obtained from the Chest Pain Evaluation by Creatine Kinase-MB, Myoglobin, and Troponin I (CHECKMATE) study performed by the Duke Clinical Research Institute in 2001. The study population included 1,005 patients with possible myocardial ischemia from six US hospitals.
Sensitivity, specificity, positive predictive value, and negative predictive value were calculated by gender for the following predictor versus outcome variables: baseline troponin result predicting MI diagnosis (using standard objective criteria); discharge diagnosis predicting MI diagnosis (using standard objective criteria); and discharge diagnosis predicting baseline troponin results.
RESULTS
The sensitivity and specificity of troponin didn't vary by gender in the diagnosis of MI, but PPV was higher in men than women (p>0.05). The sensitivity was surprisingly low for both genders. When using discharge diagnosis of MI in predicting objectively defined MI, sensitivity was higher in men (p>0.05) and specificity was higher in women (P<0.05).
CONCLUSION
No differences were found between men and women for the accuracy of the troponin assay in the diagnosis of MI, perhaps due to the low study power. The major finding was that women with similar clinical results were less likely than men to receive a discharge diagnosis of MI.
C. Smith DeWaal, K. Johnson, CSPI
An estimated 76 million cases of foodborne disease occur each year in the United States. Contaminated seafood is one of the leading causes of food poisoning outbreaks in the United States. The U.S. Food and Drug Administration (FDA), the Centers for Disease Control (CDC), and other available epidemiological studies found the risk of becoming sick from eating seafood to be one in 250,000 (NC State Univ.).
CSPI maintains a unique listing of foodborne illness outbreaks, categorized by food. CSPI's data is compiled from various sources, including the CDC, state health departments, and scientific and medical journal articles. The database contains only those outbreaks with known or suspected etiology and an identified food source. This research focuses on seafood-related outbreaks, the most common food vehicles associated with outbreaks, and the most common causes of seafood outbreaks.
Between the years of 1990-2003, there were a total of 4,486 outbreaks in CSPI's database. Sixty six percent of these outbreaks (2,954) were related to foods regulated by the FDA. The single-food category most commonly linked to food-borne illness outbreaks was seafood. 899 outbreaks and 9436 cases were linked to seafood, including finfish, molluscan shellfish, other shellfish, and seafood dishes. Scombrotoxin (321 outbreaks), Ciguatoxin (215 outbreaks), and Vibrio (78 outbreaks) were the top three causes of seafood-related outbreaks.
With such a range of benefits and risks, fish and seafood should be strictly regulated to ensure that consumers can enjoy the health benefits without fear of getting sick. While several regulations currently exist that address seafood, much more is needed to truly ensure a safer food supply for U.S. consumers. Federal food-safety agencies should increase laboratory testing for dangerous bacteria in seafood and government inspectors should monitor conditions in processing plants. With the help of governmental agencies, there is a bigger guarantee in consumers' protection against unsafe seafood. American consumers expect that they are getting the safest, highest quality food in the world - this is currently not the case. American consumers deserve to know that their government and the agencies charged with inspecting their food have sufficient resources.
C. Smith DeWaal, K. Johnson, CSPI
Salmonellosis is a bacterial infection that causes diarrhea, fever, and abdominal cramps. In the United States, approximately 40,000 cases of Salmonellosis are reported and approximately 600 people die each year. Because many milder cases are not diagnosed or reported, the actual number of infections may be thirty or more times greater. Young children, the elderly, and individuals who are immunocompromised are the most likely to have severe infections (CDC).
The Center for Science in the Public Interest (CSPI) maintains a food-borne illness outbreak database, categorized by food vehicle. CSPI's database was compiled from sources such as the Centers for Disease Control and Prevention (CDC), state and local health departments, and medical and scientific journals. The database is updated regularly, and contains only outbreaks with known or suspected etiology and food vehicles.
Twenty five (1136/4486) percent of all outbreaks in CSPI's database were related to Salmonella. Historically, Salmonella infection has been mainly associated with foods of animal origin, such as beef, poultry, or eggs, but all foods, including produce may become contaminated. During 1990-2003, there were a total of 1,136 Salmonellosis outbreaks and 46,058 cases in CSPI's database. Of the Salmonella outbreaks, ten percent (111/1,136) were related to produce, compared to fifteen percent (169/1,136) in chicken and five percent (59/1,136) in beef. The most common sources related to outbreaks include romaine and iceberg lettuce, various vegetables, and fresh fruits.
As Salmonella often originates in animals, produce outbreaks are likely due to cross-contamination or unsanitary agricultural or processing practices (contact with contaminated water during irrigation, rinsing, and processing of the product or the use of manure as fertilizer). Unlike meat, produce is often not subject to cooking and therefore, Salmonella that is transferred to fruits and vegetables during production is likely to remain on produce until it is consumed.
R.A. Sullivan, OSB, CDRH, FDA, Rockville MD
Background: Against a background of increasingly sophisticated interventional vascular procedures, a descriptive analysis of guidewire-related adverse event reports (AER) is presented.
Methods: The FDA AER database was queried for all guidewire reports received in 2005 and used in the vasculature. Procedural, device and patient-related variables were analyzed using t-test or chi-square statistical testing, with significance specified at 0.05.
Results: A total of 466 reports included 19 Deaths (4%), 204 Injuries (44%) and 243 Malfunctions (52%).Among AERs with available data, patients averaged 65 in age and included 41% females. In 66% of AERs, breakage occurred involving either guidewire tip, body or coating. Reported guidewire diameter was .014 in 75% of AERs. Guidewire fragments were unretrieved from patients in 138 (30%) of cases and among these, 19 (14%) were stented in place.In 131 cases, additional intervention included surgical retrieval or repair (35), percutaneous retrieval (66), and stent placement (10). Compared to routine cardiac intervention, the risk for death or injury was increased if intravascular ultrasound, debulking, magnetic navigation or neurovascular devices involved (p <.001 overall), whereas risk was decreased if peripheral vascular, chronic total occlusion, intravenous line or pacemaker placement. Compared to malfunction AERs, patients with injuries or death were older (p=0.016).Guidewire breaks were more likely to be related to injury while non-break events more likely involved death or malfunction (p<0.00001).
Conclusion: Increased age, guidewire breaks and some concomitant devices were associated with guidewire-related event severity. Unretrieved guidewire fragments are commonly reported and the practice of their management varies.
K. A. Thomas, S. Perry, FDA OWH, Rockville, MD
Background: OWH launched the "Take Time to Care" (TTTC) program in 1998 to provide women with FDA product information.There have been 3 program phases. Phase I: "Use Medicines Wisely" focused on safe medication use. Phase II focused on diabetes. Phase III expanded TTTC to provide information on myriad FDA topics. This poster will delineate some program outcomes which have established TTTC as an award-winning model program.
Methods: The following data were assessed: satisfaction/ program surveys by Merck-Medco and NACDS; FDA and partner-sponsored printing data; and program use reports from FDA Centers, Offices and stakeholders.
Results: Approximately 26 million consumers were reached with TTTC materials. Phase I - Distributed over 6 million medicine record-keepers with 80 groups at seven times FDA's investment. Merck-Medco surveyed 50,000 customers and found a 98.6% satisfaction rate for TTTC. Phase II - NACDS sponsored TTTC events in 22,000 drug stores and worked with ADA to identify consumers for physician referrals. Phase III - TTTC publications were distributed through unique venues such as casinos, CMS, Blue Cross/Blue Shield, IRS mailings, and Dear Abby. Across all phases, OWH developed 40 TTTC fact sheets, brochures, and guides. Partners reprinted, distributed, and translated TTTC materials at their own expense for consumers. National organizations also provided expert consumer training on drug safety, use of medical devices, and nutrition at urban, rural and reservation sites.
Conclusion: Through creative partnerships, TTTC has provided a cost effective way to reach millions of women with easy-to-read materials on the risks and benefits of FDA regulated products
A. Vega1 , S. Brajovic1 , V. Subramaniam2 , J. Lee1 , M. Vieder1 , B. Bradic1 , 1PSI International, 2VHA Pharmacy Benefits Management SHG
Introduction
The MedDRA® terminology is an international terminology employed throughout the entire drug development process and postmarketing activities.MedDRA® is extensive in size and hierarchical and multiaxial in nature.This poster session demonstrates the application and challenges encountered in the use of MedDRA® in the drug safety surveillance process by using the Veterans Health Administration Pharmacy Benefits Management Adverse Drug Event Program (VHA PBM ADE) database as a model.
Methods
The VHA PBM ADE database is a passive surveillance system which collects ADE data from all VHA medical centers and an excellent model of a comprehensive surveillance system using MedDRA® for coding, retrieving, and analyzing ADE information. We describe the drug safety surveillance process and challenges encountered in the application of MedDRA® in each phase.Data retrieval/analysis strategies demonstrated include the use of Standardised MedDRA Queries (SMQs) and multiple products. A case series involving exposure to lipid-lowering products (HMG-CoA Reductase Inhibitors) is presented.
Results
The HMG-CoA Reductase Inhibitors ADE profile identified in the VHA PBM ADE database is in harmony with that described in these products' labeling.Simvastatin had the highest number of adverse event reports containing one or more terms included in the Rhabdomyolysis/Myopathy SMQ.The two System Organ Classes (SOCs) with the highest number of reported reaction terms were the Investigations and Musculoskeletal/Connective Tissue Disorders SOCs.
Conclusion
VHA PBM ADE database is an excellent model of the usefulness of MedDRA® coded and processed ADE information that can be used for product ADE profile monitoring and safety signal generation.
A. Gupta1 , A. B. Ciavarella1 , V. A. Sayeed2 , P. J. Faustino1 , D. A. Volpe1 , M. A. Khan1 , 1DPQR, CDER, FDA, Silver Spring, MD, 2OGD, CDER, FDA, Rockville, MD
Background: Primarily due to high cost, solid oral drug dosage forms are often split by consumers and stored for extended periods time to meet individual needs. To address the impact of this practice on product quality, the stability of split tablets were compared with the intact product.
Methods: Whole and split gabapentin tablets (innovator, generic) dispensed into vials were exposed to 25°C and 30°C with 60% RH storage conditions. Samples were collected at 0, 2, 4 and 9 weeks. Dissolution, potency, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), weight and hardness experiments were used to assess the products.
Results: In general, there were no differences in hardness or weight for the whole or split tablets after storage. Potency experiments detected no significant changes for either product, intact or split. There was a statistically insignificant trend for increased related compound A in the whole and half tablets in the potency experiments at both temperatures. Storage up to 9 weeks did not alter the dissolution of the whole or split tablets as compared to the day 0 samples. The TGA and DSC profiles of all the samples showed no difference over the storage period.
Conclusions: There were no significant changes in the weight, hardness, potency, dissolution, TGA or DSC experiments for the whole or half innovator and generic tablets at both temperatures for the 9 week period. These results show that product quality attributes of gabapentin split-tablets are not changed. These results should not be extrapolated to other drug products.
S. B. Wang, B. Hasselbalch, V. Zenger, J. Gardner, CDER, FDA
Introduction
The Federal Food, Drug, and Cosmetic Act requires FDA to inspect drug manufacturing establishments at least once every 2 years. Limited agency inspection resources compel a risk-based approach to prioritizing inspections of drug manufacturers.
FDA piloted a risk-based model for prioritizing drug manufacturing establishments for CGMP inspection in FY2005 (http://www.fda.gov/cder/gmp/gmp2004/risk_based.pdf). For the FY2006 risk model, FDA re-evaluated risk factors for inclusion, and capitalized upon improved data quality from ORA's Official Establishment Inventory data systems. The purpose of the model was to systematically prioritize the establishments for CGMP inspection based on combined risk scores to maximize public health protection with limited resources.
We added 2 new risk factors and adjusted the model structure and weights to improve site selection with respect to inherent product and process risks. A list of manufacturing sites ranked by risk score was produced. FDA plans to inspect the top 500 scoring sites as a priority in this fiscal year as part of its total work plan on CGMP inspections.
The risk model permits a nationally consistent and systematic selection of firms for surveillance CGMP inspection that focuses on our current public health priorities. FDA will continue to refine the model as these public health priorities evolve.
B. Wang, D. Marinac-Dabic, G. B. Marjorie, CDRH, FDA, Rockville, MD
Background: Vacuum-assisted delivery device (VADD) is one of the major tools in operative vaginal delivery worldwide. In the United States alone, more than 200,000 births each year are vacuum-assisted deliveries (VAD). Continuously monitoring the safety of VADD is critical to ensure safety. Following the United States Food and Drug Administration (FDA) Advisory Warning about the risk of serious injury associated with VADD in May 1998, there was a dramatic increase in adverse event (AE) reports. This study provides current status of AE reports related to VADD.
Methods: The Manufacture and User Facility Device Experience database maintained by FDA was used to identify AE reports between 2003 and 2005. Current AE experience is compared with previous VADD AE reports.
Results: A total of 60 AE reports were identified between 2003 and 2005. Annual
AE reports ranged from 18 to 21. There was a continuous decrease in the number of AE reports since 1998.
Conclusion: The decreasing number of AE reports associated with VADD since the FDA Advisory Warning suggests the necessity of continuous encouragement of AE reporting to enhance safety. Through AE reports, the FDA may conduct investigations to identify the cause, update product labeling, and develop safety educational program for users and public.
C. E. Zinderman, L. Landow, R. P. Wise, CBER, FDA, Rockville, MD
Background:
Dextran 40 and 70, known as clinical dextrans (CDs), are widely used for postoperative thromboembolic prophylaxis. Severe anaphylactoid reactions caused by dextran-reactive IgG antibodies have been reported in 0.002 to 0.12% of CD patients. Dextran 1 pre-treatment significantly reduces this risk.
We estimated in-hospital use of CDs and dextran 1 in the US from 2000-2004 using discharge billing data from the Premier RxMarket Advisor™ sample of hospitals.
Results:
AARs accounted for 90 (24.6%) of the 366 adverse event reports for CDs received by FDA. Among 43 reports with time-to-onset information, all started within 60 minutes after infusion, and 26 (60.5%) began within 5 minutes. There were 15 deaths reported
The hospitalization ratio (which divides hospitalizations where a CD was administered by hospitalizations where dextran 1 was administered) was 28.4:1. The expected hospitalization ratio would be 1:1 if all CD recipients had received dextran 1 pre-treatment.
Conclusions:
Nearly ¼ of CD reports described AARs. Hospital discharge and product sales data in recent years suggest that few CD administrations have included dextran 1 pre-treatment.
R. P. Brown, W. H. Cyr, M. E. Stratmeyer, CDRH, FDA, Silver Spring, MD
Background: Residual ethylene oxide (EO) remaining on the surface of EO-sterilized medical devices has been reported to produce dermal burns and mucosal irritation in some patients. In lieu of conducting irritation tests for every EO-sterilized device, it is possible to derive an upper-bound allowable limit for surface residues of EO that are not expected to produce irritation in patients. An approach for deriving this allowable limit, termed the Tolerable Contact Limit (TCL), is described in the ISO 10993-17 standard. This method was used to derive a TCL for EO and the value was incorporated into the draft ISO 10993-7 standard that is currently under revision.
Method: Published data were evaluated to identify the highest concentration of EO surface residues that did not produce irritation in various experimental systems. This value was modified by taking into account interindividual variability in sensitivity to EO in the human population, the relative sensitivity of experimental animals and humans to the irritant effect of EO and any data deficiency that were present in the studies.
Results: Based on the data reported in four published studies, the weight of evidence suggests that a TCL on the order of 10 ug/cm2 is adequately protective for EO-induced local effects in various tissues.
Conclusions: This represents the first use of the approach defined in ISO 10993-17 for setting a TCL for a compound with irritant effects. Assuming that experimental data are available, this method has broad utility in the consensus standards process for setting allowable limits for irritant compounds.
R. Ouellet-Hellstrom, C. Kornegay, R. Johann-Liang, FDA
Introduction:As part of an ongoing study to understand gender differences in drug risks of hypersensitivity possibly modified by use of estrogen in US adolescent and young adults, we first identified drugs that were most commonly prescribed in this population by gender.
Methods: Drug use data from Verispan's Vector One® which measures retail dispensing of prescriptions was queried for the top 30 drugs used by females (F) and males (M) age 15-45 for 2004. Gender-specific comparisons were made for total prescriptions dispensed, drug class, and specific drugs used and the common drugs for both groups were examined to generate M/F use ratios.
Results: Overall, 2.8 million prescriptions were dispensed for M and 5.7 million for F. The top 30 most common drugs represented 43% of the prescriptions dispensed for F and 38% for M. Common drug categories, 17 (57%) of the 30 drugs, were anti-infectives, anti-depressants, pain management drugs, allergy treatment drugs, thyroid hormone replacement drugs and bronchodilators. Seven (14%) dispensed only to F were hormonal contraceptives whereas cardiac medications (B-blockers and lipid lowering agents), steroids, and stimulants were more common for M.
Conclusions: Females prescription use is twice that of M in adolescent/young adult population although there is a substantial overlap. The three main drug categories common for both genders in this age group are anti-infectives, anti-depressants, and drugs for pain. These results provide a gender-differential risk evaluation of the most common prescription drug exposure in the adolescent and young adult age group that can be used in pharmacoepidemiology.
CATEGORY J: COUNTERTERRORISM AND EMERGENCY RESPONSES. Caviness, FDA, Rockville, MD
During the disasters of the hurricanes in 2005 that repeatedly struck the south and the gulf coast, over a thousand Public Health Service Commissioned Corps Officers were deployed to various sites in several states, not just to New Orleans. A great number f these personnel were from FDA, which reflects highly on the agency and the professionalism of its staff. Over several months of the response and recovery phaces, these officers served in many capacities including examining safe food and water, to providing pharmacy and health care services to the flood refugees. They worked side by side with representatives of the military and the state governments. Many served as liaisons and provided management to the field disaster offices. After serving in one site in New Orleans or surrounding states, many were subsequently deployed to another site when disaster struck again, showing the versitility and "can do" attitude of the Corps, and the support of the FDA and other agencies. This poster provides a snap shot of the range of responses and shows photos of these personnel in uniform, in action at the disaster sites.W. C. Cunningham1 , D. L. Anderson1 , W. Lamont1 , P. South1 , M. Rury2 , G. Beachley2 , J. Ondov2 , 1CFSAN, FDA, College Park, MD, 2University of Maryland, College Park, MD
Background: FDA's Center for Food Safety and Applied Nutrition responds to radiological emergencies that may affect the nation's food supply.
Methods: Standardization, set-up and disassembly procedures, maintenance, analytical parameters, quality control, and analysis procedures were studied for beta- and gamma-ray analysis systems. These systems were optimized so they could be easily packed and transported to an alternate site, assumingly near the site of a radiological event, and provide for rapid and accurate sample analysis. Factors associated with blanks, standards, foods, and reference materials were studied. Standard operating procedures (SOPs) were developed with detailed instructions enabling the measurements to be performed by analysts with no prior radiological training. The gamma-ray system and SOP were tested under a variety of circumstances.
Results: The optimum conditions and procedures for beta analysis included use of adhesive-backed cloth swipes counted in close geometry for maximum sensitivity. No special equipment or modifications were needed. The optimum conditions and procedures for gamma-ray analysis included use of disposable counting containers and only trivial sample manipulations. Analytical parameters included use of a series of calibrations. One piece of apparatus needed to be designed and fabricated. Quality control procedures were very simple for qualitative beta analysis and slightly more elaborate for quantitative gamma-ray analysis.
Conclusions: Systems that augment CFSAN's ability to respond to a radiological event were developed and are available for transport to a location near the site of a radiological event. Accuracy, sample throughput, and ease of use were demonstrated.E. A. Garber1 , K. V. Venkateswaran2 , J. L. Aldrich3 , T. W. O'Brien3 , K. G. Oliver2 , 1CFSAN, FDA, College Park, MD, 2Radix Biosolutions, Georgetown, TX, 3Tetracore Inc., Rockville, MD
Numerous methods have been developed for the detection of proteinaceous toxins. However, antigen specific assays for the screening of samples containing an unidentified toxin are labor intensive, time consuming, and expensive. Alternatively, Luminex xMAP technology enabled the screening of a single sample for the presence of multiple antigens simultaneously.
Samples of beverages were spiked with varying concentrations of abrin, botulinum toxin A, ricin and Staphylococcus enterotoxin B (SEB) and analyzed using a cocktail of capture antibodies conjugated to spectrally unique coded beads. Multiple capture antibodies against different epitopes on the same antigen provided confirmatory tests within the same analysis and reduced the probability of false positives and false negatives that may occur when an epitope was inaccessible or altered. The assay also included two unique internal positive controls for antigen-antibody binding and quantitation. Several chemical and fluorimetric controls were also included such that a typical assay consisted of 17 different antibody-bead conjugates (17-plex).
Abrin, botulinum toxin A, ricin, and SEB were detected in beverages at <5 ppb in the analytical sample; orders of magnitude less than would be considered a health concern. Variations in assay configuration demonstrated that maximum sensitivity was obtained using a monoclonal capture antibody and polyclonal detector antibodies. Luminex technology provided an effective laboratory-based method for the simultaneous screening of beverages for abrin, botulinum toxin A, ricin and Staphylococcus enterotoxin B (SEB).
E. A. Garber1 , J. Dayan-Kenigsberg2 , A. Bertocchi2 , 1CFSAN, FDA, College Park, MD, 2Agence Francaise de Sécurité Sanitaire des Produits de Santé (AFSSAPS), France
Central to the utility of antibody-based assays such as lateral flow devices (LFDs) and ELISAs is the antigen specific mediated apprehension of a labeled detector antibody. Anything that may bind the detector antibody and immobilize it, without requiring the antigen, in a manner similar to the role of the capture antibody used in the LFD or sandwich ELISA, or the plate in a direct ELISA, is a concern since it may cause an increase in the background and false positives. Lectins pose such a problem through their ability to bind the glycosyl residues of antibodies. It was therefore not surprising that the lectin from Triticum vulgaris (wheat) in PBS caused false positives with LFDs designed to detect ricin produced by both Tetracore, Inc. (at > 25 ppm purified lectin or > 2000 ppm stone ground wheat) and a Governmental Supplier (at > 10 ppm purified lectin). The purified lectin also caused false positives with the LFDs produced by Tetracore, Inc. and the Governmental Supplier designed to detect Staphylococcus enterotoxin B (SEB). The inclusion of 100 mM lactose caused a slight decrease in the false positive response generated by the lectin. In contrast, the inclusion of 2.5% (w/v) non-fat milk powder in the analytical samples eliminated the false positives observed with the LFDs.
E. A. Garber1 , J. L. Aldrich2 , J. Wang2 , V. A. Brewer1 , T. W. O'Brien2 , G. Sigal3 , 1CFSAN, FDA, College Park, 2Tetracore Inc., Rockville, MD, 3Meso Scale Diagnostics, LLC, Gaithersburg, MD
Abrin is a potent ribosome inactivating protein (RIP-2) present in beans of Abrus precatorius, commonly known as rosary pea, jequirite bean, and crab's eyes. The possibility that abrin may adulterate food has made the development of assays for the detection of abrin a priority for the FDA. Rabbit derived polyclonal antibodies and mouse monoclonal antibodies were prepared against a mixture of the abrin isozymes. The specificity / cross-reactivity of the antibodies were evaluated against a challenge library consisting of 40 different grains, nuts, legumes, and foods at concentrations of 2, 0.2 and 0.02 % (w/v). ELISA and ECL-based assays were assembled and optimized, the later employing the 96-well format, Sector PRTM 100 ECL detector manufactured by Meso Scale Diagnostics (MSD). Polyclonal (capture) / polyclonal (detection) ELISA, polyclonal / monoclonal ELISA, and polyclonal / monoclonal ECL assays displayed limits of detection (LOD) of 0.1 to 0.5 ng/mL with purified Abrin C and various abrin extracts in buffer. The LOD for abrin dissolved into juices, dairy products, soda, chocolate drink, and condiments analyzed using the ECL assay ranged from 0.1 - 0.5 ng/mL.In contrast, the LODs for the ELISA assays were usually between 1 - 4 ng/mL (up to 20 ng/mL), depending on the assay configuration. In all cases, the LODs were considerably less than the concentration at which abrin may pose a health concern in food.
E.A. Garber, CFSAN, FDA, College Park, MD
D. E. Hanes1 , M. H. Kothary1 , B. D. Tall1 , S. G. Edelson-Mammel2 , D. H. Burr3 , S. D. Weagant4 , 1CFSAN, FDA, Laurel, MD, 2CFSAN, College Park, MD, 3CFSAN, Madison, WI, 4ORA, Bothell, WA
Concerns about bioterrorism require FDA to strengthen methods for the isolation and identification of non-traditional foodborne pathogens. A method for the isolation of Yersinia pestis (Yp) from foods using paramagnetic beads coated with antibodies to Yp and the Pathatrix system for immunocapture has been developed for use by the Food Emergency Response Network (FERN). This study describes the first multi-personnel evaluation of a screening method developed for a non-traditional foodborne pathogen. Personnel from 22 FERN laboratories were brought to the FDA for training and evaluation of the method. Participants were familiarized with the Pathatrix technology using a method for Escherichia coli, and then taught the procedure for isolation of Yp.Participants were given mashed potatoes, lettuce, egg beaters and infant formula spiked with 106 and 104 colony forming units (cfu) of Yp per gram of food. Foods were diluted 1:5 in buffer, and placed in the Pathatrix unit for 30 min of immunocapture. The captured beads were washed, and suspended in 300 µl of PBS. A 50 µl aliquot of beads was plated onto Sheep Blood (SBA), Yersinia Selective (CIN), and Yp Chromogenic (YpCM) plates and incubated for 48 h at 28°C. A 100 µl aliquot of beads was inoculated into 900 ul of Universal Pre-enrichment broth, incubated at 28°C for 48h, and 10 µl was then plated onto SBA, CIN, and YpCM plates. The remaining beads and pre-enrichment broths were analysed in a PCR assay. Colonies exhibiting typical morphology on agar plates were identified as Yp using standard biochemical tests including a modified Lysine Iron Agar slant supplemented with 1% arginine and 1% ornithine, and a Christensen's urea slant. Participants consistently recovered Yp from all foods spiked with 106 cfu/gm by direct plating of beads on CIN and YpCM. Although Yp could be recovered in foods spiked with 104cfu/gm by direct plating, enrichment culture improved recovery. PCR assays using beads or enrichment cultures were positive for Yp in all foods spiked with both concentrations of Yp. These results indicate that this method can be utilized by personnel from different laboratories for the isolation of Yp from foods.
A. E. Hayford1 , M. K. Mammel1 , J. Worley2 , J. E. LeClerc1 , T. A. Cebula1 , 1OARSA, CFSAN, FDA, Laurel, MD, 2JIFSAN, FDA, Laurel, MD
Background: Analysis of single nucleotide polymorphisms (SNPs) is an important genetic tool that provides molecular markers for rapid differentiation of closely-related strains. We have applied the discovery and analysis of SNPs for distinguishing each of the four Shigella sub-groups (dysenteriae, flexneri, sonnei and boydii) and for discriminating individual strains within the same sub-group.
Methods: In silico comparisons of the published genome sequences of Shigella (two sequenced strains from each sub-group) located regions of genetic variation. A BLAST database was constructed for each gene from the eight genomes and each gene was blasted against the database to find genetic matches. The matched genes were aligned and evaluated for potential SNPs sites. SNPs that differentiated the four sub-groups, and those that discriminated individual strains of the same sub-group, were analyzed by Pyrosequencing. Putative SNPs were tested in a set of 95 strains comprising all four sub-groups.
Results: Thirty-one informative SNPs derived from 14 genes for differentiating the test strains were identified. Eleven of the SNPs differentiated strains of the S. dysenteriae, flexneri, boydii and sonnei sub-groups, while 20 SNPs further discriminated strains within a sub-group.
Conclusions: The identification of SNPs in a set of diverse test strains of Shigella provided molecular markers for the rapid assignment of Shigella strains at the sub-species level. The further definition of SNPs that differentiated strains of Shigella within a sub-group, demonstrated here as a proof of principle, would make the method useful for both epidemiological and forensics investigations requiring individual strain identification.
C. T. Jung1 , V. E. Jensen2 , W. F. Pierce2 , B. Y. Danso1 , K. A. Awuah1 , 1CDER, FDA, Rockville, MD 20855, 2CDER, FDA, Silver Spring, MD 20993
The US Public Health Service's (USPHS) first response to Hurricane Katrina involved pre-deploying a strike team of Public Health Officers in anticipation of the medical needs.PHS Officers helped to establish one of the first field hospitals at the Pete Maravich Assembly Center (PMAC), Louisiana State University's (LSU) basketball arena in Baton Rouge, LA.Thousands of evacuees were assessed and treated for acute medical conditions.One critical function of the facility was to provide medications for acute needs and maintenance therapy of chronic diseases.Five USPHS Pharmacy Officers, all from FDA, were faced with setting up the PMAC Pharmacy within a few hours of arriving. Several important challenges included having a limited drug supply, finding a secure area, and communications. The PMAC Pharmacy functioned 24-hours a day, supporting the needs of PMAC patients, and providing clinical consultation to doctors on drug therapy and immunizations. The final pharmacy formulary consisted of over 500 medications.The most common treatments were for cardiovascular diseases, diabetes, and infectious disease.The FDA Pharmacists credit the successful operation of the PMAC Pharmacy due to strong cooperation and teamwork from state and federal agencies, emergency response teams, military forces, LSU facilities, regional hospitals, chain and local pharmacies, pharmaceutical companies, and local volunteers.S. M. Madson1 , E. D. Gonzales1 , L. T. Michel1 , Z. A. Miller1 , M. B. Buen1 , D. E. Farmer1 , P. L. Dexter1 , M. Z. Thomas1 , K. A. Watts1 , K. S. Kreuzer1 , C. L. Burns1 , J. N. Sofos2 , 1FDA Denver District Laboratory, Denver, CO 80225, 2Colorado State University, Fort Collins, CO 80523
A method for use by Food Emergency Response Network (FERN) laboratories to detect Bacillus anthracis in foods was developed by the Center for Food Safety and Applied Nutrition (CFSAN) of the Food and Drug Administration (FDA). This study evaluated the method for use in eight different inoculated food matrices including bottled water, cola drink, apple juice, rehydrated infant formula, tomatoes, meat-based baby food, and canned tuna salad. Six samples (plus uninoculated controls) of each product were inoculated with B. anthracis (Pasteur strain ATCC 4229) spores at low (100), medium (102), and high (105) cfu/mL or g levels. Heated (65°C for 30 min/100 rpm shaking) and unheated inoculated samples were analyzed using the Laboratory Response Network (LRN) real-time polymerase chain reaction (rt-PCR) procedure and by spread-plating onto selective agar media. Portions of heated and unheated samples were also enriched overnight in trypticase soy broth with polymixin B, plated onto the same media, and screened using the rt-PCR procedure. The results of this study indicated that the detection level of the cultural method for most foods was at least 100 spores per mL or g. However, the cultural method was not useful for isolating B. anthracis from tomatoes. Real time PCR was useful as a rapid test for direct screening of samples inoculated with high (105 spores per mL/g) levels of spores, with the exception of meat-based baby food, which interfered with the reaction. FDA's ability to detect potential B. anthracis contamination in foods is improved as a result of this study.
M.A. McLaughlin, CFSAN, FDA, USPHS, College Park, MD
Ricin, the toxin derived from the castor bean (Ricinus communis), is a potential threat to the security of the US food supply.Protocols have been developed by FDA to detect this toxin in foods by use of specific antibodies incorporated into an LFD format.This technology gives the Agency a simple, rapid, selective and sensitive test to use in the field.While these devices are simple to use, they are also subject to environmental factors which can affect their proper operation. Studies were conducted that investigated the effects of temperature and humidity on the operation of the ricin LFDs.The results indicate that the sensitivity of these devices can be enhanced at 37o C relative to room temperature while it is suppressed at 4o C.It was also determined that these devices can be exposed to conditions of 20o C with a relative humidity of 55-60% for 1 hour and still perform to manufacturer specifications.The results indicate that these devices are quite capable of producing valid results in the field but proper attention must be paid to the environmental conditions.
W. R. Mindak, J. Cheng, FDA, College Park, MD
Background:Food processing facilities are vulnerable to intentional tampering events or poisoning with chemical agents. This research project explored the possibility of detecting such an event by the change in conductivity of fruit juice and bottled water.
Method: Apple juice, apricot nectar, cranberry juice, lemonade, orange juice, passion fruit juice and bottled water were spiked with arsenite, cyanide, nitrite, monofluoroacetate and strychnine sulfate. Multiple brands and lots of each food were purchased to determine within lot and between lot variability. The change in conductivity was investigated as a means of detecting the addition of chemical agents. Tests were performed on 10 mL aliquots using 50 mL polypropylene tubes.
Results:Samples were spiked at 0.1, 0.2, 0.5, 2, and 5 mg/mL. Except for strychnine, response was not linear in the food samples indicating that reactions were taking place between agents and the sample. Several agent/food combinations caused color changes. Agent addition at concentrations equal to a lethal dose per serving were able to be detected in most cases and in many cases agent addition at one half the lethal dose per serving concentration could be detected. Results varied between brands.
Conclusion: Conductivity appears to be a means of indication of toxic chemical agent addition to fruit juice and bottled water. Because of the wide variation in conductivity between brands, each brand of a sample should be evaluated on its own. In-line conductivity sensors at food plants might be useful for monitoring for chemical agents.
H. Njapau1 , F. S. Thomas1 , A. Chowdhury2 , S. Trujillo1 , B. J. Cañas1 , D. L. Park1 , 1CFSAN, FDA, College Park, MD, 2JIFSAN/University of Maryland, College Park, MD
Background: The potential vulnerability to intentional adulteration of large quantities of some foodstuffs dictates that risk managers know the physical behavior of specific chemical agents in those foods in order to establish appropriate remedial measures. This study investigated the partitioning of α-Amanitin, Ricin, and T-2 toxin in the different phases (and products) of reprocessed whole milk and salad dressings.
Methods: Whole milk and salad dressings were spiked with the toxins and subjected to simulated industrial processing to obtain skim milk, cream, whey and cheese, and the lipid and aqueous layers of salad dressings. The toxins were quantified using specific ELISA methods. Distribution coefficients, not adjusted for volume/mass differences, were computed from the quotient of the total quantities of toxin in the two major products/ phases and expressed as log10D.
Results: The mean cream/skim milk distribution for α-Amanitin, Ricin and T-2 toxin indicated that 97, 92 and 67% of the recovered toxin, respectively, was in the skim milk.Similarly, cheese/whey partitioning indicated that 13, 19 and 56% of the recovered toxin was in the cheese. Relative to a unity (1.0) concentration in whole milk, only T-2 toxin substantially accumulated in cream (~11x) and cheese (~7x). Preliminary results for α-Amanitin distribution in the lipid/aqueous phases of various salad dressings indicated a tendency to isolate into the aqueous phase (2-55%).
Conclusion: The toxins concentrated in the aqueous or lipid phases of milk depending on solubility. However, this partitioning may not assure complete removal of the toxins from one phase or the other.
G. M. Orlowski1 , G. C. Ziobro2 , E. A. Garber2 , 1JIFSAN, University of Maryland, College Park, MD, 2CFSAN, FDA, College Park, MD
Ricin is a potent toxin derived from castor beans. The protein is a heterodimer made up of an A- and a B-chain. The non-toxic B-chain is a lectin involved in the binding and uptake of ricin, and is thus necessary for its toxicity. This study examined the effects of lactose and freeze-thawing on the detection and lectin binding ability of B-chain in ice cream.
Method:
An acid-treated Sepharose 4B column was used to develop a protocol to assess B-chain lectin binding ability. This assay was based on the volume of 0.25 M galactose needed to elute the B-chain. Initial studies focused on the effects of freeze-thawing on lectin binding ability. To complement the lectin binding study, commercial ELISA kits were used to examine the effect of freeze-thawing on the detection of B-chain in ice cream. The effects of 0.15 M lactose on detection were also examined. Further, the effects of other food matrices (dairy products, juice, pasta, baked goods and PBS), as well as baking and boiling, on the detection of B-chain were determined. Data were analyzed based on comparison to a best fit exponential curve.
Results:
Freeze-thawing had no effect on detection or lectin binding ability. Ice cream partially inhibited the detection of B-chain, while lactose-free infant formula and juice did not. Lactose, a component in the ice cream, diminished detection. The ability to detect B-chain was also reduced in uncooked pasta, while cooking severely inhibited detection.
N. Sergeev1 , A. Rasooly2 , 1FDA/CDRH/OSEL/DB, Silver Spring, MD, 2NIH-National Cancer Institute, Rockville, MD
Background: DNA-microarray technology has revolutionized microbial characterization enabling the detection of thousands of genetic markers simultaneously on a single chip. Polymerase Chain Reaction (PCR), combined with microarray detection, is proved to be an extremely sensitive, specific and high-throughput technique for detection and identification of microbial pathogens. The main shortcoming of the approach, limiting its utilization in FDA's evaluation of microbial contamination of medical devices, food and biologics, is the need for multiple specific PCR primers, rendering the technology unpractical.
Methods: To overcome this problem, in our study we evaluated the performance of several whole genome amplification strategies (WGA) in a combination with microarray detection for the analysis of three important pathogens: E. coli ETEC, Shigella dysenteriae and Salmonella enterica. Evaluated WGA approaches included both random amplification (Phi29 DNA polymerase-based, isothermal Klenow polymerase-based) and PCR-based strategies (multiplex PCR, asymmetric PCR, one-primer amplification of randomly generated genomic library). The performance of each approach was assessed by hybridizing amplicons to microarrays consisting of 20 to 50-mer oligoprobes specific to virulence factors and characteristic genes of the tested pathogens.
Results: We found that hybridization of single-stranded PCR amplicons generated a hybridization signal with specific oligoprobes of all lengths even under high stringency conditions. By contrast, WGA products hybridized well with long oligoprobes (30 to 50- mers) but did not produce any signal with shorter oligoprobes.
Conclusions: These results demonstrate that various WGA strategies have a considerable potential use in microarray-based analysis of bacterial pathogens and are compatible with various microarray platforms employing long oligoprobes.
N. Sergeev1 , A. Matviyenko2 , K. Herold2 , A. Rasooly3 , 1FDA/CDRH/OSEL/DB, Sliver Spring, MD, 2UMD-University of Maryland, College Park, MD, 3NIH-National Cancer Institute, Rockville, MD
Background: Polymerase Chain reaction (PCR) enables rapid and sensitive detection of microbial pathogens. Currently, PCR has a limited use for point-of-care diagnostics (POC) and in FDA's regulatory affairs mostly because PCR thermocyclers are designed as laboratory equipment requiring an external power source.
Methods: We developed a portable, battery powered PCR thermocycler employing a thin-film resistive heater and a regular computer fan for performing a rapid PCR in glass capillaries. Thermocycling conditions were controlled with computer-based pulse width modulation system. PCR (5µl) was performed in capillary cartridges placed on the top of the heating element. PCR amplicons were detected by in-capillary fluorescence and by gel electrophoresis.
Results: A prototype handheld, compact, rapid thermocycler was developed for multiplex analysis of nucleic acids in an inexpensive, portable configuration. The new thermocycler enable heating rates of 6-7 K/sec and cooling rates of 5 K/sec. The energy required by a nominal PCR cycle (20 sec at each temperature) was found to be 57 ±2 J yielding an average power of approximately 1.0 W (not including the computer and the control system). The 35 cycle PCR protocol using a single channel was completed in eighteen minutes. The prototype thermocycler was demonstrated for the detection of three pathogens (E. coli ETEC, Shigella dysenteriae and Salmonella enterica).
Conclusions: This prototype successfully demonstrates our concept of simple and efficient heating/cooling, low cost, rapid amplification, and low power consumption PCR thermocycler. The developed device is suitable for portable DNA amplification applications including clinical, POC diagnostics and field use.
K. Vierk1 , E. Elliot1 , J. J. Guzewich1 , G. Henderson1 , T. Hill1 , K. C. Klontz1 , P. McCarthy1 , S. McGarry1 , C. Purdy2 , M. Ross1 , J. Sanders1 , F. K. Shakir1 , D. Street1 , B. Timbo1 , 1CFSAN, FDA, College Park, MD, 2CFSAN, FDA, Atlanta, GA
Background:
Each year multiple foodborne illness outbreaks associated with FDA-regulated products are reported.
Methods:
Outbreaks are reported to FDA by CDC and State/Local Health Departments and are entered into an Epi Info™ database if an FDA-regulated product is implicated in causing human illness. Data are analyzed using SAS® software.
Results:
For 2004 and 2005, there were 66 outbreaks of FDA-regulated products implicated in causing 3,557 cases of human illness. Although the number of outbreaks decreased from 38 in 2004 to 28 in 2005, a comparable number of illnesses were reported for each year (1,799 in 2004 and 1,758 in 2005). Bacteria were implicated most often as the cause of outbreaks in 2004 (63.2%) and 2005 (60.7%), accounting for 71.4% and 41.5% of illnesses, respectively. In addition, parasitic illnesses increased from 191 illnesses (10.6%) in 2004 to 622 illnesses (35.4%) in 2005. Produce and seafood were most often associated with the outbreaks during this period. However, produce and processed food items accounted for the greatest number of illnesses.
Conclusion:
Outbreaks associated with bacterial agents have been implicated in the majority of outbreaks and illnesses in 2004 and 2005. The greatest number of illnesses was due to bacterial infection during this period, but an increase in parasitic illnesses also occurred in 2005. While a variety of products are implicated, produce and seafood accounted for the greatest number of foodborne outbreaks in 2004 and 2005.
F. K. Shakir1 , P. V. McCarthy1 , J. J. Guzewich1 , C. R. Braden2 , K. C. Klontz1 , C. W. Hedberg3 , K. E. Fullerton4 , A. Bogard5 , M. Dreyfuss6 , K. Larson7 , D. Vugia8 , D. C. Nichols9 , V. J. Radke2 , T. F. Jones10 , 1FDA, Center for Food Safety and Applied Nutrition, Washington, DC, 2CDC, Atlanta, GA, 3University of Minnesota, Minneapolis, MN, 4CDC, Atlanta, GA, Atlanta Research and Education Foundation, Atlanta, GA, 5Minnesota Department of Health, Minneapolis, MN, 6USDA, Food Safety and Inspection Service, Washington, DC, 7Maryland Department of Health and Mental Hygiene, Baltimore, MD, 8California Emerging Infections Program, Oakland, CA, 9New York State Department of Health, Albany, NY, 10Tennessee Department of Health, Nashville, TN
Background: FoodNet sites report foodborne outbreaks to CDC's national electronic Foodborne Outbreak Reporting System (eFORS). eFORS provides a standard format for reporting epidemiologic, laboratory, environmental, and contributing factor (CF) data. CFs describe how microbes contaminated food, how they increased in number or produced toxin, or how mechanisms to reduce or eliminate them failed.
Methods: We analyzed eFORS data from participating FoodNet sites from1999 through 2002 to determine the completeness of CF information and determine the CFs identified in produce-associated outbreaks.
Results: From 1999 to 2002, FoodNet sites reported 890 outbreaks; 469 (53%) had an etiology identified, 365 (41%) had CFs identified, and 457 (51%) had a food implicated. Produce was implicated in 97 of 457 (21%) outbreaks. Of the produce-associated outbreaks 63 (66%) had an etiology confirmed or suspected, and of these 43 (68%) were due to norovirus and 20 (32%) were due to bacteria. In the 43 produce/norovirus outbreaks, CFs were reported in 27 (63%); handling food by an infected person or carrier of the pathogen was reported in 18 (42%). In the 20 produce/bacterial outbreaks, CFs were reported in 12 (60%).
Conclusions: Contributing factors were reported for over 60% of produce-associated norovirus or bacterial outbreaks. In produce-associated norovirus outbreaks bare-hand food contact and food handling by an infected person were the factors most often cited. In produce-associated bacterial outbreaks contamination of raw product or ingredients by pathogen from animal or environment was the factor most often cited as contributing to outbreaks.P. Whittaker, J. B. Day, S. K. Curtis, F. S. Fry, CFSAN, FDA, College Park, MD
Rapid capillary gas chromatography (GC) with flame ionization detection was used to determine the cellular fatty acid profiles of Francisella tularensis. Two subspecies of F. tularensis, the live vaccine strain (LVS) derived from holarctica and novicida strain Utah 112 (U112), were used to compare the extracted fatty acid methyl esters (FAMEs). A data set for the two subspecies was prepared using fatty acid profiles of bacteria grown on two types of media, Mueller-Hinton and enriched chocolate agar (cysteine heart agar supplemented with 5% rabbit blood [CHAB]), and harvested at various time intervals (day 1 through day 4) with replicates prepared on different days. A total of 204 samples were analyzed. The results showed that these fatty acid quantitative profiles were unique for each of the subspecies and that they could be used as a fingerprint for the organism. It was determined by the rapid method that approximately 88% of the fatty acids in both the LVS and U112 strain included six saturated fatty acids 10:0, 12:0, 14:0, 16:0, 18:0, 20:0, and four hydroxy fatty acids 10:0 2OH, 16:0 3OH, 17:0 3OH, 18:0 3OH. Data analysis and determination of clustering were performed by principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA). Both PCA and SIMCA showed clear separation of the LVS and U112 strain and would be useful for prediction of unknowns. In summary, analysis of FAMEs from F. tularensis subspecies LVS and U112 grown on CHAB or Mueller-Hinton media, and using a rapid GC method can provide a sensitive procedure for identification of these organisms.R. M. Jacobs1 , P. T. Palmer2 , 1San Francisco District Lab, ORA, FDA, Alameda, CA, 2San Francisco State University, San Francisco, CA
Background: Energy dispersive x-ray fluorescence (EDXRF) technology to detect and quantify toxic elements has dramatically improved in the last 25 years. Over the last half decade portable EDXRF (PEDXRF) devices have emerged. These devices have impressive capabilities. In addition to being fully portable hand-held devices, their on-board functions have enabled them to accomplish an assortment of applications. The design of these devices has been focused on the non-scientific user. Some devices bear identification algorithms, the ability to display emission spectra, and the ability to quantify elements. Some are able to determine many toxic elements in ppm concentrations in foods. A Niton PEDXRF was used to evaluate a number of FDA samples (e.g., consumer complaint samples that caused injuries, samples of Asian patent medicines, dietary supplement ingredients both in field locations, e.g. Postal facilities and in the lab. Quantitative assessments of these products were conducted in the lab.
Methods:Samples that were chosen for evaluation were either ones from poisonings, ones that were Asian patent medicines with known additions of toxic elements, or samples where toxic elements were found by chance during the course of quantitative analysis by other techniques. One poisoning case involved dairy cows that had been accidentally poisoned with a chromate compound. These samples (cow hide) were obtained after the toxic substance had been identified. In field investigations, some samples, e.g., Asian patent medicines were initially tested through their packaging, e.g., blister packs. Other preparations, e.g., tablets and powders were placed in small testing vials equipped with a Mylar window and tested in "bulk mode". This mode yields several types of data outputs: algorithm based identification, the concentration of the element (when the sample is homogeneous), and the emission spectrum of the sample. Tests conducted in the lab were typically preceded by sample pulverization. The tests were conducted with a Niton XLi-728e. This model was equipped with dual radioisotopic sources, 109Cd and 241Am for target analyte excitation. Samples were typically excited for a 2 minute period using the 109Cd source. Typical target analytes for this mode were Cr, Fe, Pb, Hg, As, Se.
Results: 1. Examination of a consumer complaint sample involving a human poisoning from As in a food condiment accidentally contaminated with sodium arsenite, yielded nearly instantaneous identification of the presence of arsenic and comparable quantitative results by PEDXRF and ICP-MS, 244 mg/kg and 233 mg/kg, respectively. 2. An Ayurvedic medicine, Pushpadhanwa (a fertility medicine), was found to contain percent levels of Fe, Pb, and Hg. This product had been responsible for several serious injuries (e.g., spontaneous abortion, illnesses requiring hospitalization). While the lead content had been found by others, the product also contained undiscovered and equivalent amounts of mercury and iron. PEDXRF identified these ingredients almost instantaneously. 3. The analysis of an Ayurvedic medicine ingredient, Kapikachu powder, by PEDXRF and ICP-MS yielded highly similar results for arsenic, 203 mg/kg and 181mg/kg, respectively.4. Using the PEDXRF on a baby food sample reported to contain metallic mercury, the device quickly identified the "mercury" as mixture of aluminum and iron that was trapped in the glass. This finding averted undue concern regarding the safety of the product. 5. "Chromium Cow":While this turned out to be an on-farm accident, the identification of the causative agent took a couple of weeks. Examination of the exterior surface of the hide using the PEDXRF revealed, nearly instantaneously, extraordinary high amount of chromium was on the exterior surface compared to the very low levels normally encounter in tissue. The use of this device on the farm could have averted many of the suspicions of the identity and origin of the poison.
Conclusions:PEDXRF devices are able to rapidly identify and often quantify various ranges of toxic levels, i.e., from relatively low ppm levels to percent levels in foods and other sample types. These devices can often detect these elements through packaging and are ideal for field investigations. Laboratory use of the PEDXRF can allow much more rapid response to samples that may contain hazardous levels of toxic elements and therefore resolution of regulatory status of a sample. PEDXRF and laboratory grade EDXRFs could fill very important niches in FDA's inspectional and laboratory capabilities in detecting toxic elements in foods, Asian patent medicines, and other regulated products.
S. D. Torosian1 , P. Regan1 , A. B. Margolin2 , 1FDA, 2University of New Hampshire
Background: The Interim CT Yersinia pestis (YP) Screening Method used by the FDA requires a minimum of 4 to 6 days for results. Shortening that time would better protect the public and conserve resources. Currently the method uses a non selective enrichment broth. Incorporating a selective enrichment broth can promote an increase in YP numbers and limit food flora from blocking detection.
Methods: Three candidate enrichment broths are compared in this study. The first, HISA is comprised of heart infusion broth (HIB), Irgasan and sodium azide (SA). The second HBSAPCR has HIB, bile salts (B), SA, Phosphomycin [P] and Congo Red [CR]. The third, HPTPCR, has HIB, Potassium Tellurite [PT], P and CR. Lettuce (25g) was combined with 125ml of selective enrichment broth and approximately 1000 cfu of YP. The sample was stomached for five minutes then incubated at 26°C for 12 h. After incubation, 100ul of enrichment was plated directly onto Yersinia pestis Chromagenic Agar (YPCM) and incubated at 26°C for 48 h.
Results: The HISA enrichment yielded the best results in cfu of YP recovered and limitation of food flora. Undiluted and 1:100 dilutions of enrichment consistently and reliably identified YP. HBSAPCR was nearly as efficient but inhibited PCR assay. HPTPCR was least efficient. Levels of PT needed to effectively suppress flora also inhibited YP growth.
Conclusions: The incorporation of selective enrichment will shorten the period needed to obtain results by 36 hours. Successful enrichment of lettuce, a high flora food, indicates potential effectiveness of selective enrichment of YP from other foods.
CATEGORY K: REGULATORY SCIENCES, SCIENCE COMMUNICATION, POLICY, LEVERAGING, OUTREACHA. M. Api1 , D. A. Basketter2 , P. A. Cadby3 , M. F. Cano4 , G. Ellis5 , G. F. Gerberick6 , P. Griem7 , P. M. McNamee6 , C. A. Ryan6 , R. Safford2 , 1Research Institute for Fragrance Materials, Inc., 2SEAC, Unilever, 3Firmenich, Inc., 4LVMH, 5Givaudan France Fragrances, 6The Procter & Gamble Company, 7Clariant Producke GmbH
Some of the chemicals in common use today may have the potential to cause dermal sensitization. However, the fact that a chemical is a skin sensitizer does not mean it cannot be formulated into consumer products at safe levels. This is also the case for fragrance ingredients. Based on advances in our understanding of a range of factors associated with the induction of dermal sensitization, it is possible to conduct an exposure-based quantitative risk assessment (QRA) for induction of dermal sensitization to determine safe levels of fragrance ingredients in different consumer product types. Key steps of the quantitative risk assessment process are determination of benchmarks (No Expected Sensitization Induction Level or NESIL); application of sensitization assessment factors (SAF) and calculation of consumer exposure (CE) through product use. Using these parameters, an acceptable exposure level (AEL) can be calculated and compared with the consumer exposure level (CEL). The ratio of the AEL to CEL must be favorable to support the safe use of the skin sensitizer. This ratio must be calculated for the skin sensitizer in each product type. This poster provides an overview of the principles of exposure-based QRA as applied to fragrance ingredients and provides a practical example using a fragrance ingredient in different product types.
J. Z. Beer, W. H. Cyr, S. G. Coelho, B. Z. Zmudzka, S. A. Miller, CDRH, FDA, Rockville, MD
Background: In 1994, the American Medical Association (AMA) and the American Academy of Dermatology (AAD) requested that the FDA ban the use of sunlamps for cosmetic purposes. FDA concluded that a ban was not warranted. Recently, the AMA and AAD urged the FDA to ban the use of sunlamps for persons <18 years of age. However, FDA has jurisdiction primarily over manufacturing (not use) of sunlamps.
Regulatory activities: FDA supports state or local governments in their efforts to regulate sunlamps use. In addition, FDA monitors reports suggesting beneficial effects of UV exposure that may be associated with enhanced production of vitamin D. Some governments and the WHO recommend that humans seek supplemental vitamin D or regular small UV exposures. These developments impact on our efforts to modernize FDA regulations in this area. FDA takes an active part in the revisions of the International Electotechnical Committee standard for sunlamps and is considering adopting portions of this standard for use in the U.S.
Research: To reduce the risks taken by deliberate tanners, we conduct a clinical study to determine if the doses currently used for tanning can be reduced. The results show that a comparable visual tan can be produced with 3-4-fold smaller cumulative UV doses than currently indicated in our guidance. A reduction in the UV burden to deliberate tanners should result in a reduction of the carcinogenic and photoaging effects of UV exposure - at least in those for whom exposure to sunlamps contributes significantly to overall UV burden.J. H. Boal, D. D. Christodoulou, R. S. Harapanhalli, cder
Introduction:
Tobacco consumption is known to cause nicotine addition. Nicotine replacement therapy (NRT) is a form of smoking cessation based on substituting nicotine in a pharmaceutical product for other forms of tobacco, such as cigarettes, cigars, and smokeless forms like chewable and snuffable tobacco. Regulation of this class of products poses unique challenges.
Methods:
Several NDAs and INDs were analyzed for the nature and extent of CMC information. Additional literature surveys were carried out on the emerging approaches to smoking cessation. Unique scientific and regulatory issues were identified and assessed.
Results:
Tobacco-free blends and snus containing low levels of added nicotine (e.g. nicotine polacrilex) and genetically modified tobacco with low nicotine content were utilized in the development of NRTs. Several manufacturing issues pertaining to plant cultivation, harvesting, drying/curing, pulverization, decoction, expression, aqueous extraction, etc. were identified during the reviews. Additionally, controls to limit the presence of pesticides, microbial content, nitrosamines, polyaromatic hydrocarbons, etc. were needed to assure safety of these products.
Conclusion:
NRTs are an important line of treatment for smoking cessation. The complexity of their chemistry, manufacturing, and controls requires careful assessment and oversight. The Agency guidances on botanicals and biotechnology products may be applied, as appropriate, in the assessment of these products.
J. H. Boal, R. S. Harapanhalli, CDER, FDA
Introduction:
A mu opioid agonist, fentanyl is a highly potent opioid drug with possible narrow therapeutic range in a given individual. In addition to parenteral dosage form, oral tranmucosal fentanyl and fentanyl transdermal systems are the approved products for the treatment of breakthrough and chronic pain.
Methods:
NDAs, INDs, and literature were surveyed for the emerging dosage forms for the systemic delivery of fentanyl.
Results:
Due to its rapid onset and short duration of action and extraordinary cardiovascular safety, fentanyl continues to be developed as an opioid analgesic of choice.Recent developments have included sublingual transmucosal tablets and sprays, buccal mucoadhesive patches, intranasal sprays, MDI/DPI for inhalation, and transdermal passive and iontophoretic patches. The review of drug-device combination products of fentanyl required collaborative interaction with both the review divisions and compliance offices of the CDRH. Critical quality attributes and critical process parameters pertaining to both fentanyl and the delivery devices were identified in the review of these applications.
Conclusion:
Pharmaceutical development of highly potent drugs with narrow therapeutic range such as fentanyl poses a variety of challenges in terms of control strategies to limit the extent of overpotency, since no much room exists for error or variation.Identification and risk management of failure modes is critical to the regulation of the dosage forms containing fentanyl.
I. A. Chang1 , H. M. Luu1 , J. Hutter1 , M. Katzper2 , C. S. Kim3 , 1CDRH,FDA,, Rockville MD, 2CDER, FDA, Silver Spring MD, 3CFSAN, FDA, Laurel MD
Introduction: Modeling is used throughout the FDA. Frequently, practitioners of mechanistic methodologies find themselves isolated and are unaware of inter-center resources. We recognize that an organized forum would be more effective in bringing broad experience and technical expertise to bear on problems. Experiences and technical knowledge that bear upon regulatory issues could be shared and joint solutions reached.
Methods:We have established the Inter-Center Modeling Consortium as a forum for the exchange of modeling ideas. The purpose of this group is to serve as a contact point for FDA scientists who encounter difficult modeling issues. While informal means have been used to share ideas, our consortium represents an additional resource. Given the future physical proximity at White Oak, this intra-agency consortium will allow the FDA to take maximal advantage of joint resources and allow for increased productivity.
Results: We have had several initial meetings. The group has extensive experience in the areas of electromagnetics, system dynamics, mass balance, heat transfer, tissue injury, and physiologically based modeling. In our meetings we have been able to share new perspectives on regulatory problems.
Conclusions: We anticipate that the Inter-Center Modeling Consortium will enable us to keep pace and advance our understanding of modeling tools used to protect the public health. Participation in our consortium is invited.
D. D. Christodoulou1 , R. S. Harapanhalli2 , 1Review Chemist, ONDQA, CDER, FDA, 2Branch Chief, ONDQA Branch V, CDER, FDA
Introduction: A pharmaceutical development report (PD) is a documentation of evolution of the formulation and manufacturing process during the course of IND development. A clear documentation and justification are crucial to the bridging of formulations and processes to ensure meaningful outcomes in clinical studies.
Methods: PD was evaluated to understand its role in the assessment of a recently submitted NDA. Elements of the PD such as design of experiments, excipient selection, salt selection for the drug substance, formulation development, process development, scale up and commercialization strategy, etc. were assessed to understand the critical quality attributes and process parameters that were needed minimize risk of product performance failure. Draft ICH Q8 guidance was consulted.
Results: The product is a bilayer extended-release tablet made by high shear wet granulation. Dissolution was identified as a critical quality attribute to evaluate formulation prototypes. The test method was shown to discriminate against the use of unsuitable excipients. The formulation was optimized to achieve a pH independent dissolution profile and was made moisture-resistant. Based on the design of experiments, the criticality of physical properties of the excipients and the drug substance were evaluated and appropriate controls identified. Design space for the wet granulation process was identified.
Conclusion: The PD highlighted critical product quality attributes and manufacturing process parameters and proved useful in setting risk-based specifications. A well-documented PD is expected to facilitate a science and risk-based assessment of an NDA and help provide post-approval regulatory flexibility.
S. Gardner, M. Brady, H. Albersheim, S. Berman, J. Cope, M. Eakle, P. Jahnes, P. Jones, R. Lazerow, M. Mendelson, A. Pinkos, E. Skoda, M. Warner, M. A. Wollerton, A. A. Ciarkowski, CDRH Home Health Care Committee
When CDRH began regulating medical devices in 1976, most devices were designed for use by professionals in the hospital. Devices, such as infusion pumps, distributed 10 or 20 years ago (legacy devices) remain in use, are often complex, and without information to describe their operation.
The lack of information about these legacy devices has several roots:
To address the issue of medical device use in the home environment, the Center for Devices and Radiological Health (CDRH) established the Home Health Care Committee (HHCC). HHCC convened open public meetings with interested parties where we identified a need for providers to access information about devices used in the home.
As a pilot project to address the gap of information available for home use devices, HHCC established a repository for infusion pump manufacturers to voluntarily submit their device labeling and instructions. To make this repository of information accessible, HHCC established a public-private partnership to develop a database and a website.
To date, our partnership has developed a user friendly database, populated the database with information about infusion pumps, and provided an internet link for queries.
K. C. Dada, G. Creath, M. E. Kremzner, R. Chhabra, CDER, FDA, Rockville, MD
Introduction: The duty of the Division of Drug Information (DDI) is to support CDER's mission by assisting all inquirers and providing useful, accurate information in a timely manner.
Objectives: DDI initiated a survey to determine the timeliness and the level of satisfaction obtained through the exchange of information among DDI and the customers we serve via e-mail.
Methods: A voluntary 5-question Internet based Likert scale survey.Results: A total of 1,214 responded out of 4,273 who were sent the survey over 5 quarters (29%). 31% of the inquiries are made by patients or consumers. 12% of the inquirers were located internationally. 50.04%(595/1189) of respondents were very satisfied with the response. 64.23% (747/1189) were very satisfied with the amount of time that it took to receive a response. 58.00% (689/1188) indicated that the information was helpful. 86.90% (1022/1176) felt that the service provided was professional. 78.22% (923/1180) indicated that they would use the service again.
Conclusion: DDI is meeting mission goals and is providing outstanding quality of service to CDER's customers. DDI measured service provided by analyzing the parameters of demographics, location, satisfaction, timeliness, usefulness of information provided, professionalism, and willingness to use the service again.
Discussion:DDI assumed responsibility of responding to external e-mails in 1998. Since that time the rate of incoming e-mails has jumped by 23% each year.
As DDI moves forward with measuring our e-mail service we plan on sharing our survey with other federal agencies that want to implement a measurement tool for customer satisfaction.
J. Derbis1 , T. Toigo1 , B. Evelyn2 , 1OC, FDA, Rockville, MD, 2OC, FDA, Rockville MD
Background: Section 113 of the 1997 Food and Drug Modernization Act creates a public resource for information on studies of drug and biological products to treat serious or life-threatening diseases. The National Library of Medicine (NLM), FDA and others, developed the Clinical Trials Data Bank, ClinicalTrials.gov. Throughout 2002 and 2004, FDA educated sponsors about Section 113 and measured compliance with listing required trials in ClinicalTrials.gov. The current project examined sponsor compliance in 2005 and also investigated reasons why sponsors were not submitting trials to ClinicalTrials.gov.
Methods: FDA reviewed 122 new commercial protocols submitted to CDER's Division of Oncology Drug Products between May 1 and July 31, 2005 to determine whether the protocol met Section 113 requirements for listing in ClinicalTrials.gov. Reports prepared by NLM were used to verify whether IND protocols submitted to CDER were listed in ClinicalTrials.gov. Follow-up telephone calls were made to sponsors who had trials that met the listing criteria and did not appear to be listed in ClinicalTrials.gov.
Results: Compliance rates for the years 2002, 2004, and 2005 were 61%, 81%, and 71% respectively. Follow-up with sponsors revealed that trials meeting 113 requirements were not listed for a variety of reasons including, among others, termination of the IND, withdrawal of protocol from the IND, or lack of knowledge about the law.
Conclusions: Compliance with the legislation has improved since the FDA guidance issued in 2002 but there is still room for improvement in participation in ClinicalTrials.gov by pharmaceutical company sponsors.
B. M. Derby1 , A. S. Levy2 , 1CFSAN, FDA, College Park, 2CFSAN,FDA,College Park
Background: FDA faces First Amendment legal challenges to food labeling regulations for health claims.In response to recent court rulings, petitioned health claims that do not meet a "significant scientific agreement" standard have been allowed under enforcement discretion, provided they include a disclaimer about the strength of science supporting the claim This study examines options for conveying different levels of scientific support for label health claims.
Methods: A mall-intercept experimental study examined the effectiveness of a four-level scheme for qualifying health claim statements on food labels.Four options were tested across four types of food products.Two options used wording to convey level of scientific support and two used "report card" grades (B, C or D). A sample of 1,920 adults was randomly assigned to disclaimer/control label conditions. The study looked at the effects of disclaimers on perceptions of scientific certainty and the effects of disclaimers on product perceptions.
Results: Disclaimers that relied on wording variations to convey levels of scientific support were ineffective. Report card rating schemes were more successful, but also created some incorrect inferences. Some disclaimers resulted in more positive impressions of a food product than health claims without a disclaimer.
Perceptions of scientific certainty and product health benefits were affected by consumer prior beliefs and their familiarity with the diet-disease relationship..
Conclusions: Consumer research offers insights into how consumers react to label information and the difficult communication challenges presented by disclaimers.
S. Dhruvakumar, PETA
Before a drug enters human clinical trials, pharmacokinetic/dynamic parameters ("ADME") and toxicity must be assessed, and few non-animal methods are currently accepted. Animals are also used for quality control in drug production. Because of the global nature of the drug market, the International Conference on Harmonization (ICH) was established in 1990 to align regulatory requirements across key regions. ICH consists of regulators and industry groups from Europe, Japan, and the U.S., as well as several observers. Amongst its other activities, ICH publishes consensus guidelines for preclinical testing which have contributed to a decrease in duplicative animal testing across regions. However, the system is fallible: In 2001, Japan requested additional preclinical testing of Oxycontin in beagles, even though the drug had been on the U.S. and European markets for 30 years. In addition, ICH guidelines have largely not incorporated validated "3Rs" methods. The coalition of animal protection groups known as "ICAPO" (International Council on Animal Protection at the OECD) has formed a sister organization, "ICAPI," to address animal testing issues at the ICH. ICAPI has made a formal request for observership status at ICH meetings in order to liaise more efficiently with other global pharmaceutical stakeholders. Though ICH activity has tended to focus on the retrospective alignment of methods long-accepted in all member regions, ICAPI is well positioned to help expand this focus to harmonizing acceptance of emerging alternatives by bringing 3Rs methods to the table in a timely manner, thus facilitating their incorporation into ICH guidelines and their global adoption.H. J. Duggirala1 , S. Gola2 , T. P. Gross1 , A. Boam1 , 1CDRH, FDA, Rockville, MD 20850, 2Case Western Reserve University, Cleveland, OH, 44106
Introduction: The use of drug-eluting stents has drastically improved the outcomes of traditional percutaneous coronary interventions (PCI). However, soon after the first approval in April 2003, FDA received multiple reports of sub-acute thrombosis associated with the stent. It has been shown that the proper use of antiplatelet therapies such as clopidogrel or ticlopidine can reduce the risk of stent thrombosis. CDRH collaborated with the American College of Cardiology (ACC) to gather information on drug-eluting stent procedures, including the utilization of antiplatelet therapy.
Methods: The ACC manages the National Cardiovascular Data Registry (NCDR). This registry collects information from institutions nationwide on factors such as patient demographics, revascularization procedures, and procedural medications. CDRH obtained registry data from April 2003-April 2004.
Results:During the study period, the NCDR received information on 94,550 procedures. A total of 46.8% of patients were administered clopidogrel or ticlopidine any time during the procedure. In addition 48.5% of patients administered antiplatelet therapies within 72 hours of the procedure. Approximately 3.61% of patients were not administered therapy and therapy was contraindicated in 1.1% of patients.
Conclusion: More than 95% of patients implanted with drug-eluting stents in the study period were administered antiplatelet therapy. The product label notes that in clinical trials clopidogrel or ticlopidine was administered pre-procedure and for a period of 6 months post-procedure. It is important for providers to understand the benefit of antiplatelet therapy during the stenting procedure and that patients should continue the therapy for the course prescribed to decrease the likelihood of stent thrombosis.
B. F. Fritsch, C. T. Jung, B. M. Davit, CDER, FDA, Rockville, MD
Background: In 2003, President George W. Bush announced the President's Emergency Plan for AIDS Relief (PEPFAR) Program which provides $15 billion over a 5-year period for emergency relief to nations such as Africa, Asia and the Caribbean whose populations suffer from the high incidence rates of HIV/AIDS. In 2004, DHHS Secretary Tommy Thompson announced that FDA would implement a new, expedited review process to ensure that the United States (U.S.) could provide safe and effective antiretroviral (ARV) drugs to these developing countries.
Methods: The Office of Generic Drugs (OGD) encouraged U.S. and foreign firms who were developing generic ARV drugs to treat HIV disease to submit Abbreviated New Drug Applications (ANDAs) under the PEPFAR program. To support this important public health initiative and meet approval timelines under PEPFAR, all OGD divisions (Labeling and Program Support, Bioequivalence and Chemistry) review these ANDAs under a new expedited process. This expedited ANDA review process required the OGD to implement many process changes. For example, the OGD initiated a rolling review approach for PEPFAR applications. The PEPFAR Program presented a formidable challenge for staff and management.
Results: To date, under the PEPFAR program, 4 generic ARV drugs were fully approved and 10 ARV drugs were tentatively approved. The average approval time was approximately 6 months.
Conclusions: The impact of OGD's involvement in the PEPFAR program is seen in the greater accessibility to affordable safe and effective ARV drugs. Generic ARV drugs approved under the PEPFAR program are currently being purchased and distributed in countries through programs such as The Global Fund.
F. Fang, R. Adams, H. A. Hahm, CDER, FDA, Rockville, MD
Background: There is currently no USP method for determining disintegration times for ODT's.Therefore, FDA recently recommended using a modified form of the USP disintegration test <701>. However, this test uses a disintegration medium of about 900 mL and a vigorously oscillating apparatus which provide conditions far from being physiological. In this study, a quick and simple test was designed to aid regulatory review scientists in evaluating the ODT's.
Methods:Four over-the-counter (OTC) products were evaluated: one labeled "Dissolves in an instant," one labeled "Dissolving Tablets," and two labeled ODT's. In addition, two different formulations of ODT's submitted to the Office of Generic Drugs were tested. Using a disposable syringe, 1mL of water was delivered directly to the tablet placed on a flat surface. Completeness of disintegration of the tablet was checked by the manual palpation of the tablet at the end of 30 seconds.
Results: Of the OTC products, the two labeled as ODT's completely disintegrated, whereas, the other two did not. Of the two ODT formulations submitted to the Office one of them completely disintegrated, whereas the other one did not.
Conclusion: The proposed test method requires minimum equipment, allowing review scientists to visualize in office settings the disintegration of ODT's submitted for approval. It can serve as a quick screening tool for review scientists to decide whether or not a dosage form is appropriately labeled as an ODT. An incomplete disintegration may require further laboratory testing or justification by the firm to be labeled as ODT.
A. A. Hakim, R. S. Harapanhalli, CDER, FDA
Introduction:
A well-known anti-coagulant, Heparin (Mr 2000-50,000, Mr (av) is 14,000 D) is a polydisperse, highly sulfated polysaccharides consisting of repeating 1 → 4 linked hexuronic acid residues(L-iduronic acid and D-glucuronic acid) and glucosamine sugar residues (N-sulfated, O-sulfated and N-acetylated) that bind to coagulation proteins, complementary proteins and growth factors to regulate a variety of biological activities. Heparin fragments derived from its controlled depolymerization have shown clinical benefits of modulated anti-thrombotic and anti-coagulant activities.
Methods:
Various INDs, NDAs, and scientific publications in the literature were analyzed for the nature and extent of CMC information provided to support pharmaceutical product quality and comparability.
Results:
Information on the origin of animal and tissue sources of isolation of heparin and the experimental details of its controlled and region-selective depolymerization were described. The experimental techniques included 2D NMR and MS (electrospray, MALDI), Chromatography (SAX-HPLC, GPC, etc), electrophoresis (capillary, gel, etc), enzymatic analysis, labeling with florescent tags, disaccharide units and end units determination, determination of sulfate/carboxylate ratio, anti-Xa and Anti IIa activities, and anticoagulation tests.
Conclusion:
Being heterogeneous polysaccharide mixtures, heparin and its fragments require in-depth knowledge and experience in the pharmaceutical assessment and regulation. Comparative analysis of product identity and quality resulting from various methods of depolymerization of heparin require in depth analysis of scientific literature, results of bio-analytical methods, assessment of biopolymer to polysaccharide contents, sequence analysis of functional groups, disaccharide content, biological activity, and other parameters.
M. J. Kim, S. M. Huang, F. W. Frueh, A. N. Rahman, Y. M. Choi, K. M. Robie Suh, G. Shashaty, R. Rieves, L. J. Lesko, CDER, FDA, Silver Spring, MD
The use of pharmacogenomic (PG) information to support the dosing, safety and efficacy of drugs is emerging. Warfarin, an anticoagulant with a narrow therapeutic range, is indicated for the prevention and/or treatment of thromboembolic events. The major adverse event associated with warfarin is bleeding. The S-enantiomer (the pharmacologically most active form) of warfarin is metabolized to 7-hydroxywarfarin by CYP2C9. The frequencies of the CYP2C9*2 and 3 alleles are approximately 11% and 7%, respectively, in Caucasians. CYP2C9*2 and *3 variant alleles decrease CYP2C9 enzymatic activity by 30% and 80%, respectively. Patients with these variant alleles have lower clearance of S-warfarin compared to patients with the wild type (*1/*1) resulting in a lower daily warfarin dose requirement to achieve a given INR. In addition, these patients require a longer duration to achieve stable dosing and have an increased risk of a serious bleeding event (HR, 2.39; 95% CI, 1.18-4.86). VKORC1 has an independent effect on warfarin dose requirements and patients with certain genetic variations in the VKORC1 gene require lower warfarin doses to achieve adequate anticoagulation. Combining information from CYP2C9 and VKORC1 genotyping along with additional factors, such as age and body weight, explain up to 56% of the variability in warfarin dose requirements between patients. Thus, PG testing has the potential to better inform individual dosing decisions regarding warfarin and improve the benefit/risk ratio. Current evidence related to the influence of PG on warfarin dosing was presented at the Clinical Pharmacology Subcommittee of the Advisory Committee (AC) for Pharmaceutical Science in November 2005 (http://www.fda.gov/ohrms/dockets/ac/cder05.html#PharmScience). The AC recommends that PG information be considered to better inform prescribers about risk factors for overanticoagulation.
S. M. Huang1 , R. Temple2 , L. J. Lesko1 , 1Office of Clinical Pharmacology, 2Office of Medical Policy, CDER, FDA, Silver Spring, MD
Despite the increased understanding and documentation of drug interactions in the labeling and in letters to "dear healthcare professionals", adverse reactions resulting from well-recognized drug-drug interactions continued to be reported.The increased use in dietary supplements with significant drug interaction potential increases the propensity for adverse drug reactions. To better translate information into practice, CDER has published a final rule on the physician's labeling format. Drug interactions that are significant, or their absence when they are expected to occur, would appear in labeling HIGHLIGHTS, in addition to being included in the main body of the labeling. 1,2 In addition, a proposed revision of the 1999 drug interaction guidance2-4 includes a proposal to use a classification system for CYP3A inhibitors (including grapefruit juice) in the labeling in an effort to improve the consistency of labeling language and to highlight key drug interactions.The concept paper2 also suggests how metabolic and interaction information should become broadly integrated into many sections of labeling. Labeling descriptions of drug interactions can be based on interactions observed in clinical studies or projected interactions extrapolated from other studies. The labeling implications of drug interactions with dietary supplement, such as St John's wort, or with grapefruit juice will be discussed.5
[ References:
1: Draft Guidance for Industry: Labeling for Human Prescription Drug and Biological Products — Implementing the New Content and Format Requirements, http://www.fda.gov/cder/guidance/6005dft.pdf ; 2. Drug Interaction Concept paper: http://www.fda.gov/ohrms/dockets/ac/04/briefing/2004-4079B1_04_Topic2-TabA.pdf; 3. Draft drug interaction guidance, http://www.fda.gov/cder; 4. . Drug interactions during drug development: http://www.fda.gov/cder/drug/drugInteractions/default.htm]; 5. Huang, S-M, Temple R, Lesko, LJ, Drug-Drug, Drug-Dietary Supplement, and Drug-Citrus Fruit and Other Food Interactions- Labeling Implications, in"Herbal Supplement- Drug Interactions", Eds Lam, F, Huang, S-M, Hall, S, Taylor & Francis, 2006 ]
C. Zhan1 , R. G. Kaczmarek2 , N. Loyo-Berrios3 , 1AHRQ, Rockville, MD, 2FDA, Rockville, MD, 3FDA, Rockville, Maryland
Background: The purpose of the study was to assess the respective annual incidences of total hip replacement (THR), partial hip replacement (PHR) and revision of hip replacement (RHR) in the United States, perioperative and short-term outcomes, and factors associated with adverse outcomes.
Methods: We screened 7.5 million discharge abstracts from 994 short-term acute care hospitals across 28 states in 2003 in the Agency for Healthcare Research and Quality (AHRQ), Healthcare Cost and Utilization Project Nationwide Inpatient Sample to identify patients who underwent total hip replacement, partial hip replacement and revision hip replacement. Postoperative complications, in-hospital mortality and readmissions 30-day and 90-day post-discharge were evaluated. Patient and hospital characteristics associated with adverse outcomes were examined.
Results: There were an estimated 201,420 (95% CI = 185,875 - 216,964) total hip replacements.The estimated number of partial hip replacements was 105,400 (95% CI = 100,116 - 110,700).Finally, there were an estimated 35,851 (95% CI = 31,951 - 39,751) revision artificial hip surgeries. In-hospital mortality following hip replacement was 0.33% (95% CI = 0.27% - 0.36%) for THR, 3.04 (95% CI = 2.83% - 3.20%) for PHR, and 0.82 % (CI = 0.63% - 1.05%) for RHR. Perioperative complications for THR, PHR and RHR included deep vein thrombosis or pulmonary embolism (0.68%, 1.37%, and 1.07%) and decubitus ulcer (0.28%, 1.88%, 1.29%).
Conclusions: Hip replacement is a common procedure that can be performed with a low in-hospital mortality risk.
R. K. Kasliwal1 , E. E. Leutzinger1 , B. W. Uratani2 , 1CDER, FDA, Silver Spring, MD, 2CDER, FDA, Rockville MD
Background: Positron Emission Tomography (PET) drugs are radioactive drugs, where the radioactive atom disintegrates by positron emission that provides dual photons (511keV energy), which are imaged and used in the diagnosis of a disease. The FDA Modernization Act (FDAMA) directed FDA to regulate PET drugs under section 505 of the FD & C Act and required promulgation of cGMP requirements for these drugs.
Methods: We examined operations at many PET drug production facilities and held multiple public meetings to seek input on the scope of procedures and cGMP regulations. We also considered the unique properties of PET drugs; the short half-life of the PET isotope, which limits the useful shelf life of the product and the testing which can be performed on finished product.
Results: We published draft sample application formats for the submission of chemistry, manufacturing and controls information and data (Federal Register, Vol. 65, No. 48, pages 12999 - 13010, March 10, 2000). The sample application formats are intended to harmonize the product quality standards for each PET drug among different PET production facilities and provide detailed guidance on developing NDAs.
Proposed rules for cGMP requirements (21 CFR 212) as well as draft guidance "PET drug products -Current Good Manufacturing Practices" were published in the Federal Register, Vol.70, No. 181, pages 55038 - 55062, September 20, 2005.
Conclusion:Our approach to the development of application formats and proposed rules for cGMP requirements and guidance for cGMPs for PET drugs is based on scientific understanding of PET drugs, input from the PET drug community and smart application of existing regulations. After reviewing comments, the proposed rule and guidance will be finalized.
M. Katzper, S. Sobel, CDER, FDA, Rockville, MD
Background: Systems Biology which combines computational and physiological approaches is being put forth as a paradigm for improved understanding of biological phenomena. Particularly in understanding diseases and drug intervention for the individual this seems to be the way forward. The combined power of all the "omics", animal experimentation and modeling are seen as predicting human risk and benefit. This has implications for the review process.
Methods: The major ideas, directions and approaches of Systems Biology were studied. The question we address is how this scientifically sound approach would fit with the current totally empirical reliance on clinical trials for drug approval? How feasible does use of advanced scientific knowledge now appear with current resources?
Results: A Systems Biology approach is extremely resource intensive. Furthermore, the agency does not currently possess the needed capabilities, even in its Interagency Modeling Consortium, to carry out such investigations. There have been some efforts in this direction in toxicogenomics.
Conclusions: The Agency should track these developing methodologies which include genomics, proteomics, trancriptionomics and metabolomics. These have implications for the approval process. Particular attention should be paid at this time to the development of biomarkers employing the methodologies of Systems Biology.
M. Katzper, CDER, FDA, Silver Spring, MD
Introduction: Diseases and drugs interact dynamically. When looking at the end point results of a controlled clinical trial, there is a tendency to forget this fact. Modeling the process and exploring its dynamics can provide an integration of these interactions. A number of examples are given to demonstrate the overview provided by modeling.
Methods: A Systems Dynamics approach is used. Such an approach assumes that we can explicitly specify the main elements of concern and their interaction. In cases of uncertainty, a number of alternate models may have to be investigated. The major tool used is STELLA©, a graphically oriented modeling tool. Models are built to examine diverse conditions. Models and results are shown in the poster display.
Results: Models deal with issues such as the rebound effect upon drug cessation, assays of substances with circadian rhythms and developmental factors in dose size selection. Effects are studied in the presence and absence of feedback which maintains homeostasis. Acute conditions as well as chronic diseases are modeled. Studies yield a better understanding of what can be expected under a variety of circumstances.
Conclusions: Long term consequences of drug usage provide examples of aspects which cannot be studied in the drug approval process. Often these consequences can be modeled to inform us of possible outcomes. In the many circumstances where we would like to explore alternatives but cannot, dynamic modeling lets us explore these possibilities.
N. Alderson1 , U. S. Babu2 , P. Chu3 , T. Crone4 , C. Elkins5 , R. M. Fahmy3 , J. V. Gobburu6 , J. Johannessen1 , C. Kavanaugh2 , S. Kumar7 , M. Major7 , M. Manjanatha5 , R. A. Bright8 , H. Trinh1 , S. D. Torosian4 , R. S. Uppoor6 , T. Woods8 , 1OC, 2CFSAN, 3CVM, 4ORA, 5NCTR, 6CDER, 7CBER, 8CDRH
The Committee for the Advancement of FDA Science (CAFDAS) serves as an internal advisory committee to the Commissioner, Associate Commissioner for Science, and the Senior Science Council. Functioning independently of any Center or discipline, CAFDAS addresses FDA-wide science issues from a working scientist's perspective. It is composed of two members from each Center and the ORA who represent research, review and compliance aspects of the FDA mission; each member serves a three-year term. The primary objective of CAFDAS is to aid in enhancing the agency's science infrastructure by: serving as a medium for advancing ideas and concerns from scientists to senior FDA management regarding the state of science in the FDA, providing comments to the Commissioner on science policy questions and planning, and offering practical suggestions and constructive solutions to achieve Agency-wide scientific excellence. Some CAFDAS activities include reviewing OSHC collaborative science project applications, disseminating leveraging information on competitive research funding sources and mechanisms, and, most recently, providing input for potential future changes of the peer review system for FDA scientists.In cooperation with the Office of Science and Health Coordination (OSHC) and the Office of the Commissioner (OC), CAFDAS initiated "The Commissioner's Seminar Series."This internal FDA seminar series highlights FDA science pertaining to the "Critical Path" and promotes the recognition of mission-based science through plain-language presentations to audiences with diverse backgrounds and experience.More information regarding CAFDAS' history, meeting minutes, leveraging activities and The Commissioner's Seminar Series is available at: http://first.fda.gov/cafdas.
K. Randolph1 , H. C. Chang1 , E. Waldron1 , P. Turowski1 , K. A. Smeds1 , V. Gilliam1 , A. Shanklin1 , E. Sanchez1 , M. E. LaVecchia1 , A. M. Hollander1 , J. Ziyad1 , A. L. Lipman1 , M. Cheeseman1 , A. Waldo2 , K. Kenny2 , L. Marchant2 , 1FDA, 2Decernis LLC
Background: Determining a manufacturer's or processor's obligations with regard to requirements applicable to the safety of food additives and food contact materials in a global market is a complex task for a large company, but especially challenging for a small or medium-sized organization. It can be equally challenging for regulators to assist stakeholders in determining compliance. Such requirements may differ in procedure and substance between countries of use. In addition, these regulations are in flux, making it difficult to keep current. Regulatory compliance is a critical control point in safety of the food supply chain of a global marketplace.
Methods: FDA/OFAS and Decernis have established a collaborative agreement and developmental project to make available an international regulatory compliance reference system. Through this cooperation between government and industry, FDA has developed and shared databases of critical information regarding food safety and FDA requirements. Using its multi-lingual document and data management technologies, Decernis has integrated this information together with other national requirements into a consistent system for keeping up to date with global food safety requirements.
Results: FDA and Decernis have developed a collaborative system - called gComply -- that is today used by both government and industry. FDA Consumer Safety Officers can use the integrated system in answering questions more efficiently. Further, FDA links the system to its internal databases, allowing greater depth in review and analysis. Domestic manufacturers, importers, and non-U.S. companies access the Decernis system, obtaining better information for assuring compliance for the development and shipment of food products within and to the United States. The project makes available more comprehensive, current information to the regulated public in an accessible form, and provides a consistent approach to determining compliance on a global basis. It also aids FDA in having more comprehensive information.
Conclusions: The collaboration of the FDA/OFAS and Decernis has resulted in a reference system that can reduce the complexity and costs of compliance, and improve the effectiveness of decisions made in marketing foodstuffs in a global marketplace.
N. C. Mezu-Nwaba1 , O. J. Mezu-Ndubuisi2 , K. R. Mezu3 , 1CDRH, FDA, 2LSU, Shreveport, Louisiana, 3Morgan State University, Baltimore, MD
Objective: To propose guidelines for the management of Sickle Cell Disease (SCD) based on existing therapies that have proven effective.
Background: SCD Anemia is an autosomal recessive disorder of the hemoglobin gene, the presence of two defective genes (SS) is needed for SCD. SCD affects millions of people in the world and about 72,000 in the US, affecting 1 in every 400 African-American births. These patients suffer excruciating pain during sickling crisis, and are at increased risk of developing life-threatening infections, largely due to autosplenectomy, accounting for over $475million dollars in healthcare cost. Other complications include: stroke, aplastic crisis, gallstones, acute chest syndrome, splenic sequestration crisis, and delayed growth.
Methods: We conducted a literature search of articles on recent guidelines for managing SCD that focused on infection prevention, pain management, acute chest syndrome and management of iron overload. We evaluated the effectiveness of the parameters in improving care.
Results: Based on the data reviewed, infection-prevention, adequate pain management, use of Desferal® for iron overload, and hypertransfusion, appeared to reduce the SCD crises. Penicillin prophylaxis is recommended in children. Other therapies include; Exjade®, hydration, folic acid, and hydroxyurea which showed a 50% reduction in pain episodes and hospitalization. World Health Organization's pain protocol is widely accepted and effective. Recommended immunizations include; Pneumococcal, Haemoephilus Influenza and Hepatitis B Vaccines.
Conclusion: Management of SCD with guidelines that address; pain management, iron overload, infection prevention, and education of patients and healthcare providers can improve the quality of care of patients with this chronic disease.
S. A. Miller, B. Z. Zmudzka, S. G. Coelho, J. Z. Beer, CDRH, FDA, Rockville, MD
Background: The practice of indoor UV tanning raises serious concerns, since UV is carcinogenic and also carries other risks.Nevertheless, millions of people frequent tanning facilities or use sunlamps at home. FDA discourages consumers from using UV-emitting devices for cosmetic reasons. To establish if the UV burden to those who choose to use sunlamps can be reduced without compromising the desired effect, we are comparing the effects of 3 tanning schedules, each requiring much lower UV exposures than thoserecommendedin the current (published in 1986) FDA guidance.
Methods: Here we report preliminary observations on 20 subjects (phototypes 2 or 3).Small, 3x3 cm, areas on the backs of these subjects were exposed ~10 times over 8 weeks to accumulate, respectively, 1900 J/m^2 (schedule A), 2900 J/m^2 (schedule B), and 4300 J/m^2 (schedule C).The intensity of tan was assessed by diffuse reflectance spectroscopy.
Results: We find that while schedule A produces only a light tan, schedules B and C produce a moderate to dark brown tan. The difference between tanning effects (by visual evaluation) of schedules B and C is small. The current policies would allow accumulation of up to 12000 J/m^2 during the same period of time, which is a factor of 3 to 4 higher than our experimental schedules B and C.
Conclusions: These results confirm that the current recommended exposure schedules permit excessive doses of UV that may not be necessary for producing a tan. Upon completion of this study, we will propose appropriate changes in the recommendations for exposure schedules.A. K. Mitra, E. P. Duffy, ONDQA, OPS, CDER, FDA
Background: Transdermal drug delivery systems are self-contained discrete dosage forms that, when applied to intact skin, are designed to deliver drug(s) through the skin to the systemic circulation. The common structural elements of a transdermal system include the following: 1) backing layer; 2) drug layer or reservoir; 3) adhesive layer; and 4) protective or release liner. In some instances the drug and the penetration enhancer(s)/solubilizer(s) are included in the adhesive layer. In other cases, the drug in a reservoir with penetration enhancer(s)/solvent(s) is incorporated inside a microporous or dense polymeric membrane followed by an adhesive layer for skin contact.
FDA is aware of adverse events in which patients who were wearing Nicotine transdermal patch during MRI experienced burns. The burns were traced to induction heating of the aluminized backing used in the Nicotine patch.
Method: FDA's database was searched for approved transdermal systems with aluminized backing and a list of transdermal systems with aluminized backing was prepared. Based on this list various clinical divisions where the drug products were approved were contacted.
Results: The FDA data base contained the following approved transdermal systems with aluminized backing: 1. Androderm (Testosterone transdermal system,); 2. Catapress-TTS (Clonidine transdermal system); 3. Nicoderm (Nicotine transdermal system); 4. Nicotrol (Nicotine transdermal system); 5. Prostep (Nicotine transdermal system);6. Habitrol (Nicotine transdermal system); 7. Nicotine transdermal system (generic nicotine transdermal system); 8. Transderm Nitro (Nitroglycerin transdermal system); 9. Trasnsderm Scop (Scopolamine transdermal sytesm).
Conclusions: The labeling change was recommended by several divisions. The following common language was adopted in the package insert and patient package insert for the drug products. "Skin burns have been reported at the patch site in several patients wearing an alumnized transdermal system during a magnetic resonance imaging scan (MRI). Because the drug product contains aluminum, it is recommended to remove the drug product before undergoing an MRI". Further work is ongoing at other affected divisions to adopt the change in the package insert and patient package insert with similar language.
S. C. Nallani1 , L. D. Coles2 , 1OCP, CDER, Silver Spring, MD, 2College of Pharmacy, University of Maryland, Baltimore, MD
Background: Providing pregnant and lactating women with the benefits of scientific evidence-based treatment is an unmet medical need. We attempted to derive meaningful clinical pharmacology, dosage adjustment information for drugs used in pregnancy and lactation.
Methods: Regulatory submissions and public domain literature were searched for information on pharmacokinetics and pharmacodynamics of parent drug and metabolite(s) during pregnancy and lactation. Data were critically reviewed with regard to the analytical method validation, pharmacokinetic and statistical data analysis methods, validity of pharmacodynamic endpoints, adverse event reports in pregnant and lactating women.
Results: A searchable clinical pharmacokinetics database was created consisting of over 450 published articles and over 50 product labels. The drug classes evaluated include antiepileptic, antidepressant, cardiovascular, antiretroviral, and anesthetic and analgesic agents. Changes in absorption, distribution, and elimination were noted for several drugs used during pregnancy. Data were summarized with emphasis on specific mechanistic changes in ADME that occur during pregnancy.
Conclusions: A clinical pharmacokinetics database has been created and the information is being used to improve the "Pregnancy" Section of several product labels. It is also a convenient aide for the FDA reviewers in providing quick access to clinical pharmacokinetics data for specific drugs used in pregnancy
S. E. Norris, D. I. Freedberg, CBER,FDA,Rockville, MD
Neisseria meningitidis, Haemophilus influenza type b and Streptococcus pneumoniae are Gram negative bacteria that cause more than two dozen potentially fatal diseases in humans, particularly infants and young adults. The World Health Organization reported approximately 50,000 deaths worldwide annually from meningococcal meningitis alone. Each serogroup is known to have structurally distinct extracellular capsular polysaccharides (CPS), the presence of which is related to their ability to cause invasive diseases . This encapsulation has been identified as an important, but not sole condition for pathogenicity, but it has long been known that CPS can be immunogenic. As a result CPS vaccines have been in use for more than 50 years. However, the CPS vaccines' T-cell-independent characteristics lead to a poor response in infants less than 18 months of age, which is in many cases a more susceptible population. Conjugating representative polysaccharides to a carrier protein was initiated in the late 1980's as a way to convert a T-cell-independent immune response to one of T-cell dependence, providing enhanced efficacy with prolonged immunological memory. Nuclear Magnetic Resonance (NMR) is a valuable tool for "fingerprinting" and verifying purity, concentration and structure of bacterial polysaccharide vaccines. It is currently used as an identity check in lot release for many CPS vaccines. NMR is more powerful than just for use as a lot release test:it can be used as a quantitative tool.As an example, in this poster we show NMR techniques that we have adapted for a better understanding of Neisseria meningitidis group W-135 polysaccharides in preparation for conjugate vaccines.
D. Obias-Manno, P. Scott, J. Kaczmarczyk, E. Pinnow, K. Uhl, OWH
Background: In 1994, the FDA Office of Women's Health (OWH) was created to provide leadership and policy direction for the Agency regarding issues of women's health. Within its first year, OWH established a science program to fund women's health research.
Methods: We contacted investigators of 147 (intramural n=102, extramural n=20, special funding initiative n=25) studies funded by the Office of Women's Health from 1994-2005 to ascertain study-related publications, presentations, abstracts, scientific outgrowth and regulatory documents. The purpose of this study was to describe the impact of OWH funded research on academic, scientific and regulatory outcomes.
Results:Thirty-four percent responded to our inquiry, providing additional data to that already drawn from OWH study files. OWH studies resulted in >120 publications in peer-reviewed journals and >125 abstracts/presentations at national meetings. Some studies resulted in scientific outgrowth providing a basis for other related studies. Several studies have had significant bearing on regulatory policy.
Conclusions: The Office of Women's Health has provided over $14 million to fund 147 women's health research studies covering a broad range of health topics e.g. cardiovascular diseases, osteoporosis, PK/PD, allergies, sex differences, dietary supplements and counterterrorism, including in-vitro, animal and human studies. The Office of Women's Health science program promoted women's health through the advancement of education and science and enhancement of regulatory policy including cross labeling initiatives for drug-dietary supplement interaction, testing standards for product quality and guidance documents for industry and FDA (e.g. non-clinical evaluation of the potential for delayed ventricular repolarization, PK in pregnancy, breast implants).
J. Y. Park1 , M. J. Kim1 , S. Ortiz1 , V. R. Jarugula2 , A. Parekh1 , 1DCPIII, OCP, CDER, FDA, SILVER SPRING, MD, 2DCPIV, OCP, CDER, FDA, SILVER SPRING, MD
Backgound: Combined hormonal contraceptives contain estrogen and progestin. They come in various doses of ethinyl estradiol (EE) ranged from 15 to 50 mcg as well as three different routes of administration including oral, vaginal ring (VR), and transdermal patch (TP). The purpose of this study is to compare the pharmacokinetic (PK) parameters of EE in these contraceptive products.
Methods: The products containing EE were identified using Drugs@FDA and Physicians' Desk Reference (PDR). Doses of EE, routes of administration, and PK parameters (AUC, Cmax, Css, Cavg) of each product were collected from the labels extracted from Drugs@FDA and the PDR. Searches of PubMed were conducted if PK data were not available in the label.
Results: We identified approximately 95 contraceptive products containing EE. The PK data of 23 products were attained. Of these, 21 products are oral contraceptives (OCs) with varying doses from 20 to 50 mcg of EE. The VR product has the lowest daily dose of EE (15 mcg) and the TP with 20 mcg of EE. AUC of EE from the TP is generally higher compared to the OCs/VR products.
Conclusions: Further investigation is ongoing to understand how different routes of administration of contraceptive products containing EE have an effect on EE exposure.
D. Patel, B. M. Davit, S. H. Haidar, X. Jiang, B. Fabian-Fritsch, L. Yu, D. P. Conner, Office of Generic Drugs, CDER, FDA
Background: Drug products that are highly variable (HV) in the pharmacokinetic (PK) parameters AUC and Cmax can fail to meet the FDA's bioequivalence (BE) limits of 80-125% in a BE study that enrolls the usual number of subjects. Generic drug applicants claim that applying the usual BE limits to these drugs places a high economic burden on them since they believe that they must use large numbers of subjects to demonstrate BE. We evaluated the scope of this problem by collecting data from BE studies of generic HV drugs.
Methods: We collected data from 752 acceptable BE studies in 370 Abbreviated New Drug Applications (ANDAs) submitted in 2003-2004. We analyzed the ANOVA Root Mean Square Errors (RMSEs), point estimates and 90% confidence intervals (CIs) for generic/innovator AUC and Cmax ratios. Drugs were considered HV if Cmax and/or AUC RMSE >= 0.3.
Results: In 85/752 studies, AUC and/or Cmax met HV criteria. Cmax was HV more frequently than AUC. Of these 85 acceptable HV drug studies, 7% enrolled from 81-135 subjects, 7% enrolled from 61-80 subjects, and 86% enrolled from 22-60 subjects. In 16 cases in which multiple ANDAs were submitted for the same drug, PK variability was high in one ANDA but not in the other ANDAs.
Conclusion: In 2003-2004, 11% of all BE studies submitted in ANDAs were for HV drugs. Most studies of HV drugs met BE limits without excessively large numbers of subjects. For many multi-source drug products, PK variability was not consistent.
E. Pinnow1 , P. Scott1 , J. Derbis2 , T. Toigo2 , K. Uhl1 , 1OWH, 2OSHI
Background: The FDA requires clinical trials to include a representative sampling of the population that will likely use the drug.Although women may not be explicitly excluded, restricting inclusion to women of non-childbearing potential (non-CBP) may hinder female participation.These exclusions persist despite the contraceptive requirements that can be imposed for trial participation. The purpose of this study is to describe the contraceptive requirements for women and men participating in clinical trials.
Methods: The study used an OSHI database that included information (e.g., indication, and inclusion by gender) on 883 new commercial protocols submitted to CDER between January 1 - April 30, 2002. A subset of 711 protocols for non-sex specific indications was reviewed by OWH to abstract information on contraception requirements.
Results: Thirty-eight protocols included only men, 18 included only women (all non-CBP), 56 included men and women of non-CBP, and 599 included both men and women. Of the 599 protocols that included both men and women, contraception for female participants was required in 551 (92%) and in 74 (12%) of male participants.Of the protocols with a contraception requirement for women, 196 contained vague language (e.g. "acceptable", "effective" or "adequate") and 355 contained a list of acceptable method(s). Of the 355 protocols, 309 required one or more approved contraception method and 46 required at least two combined methods of contraception.
Conclusions: Contraception for women is generally required, and may involve more than one method.Men of reproductive age are less likely to have similar restrictions.
E. A. Reinhold1 , D. M. Schmit1 , L. R. Dusold2 , 1Computer Technology Services, Inc. (CTS), Rockville, MD, 2OIT-CFSAN, FDA, College Park, MD
Information on the CFSAN Website http://www.cfsan.fda.gov contains specialized, technical vocabulary. Most users of the website search for information using specific terms. However, regional variations in word use present a barrier to searches. For example, a user might use "ethene" for "ethylene", or "maize" for "corn", or "ddt" for "dichlorodiphenyltrichlorethane". We can utilize the taxonomies in the CFSAN Thesaurus to present more complete search results. Selected portions of the CFSAN Thesaurus were extracted into a synonym list. This list was incorporated into the front end of a search engine. The result was an increase in relevant hits returned from searches. Additionally, the CFSAN Lexicon was expanded, and the larger list used in the spell checker component of the search function. Utilizing the CFSAN Thesaurus in electronic and web applications expands access to technical vocabulary and helps improve stakeholder access to FDA information. Reference: http://www.cfsan.fda.gov/~frf/forum05/Q-04.htmL. Holness, M. Knippen, L. Simmons, CBER, FDA, Rockville, MD
BACKGROUND: Fatalities due to transfusion related acute lung injury (TRALI) have been increasing in recent years. Currently there is discussion but no agreement on donor management strategies to reduce the incidence of TRALI. Analysis of donor characteristics in fatalities attributed to TRALI from 1995 to 2004 may help suggest options for donor management.
STUDY DESIGN: Retrospective review of donor investigations in 110 fatalities attributed to TRALI.
RESULTS: Multiparous female donors implicated in TRALI fatalities exceeded nulliparous women and previously transfused male donors. Most multiparous donors were found to have Human Leukocyte Antigen (HLA)- and/or neutrophil-specific antibodies passively transferred to recipients who later died; thirty-nine percent had multiple antibodies. Most blood centers no longer consider male donors at risk for causing TRALI but our data show males, reportedly never transfused, may have these antibodies and were implicated in 5 deaths. In 6 cases, the implicated donors tested negative for HLA- and/or neutrophil-specific antibodies but the recipients tested positive apparently reacting to residual white cells in the components. Recipients in 26 cases experienced symptoms of TRALI but the donors tested negative for the specific antibodies; these recipients were not tested.
CONCLUSIONS: Donor management strategies that do not negatively affect the blood supply are needed to prevent TRALI. Criteria for evaluation of implicated donors should be established by blood centers. Quick inexpensive tests are needed to identify HLA Class I and Class II, and neutrophil-specific antibodies. Recipient samples should be included in the testing protocol in cases of TRALI fatalities.
L. Holness, P. Jones, M. Knippen, L. Simmons, M. T. Niu, CBER, FDA, Rockville, MD
BACKGROUND: Contamination of blood or blood components with bacteria or parasites occurs infrequently but when present, has the potential to cause serious infection in the recipient. Transfusion associated infection from these microbes may cause serious sequelae which if not recognized and treated can be fatal. FDA received 1063 fatality reports related to blood transfusion and collection between January 1, 1986 and September 30, 2004. One hundred twenty-eight (12.0%) were associated with bacterial or parasitic infection.
STUDY DESIGN: 19-year retrospective review of fatalities resulting from contaminated blood and blood components.
RESULTS: Deaths from contaminated blood components found platelet products and Red Blood Cells most often implicated. Transfusing contaminated platelets resulted in fatalities more than twice (71.1%) as often as deaths from contaminated Red Blood Cells (28.9%). Storing platelets at room temperature may impact that outcome. Recipients of random platelets administered singly or in platelet pools more often received a contaminated product than patients receiving pheresis platelets. Gram negative organisms were identified in 57.8% of these fatalities; Gram positive in 39.1%; 1.5% were anaerobes. Babesia parasites identified in blood smears from previously asymptomatic recipients were responsible for 1.5% of these fatalities. Where both the recipient and the implicated blood product were cultured, the same organism was often isolated in both.
CONCLUSIONS: Because of potentially fatal outcomes in recipients, donors must be adequately screened and infection control measures strictly followed while collecting, processing, and handling blood and blood components. Development of simple and effective testing procedures for detecting contamination in blood is critical.
S. Elyashiv-Barad, K. A. Smeds, DFCN, OFAS, CFSAN, FDA, College Park, MD
Consumption factors (CFs) for food packaging describe the fraction of the daily diet expected to contact a specific packaging material. Many of the currently used CFs for packaging categories (e.g., metal, paper, polymers, etc.) and specific food-contact polymers (e.g., polyolefins, polystyrene, etc.) were developed from market survey data collected in 1980. In our continual effort to evaluate safety, we have reexamined several established CFs. The CF for polystyrene (PS) was previously determined to be 0.08 (i.e., approximately 8% of all food contacts PS packaging). This CF was rounded to 0.1 in 1995 and further subdivided into CFs for impact PS (0.04) and non-impact PS (0.06). Because the use of PS in food-contact and disposable applications has expanded since 1980 and is expected to continue to increase, it is important to revise the PS CF to account for increases in consumer exposure to substances migrating from styrenic food packaging. We have revised this CF utilizing three different sources of market data. Using these poundage data, the weight of food contacting PS on a daily basis (WPS) was determined. From population data, the weight of food consumed per day contacting all food packaging (WAF) was estimated. We then calculated a new CF of 0.14 for PS by dividing WPS by WAF. This CF has been further subdivided to allow for the refinement of exposure estimates for uses limited to specific subcategories of PS packaging. The increase in the PS CF may affect the safety assessment of substances migrating from styrenic food packaging.
J.A. Spearmon, CDER, FDA, Silver Spring, MD
The FDA protects the public's health by assuring the safety, efficacy, and security of human and veterinary drugs, biological products, medical devices, our nation's food supply, cosmetics, and products that emit radiation. This is accomplished by using the highest standards for safety and effectiveness in approving medication labeling because the entire adverse event profile of a medication may not be known at approval and initial marketing because of the limited size and controlled nature of pre-marketing clinical trials.R. Pazdur, K. Weiss, D. Ross, G. Jones, D. Spillman, O. H. Suleiman, CDER, FDA, Silver Spring, MD
In July 2005, FDA established the new Office of Oncology Drug Products (OODP) within the Center for Drug Evaluation and Research. This office will improve consistency of review and policy regarding oncology drugs, and bring together a critical mass of healthcare professionals and scientists who will help facilitate development of new therapies.
The review of drugs and therapeutic biologics used to diagnose, treat, and prevent cancer are now all in the new Office of Oncology Drug Products. OODP is also responsible for the review of drugs and certain therapeutic biologics used in hematology and medical imaging, many of which are used for cancer support, detection, or treatment. The office is also responsible for the review of clinical studies conducted under investigational new drug applications (IND), marketing applications, i.e. new drug applications (NDA) and biologics license applications (BLA), and basic science research programs conducted under the Radioactive Drug Research Committee (RDRC) regulations (21 CFR 361.1).
OODP also manages a cross-cutting, inter-Center Oncology Program within FDA, whichcoordinates work performed throughout the FDA Centers (CDER, CBER, CDRH) related to the prevention, diagnosis, and treatment of cancer. The Oncology Program also facilitates cross-agency expert consultation, devises and promotes Critical Path initiatives relevant to oncology, provides a forum to discuss and develop regulatory policy and standards, and serves as a focal point for agency interaction and collaboration with oncology professional societies, the National Cancer Institute (NCI), and other important stakeholders. This program will also coordinate cross-cutting training and oncology educational activities.
M.E. Van Hoef, Transplant Creations, Amsterdam, The Netherlands
In acute leukemia the highest probability of disease free survival can be obtained by intensive treatment from diagnosis. Combinations of cytotoxic cytoreductive compounds induce rapid clinical remission; for consolidation of clinical into cytogenetic or molecular responsecombinations of cytotoxic cytoreductive compounds are used again, sometimes supported by autologous hematopoietic cell transplantation; an allogeneic transplant, consisting of combinations of cytotoxic cytoreductive compounds and among others immune competent cells, is finally used to eradicate residual occult malignant cells. Effects of treatment that induce side effects are to be controlled to reduce treatment risks and achieve optimal overall survival. At each level of therapy, induction, consolidation and eradication, combinations of different treatment modalities are to be carefully selected with attention to detail to prophylaxis and supportive care. The method of integration of independent treatment combinations will be described in detail and product development consequences addressed.L. Verrill, C. Choiniere, A. Lando, CFSAN, FDA, College Park, MD
The food label has long been frustrating for people seeking information about allergenic ingredients. The Food Allergen Labeling and Consumer Protection Act of 2004 requires manufacturers to declare the presence of ingredients that are or contain protein derived from the eight most common allergens. The Act does not address use of advisory statements, i.e, statements that alert consumers about the possible presence of a food allergen due to cross-contact. Examples of these statements are "May Contain…" and "Processed in a facility that also processes...."
Two methods were used to assess consumer attitudes and behaviors in response to advisory statements. A survey was conducted to gauge consumer preferences for four kinds of advisory statements, three of which are commonly found on food labels. The fourth statement was created by FDA researchers and contains more information about food processing than is usually available in an advisory statement. An experiment was used to determine whether any of the four statements more effectively provides consumers with information that would help them make food allergen-related consumption decisions. Results indicate that although consumers prefer certain advisory statements, none of the four statements is substantively more or less effective than the others at conveying allergen risk information to the consumer.
L. Zhang, J. M. Strong, W. Qiu, L. J. Lesko, S. M. Huang, CDER, FDA, Silver Spring, MD
Background: Pharmacokinetic drug interactions can lead to serious adverse events. Despite increased understanding of metabolism-based drug-drug interactions, unexpected drug interactions still occur. One of the confounding factors may involve interactions mediated by transporters. A new drug interaction draft guidance that has discussion on emerging areas such as drug transporters will soon be available for public comments.
This poster will focus on the potential role that drug transporters may play in drug interactions and what information may be needed during drug development and new drug application (NDA) submissions to address potential drug interactions mediated by transporters.
Methods: Major transporters and their clinical relevance were reviewed. Current status and challenges in predicting in vivo drug interactions and latest scientific consensus on drug transporters were considered.
Results:Tools to study P-glycoprotein (P-gp) transporters are more advanced compared to other transporters. In vitro methods and criteria for determining whether a new molecular entity is a P-gp substrate or inhibitor are discussed. Decision trees for suggested in vivo interaction studies based on in vitro results are proposed. In addition, methods for determining P-gp induction and recommendations for evaluation of other transporter-based interactions are discussed.
Conclusions: The poster includes current recommendations for in vitro and in vivo studies to evaluate potential drug interactions mediated by transporters (especially P-gp) during drug development.
Acknowledgement: The authors acknowledge members of the FDA Drug Interaction Working Group who contributed to the development of a concept paper entitled "Drug interaction studies — Study design, data analysis, and implications for dosing and labeling" and a draft guidance.
G. C. Ziobro1 , L. A. D Hoostelaere2 , 1CFSAN, FDA, College Park, MD, 2ORA, Rockville, MD
Despite the great strides made in modern food processing technology, adulteration of the food supply by filth and extraneous materials is still problematic.Review of the laboratory results recorded in FACTS (Field Accomplishments and Compliance Tracking System) of nearly to 10,000 samples tested for filth and extraneous materials between FY03-FY05 shows that 81.4% were classified as lab class 1 (in compliance); 6.58%, lab class 2 (regulatory action not indicated); or 12.04%, lab class 3 (adverse findings).At least 1.5% of all the samples were contaminated with known vectors of food-borne pathogens, which there are no tolerances under the revised filth strategy of 1996. Rodent contamination (either excreta or hair) was found in 13.6% of the samples.Five commodity groups accounted for close to 50% of the analyzed foods: fishery/seafood products; vegetables and vegetable products; fruits and fruit products; bakery products, doughs, bakery mixes and icings; and whole grains, milled grain products and starch.
Review of the data collected from routine sampling enables the Agency to develop correlations between specific filth elements and a particular food category.Such knowledge in turn, allows the Agency to allocate resources to monitor and take regulatory actions against problematic foods.Y. A. Hsieh, X. H. Chen, S. Pope, R. S. Harapanhalli, ONDQA, CDER, FDA, Silver Spring, MD 20993
Background
Tyrosine kinase inhibitors inhibit cell growth by blocking the ability of protein tyrosine kinases to function. This class of drugs provides valuable therapeutic options to cancer treatment.
Methods
Tyrosine kinases are enzymes within the cell that phosphorylate the amino acid tyrosine. Mutations in genes encoding tyrosine kinases may alter or disrupt the signaling pathway which leads to unregulated cell proliferation. Tyrosine kinase inhibitors act by competing with ATP for binding to the kinase. Inhibitors with high specificity have shown to be beneficial, since they can be directed to kill tumor cells while causing minimum harm to normal cells. Several NDAs and INDs were reviewed in addition to the literature surveys to identify various chemical structures possessing tyrosine kinase inhibitory activities.
Results
Tyrosine kinase inhibitors approved as cancer treatments include Herceptin (trastuzumab) indicated for metastatic breast cancer, Gleevec (imatinib) for chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST), Iressa (gefitinib) for non-small cell lung cancer and Erbitux (cetuximab) for metastatic colorectal cancer. Many more candidate drugs are in various phases of clinical trials.
Conclusion
Tyrosine kinase inhibitors are an important class of anticancer drugs with minimal cytotoxicity to normal cells. Consistent with the Agency's critical path initiative, regulatory options such as fast track and rolling NDA were utilized in speeding up approval of this important class of anticancer drugs.
K. M. Wu, J. G. Farrelly, DAVP/ODEIV/OND/CDER/FDA
Many pharmacological agents are prepared in prodrug forms. Prodrugs can be classified into two types based on their sites of conversion into the final active drug form: (1) Type I, that is converted intracellularly (e.g., anti-HIV or anti-HBV nucleoside analog reverse transcriptase inhibitors); and (2) Type II, that is converted extracellularly, especially in digestive fluids or systemic circulation (e.g., valaciclovir). Many of the Type II prodrugs resulted pharmaceutically from rational drug design, to strategically circumvent bioavailability issues, including poor permeability and vulnerability to gastric acidity. Recently, an increasing number of INDs have appeared as Type II prodrugs. This manuscript addresses common characteristics of Type II prodrug submissions and regulatory insights derived from evaluation of these INDs. Because final therapeutic activity of a Type II prodrug is expressed through final active drug, the following pharmacokinetic information becomes critical to the IND: (1) Site and kinetics of conversion of the prodrug into active drug and the converting enzymes involved, (2) Extent of the transition or duration of prodrug molecule or its intermediates, if any, appearing in the systemic circulation, as expressed by the detectable plasma concentrations. The contributory role of prodrug or intermediates that are transiently present in the body to the overall toxicity profile of prodrug can be analyzed through evaluating comparative toxicology between prodrug and active drug. Comparative toxicology reflects a unique and identifiable feature existing in the Type II prodrug IND package, in which the preclinical safety information often contains dual tracks of toxicity profiles respectively, one on the prodrug and other on the active drug. Dual-track toxicology is developed when the active drug was investigated originally early and found inadequate, and later advanced to a prodrug format due to reasons delineated above (e.g. bioavailability). Because the converting enzymes are both ubiquitous (e.g., esterases or phosphatases) and fast-acting, the active drug's toxicity profile is often claimed by the sponsor to reflect the prodrug's toxicity profile. A comparative toxicology table is designed here on prodrug and active drug to provide a useful means for reviewers to evaluate the dual toxicity profile effectively and helps to provide regulatory insights. Differences in target organs and profiles of toxicity that are apparent, as emerged upon comparative toxicology evaluation, could result from (1) Additional toxicity contributed by the prodrug itself or its intermediates (this likelihood would be supported by measurable plasma levels of these compounds) or (2) Difference in final active drug exposure levels between prodrug-group and active drug-group animals. In the latter case, additional toxicity produced is mostly likely due to higher systemic exposures.In summary, employing comparative toxicology has proven useful in evaluating type II prodrug INDs in the determination of target organ of toxicity and toxicity profile produced by prodrug alone or active drug itself. CATEGORY L: VALIDATION, TESTING, STANDARDIZATION, AND QUALITY ASSURANCEF. Abbasi1 , V. Zenger2 , L. Wang3 , A. Giagalas3 , R. Vogt4 , G. E. Marti1 , 1CBER, FDA, Rockville, MD, 2CDER, FDA, Rockville, MD, 3NIST, Gaithersburg, MD, 4CDC, Atlanta, GA
Flow cytometry is used to quantitatively measure cell parameters in many diseases. This clinical data, from many different machines, is then used for therapeutic decision making, drug applications, lot release and potency assays. Accuracy of flow results is based on the calibration of the flow cytometers. In recent years flow cytometers are becoming more complicated with resolution of up to 22 fluorochromes at one time. Rainbow Calibration Particles (RCP-Spherotech, Inc.) are typically used to develop calibration curves. RCP consist of eight populations of hard-dyed microbeads that can be excited at 365 nm to 650 nm and emit between 500-700nm. We examined the ability of 13 instruments to develop calibration curves in all detection channels. Each instrument was set up in accordance with the routine procedures of each laboratory in which they were located and data collected on all available channels. Data were displayed and analyzed with FlowJo. In the first three channels of all instruments, all eight bead populations were detected with baseline separation. For higher channels resolution was suboptimal,this was especially true for UV channels. Calibration curves for higher channels and UV channels were not parallel suggesting that dynamic range has been compromised. It would also suggest that the algorithm used for automatic spectral overlap correction would perform suboptimally since spectral overlap correction depends on the linearity of the instruments. This suggests a need for new calibrator that would be excited across all lasers and fluoresce along a wider range.
E. B. Asafu-Adjaye, A. S. Carlin, E. H. Jefferson, A. R. Bryant, P. J. Faustino, B. Rothman, CDER, FDA, Silver Spring, MD
Background: The stability of repackaged drugs and the extension of expiration from 6 months to 12 months are of concern to regulators.This study is part of an effort, in collaboration with the Office of Compliance, to address these concerns and to identify stability issues associated with repackaging.
Methods: The stability of 40 mg furosemide tablets packaged in their original HDPE containers versus repackaged in USP Type A unit dose blister packs was investigated. Controlled stability studies were conducted at 25°C/60% relative humidity (RH) (1-12 months) and 40°C/75% RH (1-3 months). The tablets were analyzed for potency, dissolution rate, water content and hardness. Tablets were also analyzed nondestructively by near-infrared (NIR) spectroscopy and NIR chemical imaging.
Results: Neither stability conditions significantly affected the attributes measured for tablets in the original containers or the blister packs. Tablet potency over a 13-week period was 92 - 105 % labeled strength (LS) for the 25°C/60% RH condition compared to 94 - 103 % LS for the 40°C/75% RH storage condition. Similarly, no significant differences were observed for the spectroscopic and other attributes measured.
Conclusions: Results from this study show that both original and the repackaged products were stable under the long term and accelerated conditions. However, this should not be extrapolated to other products as the stability is product dependent.
S. Audet, M. L. Virata-Theimer, J. Beeler, D. E. Scott, D. J. Frazier, M. G. Mikolajczyk, N. Eller, F. M. Chen, M. W. Yu, CBER, FDA, Rockville, MD
Background: Immune globulin intravenous (IGIV) lot release is partly dependent on measles antibody (MeAb) potency.Because the proportion of blood donors with vaccine-induced measles immunity is increasing, this study was conducted to determine whether this shift in demographics had an effect on MeAb potency. Effects of plasma type and manufacturing procedures were also evaluated.
Methods: 166 lots of 7 IGIV products were tested by measles neutralization test (PRNT).Representative lots for each product were evaluated by ELISA to determine IgG subclass distribution. FPLC-purified IgG subclasses from one lot of IGIV were tested by PRNT as were HPLC-purified IgG monomer, F(ab')2 and Fab fragments. PRN titers for each product were used to calculate peak and trough serum levels of MeAb to determine if potency of IGIV would maintain levels at or above that associated with protection.
Results: There was a progressive decrease in MeAb potency for lots manufactured during 1999-2002.Products made from Source Plasma had lower potency than those from recovered plasma which may reflect the younger age and greater proportion with vaccine-induced immunity among Source Plasma donors.Manufacturing procedures affected IgG subclass distribution in IGIV products.Tests on IgG subclasses and fragments showed that IgG1 and bivalent antibodies had the highest PRN activity; products with the lowest levels of IgG1 and IgG3 had reduced potency. Calculated MeAb serum levels for IGIV products remained >120mIU/mL for one month after administration.
Conclusions: Decreased MeAb potency of IGIV may reflect donor demographics and can be affected by plasma type and manufacturing procedures.
A. Khurana, G. Awuah, L. Weddig, C. Balestrini, Food Products Assocation, Washington, DC
Background: FDA regulations 21 CFR 108 and 113 require thermally processed low acid foods packaged in hermetically sealed containers to be commercially sterile and free of viable pathogens. Processing authorities use time-temperature data from the coldest location of the container to establish a scheduled process that assures product safety and provides commercial sterility.
Methods: Traditionally, thermocouples (connected by wires to a datalogger) are mounted in containers for recording time-temperature data. Small self-contained sensors that allow data to be remotely captured and recorded are now commercially available and may offer opportunities for heat penetration and/or thermal process validation studies in retort systems where the use of thermocouples is limited. Using bentonite as model food, we compared thermocouples and remote sensors mounted in one or more can sizes processed in different retorts using steam or water as the heating medium. Heat penetration parameters (fh and jh) and the Ball process time were calculated for comparison.
Results: Both fh and jh values varied depending on the type of system, rpm and headspace. Generally, remote sensor heating factors were comparable and statistically insignificant. However, calculated Ball process times for the remote sensors were noticeably shorter than the thermocouples.
Conclusions: The remote sensors have the potential to be used for heat penetration tests. However, because several operating parameters could influence their performance in containers, the processing authority must establish conditions that prevent these sensors from under-estimating process times and potentially impacting public health.
J. Graham1 , N. Hai2 , B. D. Buch3 , 1OSEL, FDA, Rockville, MD, 2George Washington University, Washington, DC, 3CDRH, FDA, Rockville, MD
Introduction: Vertebral compression fractures are estimated to affect 26% of women over age 50, causing pain, disability, and increased mortality risk [1]. An emerging surgical treatment is vertebroplasty, or injection of reinforcing acrylic bone cement into the vertebral body. Previous work has suggested that bone porosity can have a significant effect on the integrity of cement fixation in joint replacement [2], so the potential benefit of vertebroplasty surgery may also depend on a patient's degree of osteoporosis. Other authors have investigated the relationship between bone density and mechanical behavior after vertebroplasty, but only using intact vertebrae [3] or vertebrae from an exclusively osteoporotic patient sample [4]. Our hypothesis was to test whether bone mineral density (BMD) can be used to predict improvements in mechanical strength of the vertebral body after cement injection over a range of normal and osteoporotic bone densities. A secondary hypothesis was to test whether the relationship between mechanical strength and BMD varied with volume of cement injected.
Materials and Methods: The spinal columns (T8-S1) of eight Caucasian female cadavers (age range 53 to 90, median age=62) were scanned for bone mineral density and T-score using a dual-energy x-ray absorptiometry (DEXA) scanner (GE Lunar Prodigy, GE Healthcare, Waukesha, WI) at a clinical osteoporosis screening facility. All forty lumbar vertebrae were dissected, the discs and soft tissues were excised and the posterior elements were removed to create isolated vertebral body specimens. The volume of each vertebral body was calculated by measuring the width, depth and height at each endplate and at the sagittal midplane with digital calipers. The areas of the vertebral endplates were calculated by tracing the endplates onto transparent acetate film, cutting out these traced areas and correlating the mass of each film with its area. The superior and inferior ends of each vertebral body were then potted in acrylic dental cement (Instant Tray Mix, Lang Dental, Wheeling, IL) in order to allow even load distribution on the endplates during mechanical testing. All vertebral bodies were wrapped in 0.9% NaCl saline-soaked gauze and stored at -20°C when not in use, and were subjected to exactly four cycles of freezing and thawing.
The five lumbar vertebrae from each cadaver were distributed into five experimental groups using a randomized block design. The first experimental group (control) received no cement treatment. The remaining four groups (0% fill, 4% fill, 12% fill and 24% fill) were first loaded asymmetrically in order to induce a wedge compression fracture. This wedge fracture was created by loading each sample with a pin placed two-thirds of the way between the posterior and anterior walls of the vertebral body until yielding was observed on the load-displacement curve. These vertebral bodies were then injected with a dose of surgical grade acrylic bone cement containing 30% barium sulfate radiopacifier (Spineplex, Stryker Interventional Pain, Kalamazoo, MI). The volume of cement injected for each specimen was determined based on the volume of the vertebral body and the experimental group to which it was assigned. The powder and pre-chilled liquid components of the cement were hand-mixed for 45±10 seconds before being poured into 5 mL syringes and injected into the vertebral body through 11-gauge bone biopsy needles. For every specimen, a needle was inserted into the vertebral body using a transpedicular approach through the left pedicle and the needle tip was positioned in the anterior third of the vertebral body near the sagittal midline and axial midplane. The cement was allowed to cure at -20°C for 18±2 hours, after which the vertebral bodies from all five experimental groups were tested in parallel-plate axial compression to measure the yield strength. All mechanical testing was performed using a motor-driven universal testing machine (Instron 4400R, Instron Corporation, Canton, MA). All specimens were preconditioned with ten cycles of loading and unloading from 100N to 250N of compression at a rate of 3 mm/min, and then loaded to failure at a rate of 5 mm/min. Yield strength (in N/cm2) was calculated as the load at the first inflection point on the load-displacement curve, normalized by the mean area of the superior and inferior vertebral endplates. Linear regression analysis was used to look for statistically significant (p<0.05) relationships between yield strength and bone mineral density for each of the five experimental groups, and analysis of covariance was used to compare the slopes of regression lines among the five groups.
Results: DEXA scanning showed that the eight cadavers represented a broad range of bone mineral densities with two normal subjects, two osteopenic subjects and four osteoporotic subjects (individual bone mineral densities of 0.342 to 1.411 g/cm2, T-scores of -6.5 to +1.25). The control and 12% fill groups showed statistically significant positive correlations between yield strength and bone mineral density (p=0.005 and 0.007, respectively), while the 0%, 4% and 24% fill groups showed positive correlations that fell short of statistical significance (p=0.22, 0.10 and 0.13, respectively). When the slopes of the regression lines were compared among groups, the 12% fill slope was found to be statistically greater than the 0% fill or control groups (p<0.05).
Discussion and Clinical Relevance: Vertebroplasty has been reported to have high success rates in providing pain relief [5], but the procedure is not without risks. Serious adverse events associated with cement leakage have been reported including persistent pain, paralysis, loss of sensation and death. Another secondary complication is adjacent vertebrae fracture due to redistribution of loads following surgery. Use of smaller volumes of cement may therefore be desirable to reduce the risk for these potential adverse events. Our results suggest that there may be significant differences between osteoporotic and non-osteoporotic patients in terms of the relative improvement in strength that larger cement volumes can offer them. In our study, highly osteoporotic samples did not show as large an improvement in strength as non-osteoporotic samples when cement volume was increased. This study suggests that clinicians may be able to use DEXA to select an appropriate cement volume and to determine the expected improvement in mechanical stability after vertebroplasty for a specific patient based on his or her bone mineral density.
References:
[1] Silverman, S., Bone 13:S27-31, 1992.[2] Graham J et al., J Bone Joint Surg 85A:1901-1908, 2003.[3] Higgins KB et al., Spine 28(14):1540-1548, 2003.[4] Molloy S et al., Spine 28(14):1549-54, 2003. [5] Garfin SR et al., Spine 26:1511-1515 (2001).
V. Buzduga, D. Witters, J. Casamento, P. Ruggera, W. Kainz, S. Seidman, G. Mendoza, CDRH, FDA, Rockville, MD
Background: The implantable medical devices should be protected from the effect of magnetic fields in certain limits. The actual standards check this protection with voltages which simulate the effect of magnetic fields on device circuitry. At CDRH we developed a system and methods for testing medical devices with magnetic fields instead of voltages.
Test system and methods: We designed and prototyped a test system which produces magnetic fields of 150 A/m up to 100 kHz and strengths decreasing as 1/f above 100 kHz as required in several standards. The implantable device for test is placed together with its leads in a saline tank which is immersed in the magnetic field of an induction coil. The signal delivered in saline by the device under test is monitored on oscilloscope.
Results: We tested pacemakers and neurostimulators of several models and makes at different operating modes and sensitivities. The results of tests reveal possible interference in medical devices due to magnetic fields. The test system was presented to standardization working group members for ISO14708 and PC69. They expressed the intention to introduce this test method in standards.
Conclusions: The system and methods developed at CDRH are very useful for testing the immunity of pacemakers and neurostimulators to magnetic fields. The results of the research performed at CDRH will help the manufacturers to improve the electromagnetic compatibility of medical devices within modern environments.
B. Cai1 , F. McMaster2 , J. McDonough2 , N. Sadrieh3 , S. Brown4 , A. M. Wokovich5 , W. H. Doub5 , L. F. Buhse5 , P. Schwartz1 , R. Patel1 , M. L. Chen3 , 1OGD, OPS, CDER, FDA, Rockville, MD, 2SouthWest Research Institute, San Antonio, TX, 3OPS, CDER, FDA, Silver Spring, MD, 4CDRH, FDA, Rockville, MD, 5OTR, OPS, CDER, FDA, St Louis, MO
Background: Currently there is no generally accepted method for evaluating adhesion-to-skin performance of transdermal drug delivery system (TDDS). The aim of this study was to develop in vitro adhesion testing methods for quality control and/or for product comparison.Conclusions: The modified peel adhesion testing method with 2 µin SS appears useful for quality control whereas HDPE or Vitro-Skin® may provide a better correlation with in vivo human skin. Further studies are needed to evaluate the applicability of this method to TDDS.In addition, in vivo studies will be required to establish the correlation between in vitro and in vivo peel adhesion testing results.
S. A. Colburn, G. G. Gantt, M. D. Brooks, J. F. Lipman, FDA/CDRH/ODE, Rockville, MD
Subject: Requirements of Devices used for Power Injection of Contrast Media:
Device Focus: Short- and Long-term Intravenous Catheters, Implanted Ports, Intravascular Administration Sets, and Accessories
Background: Over the past several years, the Agency has received over 250 adverse event reports in which vascular access devices have ruptured when used with power injectors to administer contrast media.The pressure developed during contrast injection depends on many factors including flow rate, contrast media viscosity, tube diameter, tube length, and any obstruction to flow. Device failure occurs when the pressure from the injection exceeds the maximum allowable pressure of any component between the power injector and vein.A FDA Postmarket Issue (PMI) Action Team has circulated a notice entitled "Reminders from FDA Regarding Ruptured Vascular Access Devices from Power Injection" to various organizations to raise awareness and encourage safe use of these devices.
Premarket Review Issue: Due to the many factors that can cause device failure, it is difficult to develop appropriate test methodologies and labeling to ensure safe use.
Current Premarket Approach: To support an indication of power injection of contrast media, FDA requests that manufacturers perform device testing under worst-case conditions (i.e., maximum flow rate, device use conditions, contrast media viscosity).
FDA is conducting a study to determine the most practical and informative testing methods and hopes to make this information public at some point.
E. Rorer, D. Calogero, B. Drum, CDRH, FDA, Rockville, MD
Background: Multifocal intraocular lenses (MIOLs) increase the distance range of useful vision, but at the cost of blurring best-corrected vision at all distances. It is not clear that current clinical tests can predict how these changes will affect the performance of real-world tasks. The FDA therefore requires MIOL manufacturers to evaluate night driving visual performance to assure MIOL safety and effectiveness. In collaboration with the University of Iowa, we are using the National Advanced Driving Simulator (NADS) to compare visual performance during simulated night driving with clinical tests of vision that may replace the expensive and burdensome driving requirement.
Methods: Fifty-five subjects aged 30-60 years completed a battery of clinical tests (visual acuity, contrast sensitivity, and optical quality of the eye) and simulated night driving tests on the NADS (maximum identification distances for road signs and hazards). The tests were repeated for clear vision and vision "fogged" by diffusing goggles.
Data Analysis Design: Data collection has just been completed, and preliminary processing is in progress. We will compare the clinical data to the driving simulation data via correlation and regression analyses and analysis of variance. We will look for parallel effects of age, pupil size, and fogging level in the driving vs. clinical results and for strong correlations between driving performance and clinical parameters.
Goals: We hope to identify clinical vision tests that can accurately predict visual performance under adverse viewing conditions, and to use them to replace the night driving requirement in MIOL clinical trials.
R. Zapata, C. N. Wendakoon, C. Carrillo, P. Browning, W. M. Fedio, Food Safety Laboratory, New Mexico State University
Background: Detection of Salmonella spp. in foods is based on conventional enrichment and isolation on selective media. AOAC International has approved a rapid automated method using the VIDAS® Immuno-Concentration Salmonella (ICS), which captures Salmonella cells for subsequent detection by the VIDAS immunofluorescent (SLM) assay. The reliability of the VIDAS ICS/SLM method was compared with the FDA-BAM procedure for Salmonella detection in selected foods artificially contaminated with Salmonella Typhimurium.
Methods: Whole milk, powdered infant formula, almonds, strawberries, cilantro, parsley, bok choy, lettuce, cantaloupe andmung bean sprouts were artificially contaminated with Salmonella Typhimurium (ATCC 14028) at low (1-5 CFU/25g) and high (10-50 CFU/g) levels. At each level, 20 samples were analyzed for Salmonella spp. by the two methods. For each food, five uninoculated control samples were also tested. McNemar's χ2 test was used to determine significant differences between the two methods at each inoculum level.
Results: No statistical differences (p ≥ 0.05) were found between the two methods except for mung bean sprouts inoculated at the low level. The VIDAS method failed to detect 15 inoculated mung bean samples shown to contain Salmonella spp. by the BAM procedure (100% false negative rate). Parsley, bok choy, cilantro and cantaloupe inoculated at low levels also gave false negative results with the VIDAS method.
Conclusion: The VIDAS ICS/SLM method was shown to be convenient and considerably faster than the BAM procedure for Salmonella detection. However, for foods with a high background microflora, false negative results were seen, particularily at low levels of contamination.
C. N. Wendakoon, R. Zapata, W. M. Fedio, Food Safety Laboratory, New Mexico Mexico State University
Background: The FDA-BAM cultural method for detecting Shigella spp. requires anaerobic incubation followed by presumptive and confirmatory tests. This study was conducted to evaluate aerobic enrichment for the isolation and detection of low numbers of Shigella spp. by selective plating and by PCR.
Methods: Artificially contaminated Shigella broth, potato salad and cilantro rinse samples were enriched in Shigella Broth (SB), SB with 0.5% novobiocin and SB with 3% novobiocin at 40°C. Evaluations were performed with S. sonnei, S. flexneri, S. boydii and S. dysenteriae at four inoculum levels (0-1, 1-10, 10-100 and 100-1000 colony forming units). After enrichment for 20 h, samples were streaked onto selective agar and the presumptive shigellae were confirmed by biochemical tests. Enriched samples were also used to detect Shigella spp. by nested PCR.
Results: In inoculated SB, both PCR and selective plating detected shigellae at the lowest inoculum. For potato salad enrichments, the PCR procedure was found to be approximately 10 times more sensitive than the selective plating procedure for detecting Shigella spp. While for cilantro, a 100-1000 fold increase of sensitivity was seen for the PCR procedure. Different novobiocin levels in the enrichments did not improve isolation on the plates.
Conclusion: Followingaerobic enrichment, the PCR procedure detected Shigella spp. at very low levels of contamination. Competing microorganisms in the cilantro rinse enrichments made the isolation of shigellae difficult on the selective agar plates. Use of more than one selective agar improved the isolation of Shigella in highly contaminated food matrices.
W. M. Fedio, C. N. Wendakoon, R. Zapata, C. Carrillo, P. Browning, Food Safety Laboratory, New Mexico Mexico State University
Background: Staphylococcus aureus is one of the most common agents implicated in foodborne disease outbreaks. The FDA-BAM method uses Baird Parker agar for direct enumeration of S. aureus in foods. The PetrifilmTM Staph Express Count system (STX) is a rapid test for the identification of S. aureus and has been approved by AOAC for enumeration of S. aureus This study was undertaken to evaluate the performance of the Petrifilm method for the detection and enumeration ofS. aureus in artificially contaminated hard cheese.
Methods: The 3M PetrifilmTM Staph Express Count System (STX) was compared with the FDA-BAM direct plate count method for the enumeration of Staphylococcus aureus in six types ofartificially contaminated hard cheese (Asiago, Cheddar, Gruyère, Parmesan, Romano and Swiss). Five different samples of each cheese type were inoculated with S. aureus (ATCC 25923) to 10-100CFU/g (low), 100-1000 (medium) and 1000-10,000 CFU/g (high) inoculum levels. S. aureus was enumerated by both methods and the results were compared using paired t tests.
Results: Of the 24 comparisons for S. aureus enumeration, only Romano cheese inoculated at the low and high levels showed significant (P<0.05) differences between the two methods. However, the differences in the counts were within one log cycle.
Conclusion: The PetrifilmTM Staph Express Count System compared favorably with the BAM procedure for S. aureus enumeration in hard cheese. However, the rapid method was more convenient to use, considerably faster, and less expensive to perform than the BAM method.
S. L. Foley1 , V. Call2 , P. R. Kaldhone3 , C. D. Tyler3 , L. Potter2 , G. Anderson2 , S. Phillips2 , K. Kerdahi2 , R. Nayak4 , 1Marshfield Clinic Res. Fndn.; Univ. of Central Arkansas and Arkansas Regional Laboratory, ORA, 2Arkansas Regional Laboratory, ORA, 3Marshfield Clinic Res. Fndn. and Univ. of Central Arkansas, 4Division of Microbiology, NCTR
Background: Disk diffusion and broth dilution assays are conventionally used for antimicrobial susceptibility testing (AST) of bacteria. Within ORA, different laboratories have varied resources to carryout AST. The goal of this study was to determine whether the results of different AST platforms correlate with one another to facilitate inter-laboratory comparison of results.
Methods: Salmonella Heidelberg (n=104) were tested using 4 different AST methods: disk diffusion, broth microdilution using Sensititre (Trek) with the NARMS (CMV1AGNF) panel, manual broth microdilution, and Vitek (BioMerieux) with GNS-207 cards. AST was performed using CLSI interpretive guidelines and recommended quality control organisms. Eight drugs were common to all testing methods; including amikacin (Ami), amoxicillin/clavulanic acid (Amc), ampicillin (Amp), chloramphenicol (Cml), ciprofloxacin (Cip), gentamicin (Gen), tetracycline (Tet), and trimethoprim/sulfamethoxazole (Sxt).
Results: No resistance to Ami and Cip was detected. Resistance to Amc (n=2), Amp (n=3), and Cml (n=3) was only detected by the Sensititre method. Resistance to Gen (n=27), Tet (n=10) and Sxt (n=5) were detected by multiple methods. AST result agreement among all of the methods tested was: Ami (100%), Amc (98%), Amp (97%), Cml (97%), Cip (100%), Gen (80%), Tet (90%), Sxt (95%). When resistance was detected by 3 of 4 methods (n=13) it was typically the Vitek that called the organism susceptible (n=9).
Conclusions: The study indicated that AST methods correlated with one another, with a few exceptions. In general, when discrepancies among the methods occurred, it was due to an increased number of resistances detected with Sensititre or a lower number with Vitek.
C. D. Frazar, P. A. Orlandi, CFSAN
Immunomagnetic separation (IMS) techniques are a well established method for the recovery and detection of Cryptosporidium parvum in water. Its use in detecting C. parvum oocysts in food commodities however, has been limited. Here we compare the recovery of C. parvum oocysts from various commodities by IMS followed by three different means of detection, two PCR based methods and a fluorescent microscopy protocol.Seven different products were spiked with C. parvum oocysts including four beverages, two leafy vegetables and one fruit. Oocysts were directly isolated from beverages by IMS. Vegetables and fruit were washed in a bag filter prior to IMS recovery of oocysts from the resulting rinses. DNA templates were then prepared for PCR using either FTA filters or total DNA extraction by a commercially available kit. Oocysts were also prepared for microscopic detection using the HydrofluorTM Combo kit.Preliminary results suggest that total DNA extraction is a more efficient and consistent method for preparing template for PCR based detection than FTA filters. DNA extraction did, however, lengthen the analysis time compared to FTA filters. More complex matrices, such as high pulp orange juice reduced recovery efficency. Of the three protocols, microscopy was slightly more cumbersome and time consuming but nonetheless performed reliably.In general we found that immunomagnetic separation techniques in conjunction with either microscopy or PCR, using total DNA extraction for template preparation, maximized detection sensitivity for C. parvum in foods and beverages.T. J. Fu1 , N. Maks2 , 1CFSAN/NCFST, FDA, Summit-Argo, IL, 60501, 2NCFST, Illinois Institute of Technology, Summit-Argo, IL, 60501
Background: The use of allergen detection methods helps to ensure the absence of undeclared allergens in foods as well as to validate/verify allergen control measures. Many commercially available ELISA kits employ antibodies that are reactive to all protein components in allergenic foods; others use antibodies that are specific towards single allergenic components or marker proteins.This study compared the performance of both types of test kit for detection of heat-treated egg proteins.
Methods: NIST whole egg powder standard reference material #8415 was heated in the presence of water at 60 and 100°C or was dry-heated at 60, 100, 120, 176, 232 and 400°C for 10 min. The amounts of extractable proteins in the heated samples were assayed using the BCA method as well as two commercial ELISA kits.
Results: Elevated heat treatment resulted in a lower level of proteins being extracted. Boiling resulted in more than a 70% decrease in the amount of extractable proteins whereas dry heating at 100°C did not affect the solubility of egg proteins.While the decrease in the amount of extractable proteins in dry-heated samples was generally reflected in the readings observed using both test kits, the change in the amount of extractable proteins in the boiled samples was not proportionally indicated in either test kit. The Neogen Veratox® kit, which is reactive to whole egg proteins, underestimated the amount of residual proteins in boiled samples.On the other hand, the Tepnel Biosystems Biokit® assay, which employs antibodies specific to a heat stable marker protein (ovomucoid), overestimated the amount of protein remaining.
Conclusions: Heat-induced changes in the solubility and structure of proteins need to be considered when commercial ELISA assays are used for the quantification of allergens in thermally processed foods.
T. J. Fu1 , N. Maks2 , B. Parisi2 , P. Slade2 , 1CFSAN/NCFST, FDA, Summit-Argo, IL 60501, 2NCFST, Illinois Institute of Technology, Summit-Argo, IL 60501
Immunomagnetic separation (IMS) technology has been increasingly used for the selective isolation of pathogens from complex food matrices and culture enrichments. An automated IMS system, the Pathatrix™ (Matrix Microscience, CO), has become commercially available and has been performance certified by the AOAC International for detection/isolation of E. coli O157, Salmonella spp., and Listeria spp. at levels of 1 cell/25g. In this work, the efficacy the Pathatrix™ system for isolation of E. coli O157:H7, Salmonella and L. monocytogenes from spent irrigation water collected during sprouting of alfalfa seeds was evaluated. The feasibility of combining sample pre-concentration with IMS for pathogen detection in greater volumes of sample was also evaluated. Twenty five ml of un-concentrated and concentrated sprout water were inoculated with 0, 1, 10, or 100 cfu of the pathogens and subjected to the enrichment and capture protocols recommended by the manufacturer. The efficacy of the Pathatrix™ system was determined by plating the captured IMS beads on selective media and looking for typical colonies. Using the recommended protocols, the system was able to isolate Salmonella and L. monocytogenes from sprout water inoculated at levels of 10 cfu or higher but failed to isolate E. coli O157:H7 due to interference from background flora. Modifications to the enrichment protocols can improve the detection limit for all three pathogens to as few as 1 cfu per 25 ml for both unconcentrated as well as concentrated samples. In summary, with modified enrichment protocols, integration of sample pre-concentration with IMS would allow isolation of E. coli O157:H7, Salmonella, and L. monocytogenes from spent sprout irrigation water at levels as low as 1-10 cfu per 2.5 L.
T. J. Fu1 , K. Reineke1 , T. Krupinski2 , O. VanPelt2 , N. Maks-Warren2 , B. Parisi2 , P. Slade2 , 1CFSAN/NCFST, FDA, Summit-Argo, IL 60501, 2NCFST, Illinois Institute of Technology, Summit-Argo, IL 60501
Microbiological testing of spent sprout irrigation water has been recommended as part of an overall strategy to enhance the safety of sprouts. The extra cost required, however, has hindered the full implementation of this recommendation. Sample pooling has been proposed to lower the cost of testing. Concerns exist that pooling may reduce the detection efficiency due to dilution of target pathogens. One way to alleviate this problem is to institute a sample pre-concentration step that would allow the entire pooled sample to be analyzed by a single test. We have developed a tangential flow filtration (TFF) system capable of concentrating 10 L of sprout water by 100 fold within 2 h. This study examines the feasibility of integrating sample pre-concentration with existing rapid tests for detection of low levels of E. coli O157:H7, Salmonella and L. monocytogenes in large volumes of sprout water. Two aspects of system performance were evaluated. The first was to determine whether the increase in background microflora in the concentrated samples affects the enrichment and subsequent detection by commercial tests, and the second was to determine the lowest level of pathogens that the system can recover. All the test kits evaluated were able to detect the presence of 1 cfu of target pathogens in 10 ml of concentrated samples (corresponding to 1 L of sprout water prior to filtration), and the system could recover as few as 1 - 10 cfu of each pathogen from 10 L of sprout water. These results suggested that incorporation of TFF can improve the sensitivity of existing rapid tests to 1 cfu/L and allow 10 L of sprout water to be analyzed by a single test.
T. Geng, J. M. Yeung, Food Products Association
Background: Prevalence of celiac disease caused by glutens has been reported to be 1:133 in the United States. Strict avoidance of harmful concentrations of glutens in the diet is the only effective way to manage the disease. In order to protect sensitive consumers, accurate quantitatively determination of glutens is needed. Currently, some commercial test kits provide two different extraction procedures in the same kit without guidance on criteria of use.
Methods: Six commercial immunochemical kits were evaluated to investigate effects of extraction methods: RIDASCREEN® Gliadin, RIDASCREEN® FAST Gliadin, RIDAQUICK Gliadin, Veratox® for Gliadin, Tepnel BioKits Gluten Assay, and Morinaga Wheat Protein ELISA kit. The gluten free foods were spiked with different concentrations of gluten standard reference material (SRM 8418). Two extraction methods: ethanol or cocktail buffers were compared and used according to kit manufacturers' instructions.
Results: Results obtained with 6 assay systems and extraction procedures differed considerably. Extraction with cocktails gave almost two times higher concentrations of gluten than that of ethanol extraction in some test kits. One of the thirty-one gluten-free commercial foods was found to be contaminated by wheat gluten, which was confirmed by PCR method.
Conclusions: The discrepancy in results generated from two different extraction procedures creates uncertainty for interpretation as to which result is valid. There is an urgent need for harmonization for the use of reference materials in control standards, and validation of extraction methods in order to ensure meaningful results.
S. Maruvada1 , G. R. Harris1 , B. A. Herman1 , R. L. King2 , 1CDRH, FDA, Rockville, MD, 2King Acoustic Technologies, LLC, Washington DC
It is essential to know the acoustic power radiated by transducers used in high intensity focused ultrasound (HIFU) surgery devices for both safety and effectiveness considerations. The power radiated by medical ultrasound transducers usually is measured via radiation force balance (RFB) methods. However, for the high power, focused fields encountered in HIFU applications, such measurements can be difficult due to the need for short measurement times to prevent transducer damage, heating of the RFB target, bubble formation, and acoustic streaming effects. To address these challenges, a procedure based on pulsed measurements was formulated. High output, focused ultrasound transducers were characterized in terms of an effective duty factor, which was then used to calculate the power during the pulse at high drive levels. Two absorbing target designs were used, and both gave reliable results and displayed no damage and minimal temperature rise if placed near the HIFU transducer and away from the focus. The procedure was reproducible up to the maximum power generated of approximately 230 W, well within the HIFU range, thus allowing the radiated power to be calibrated in terms of the peak-to-peak voltage applied to the transducer.
Y. Liu1 , B. A. Herman2 , R. L. King2 , S. Maruvada2 , G. R. Harris2 , 1Duke University, Durham, NC, 2CDRH, FDA, Rockville, MD
High intensity focused ultrasound (HIFU) is being studied as a minimally-invasive surgical technique for the thermal ablation of both malignant and benign tumors and cessation of internal bleeding in injured vessels and organs with little damage to the surrounding tissue. Although some clinical success has been achieved, the lack of standardized methods to assess the acoustic and thermal characteristics of the focused fields is one factor that has hampered general understanding and acceptance and has slowed the regulatory review process, especially in the pre-clinical phase. The development of standard measurement instruments and methods, including tissue mimicking test phantoms, would be a significant step in the advancement of HIFU surgery. To that end, tissue mimicking materials (TMMs) have been developed for the acoustic and thermal characterization of HIFU devices. The phantoms comprise a hydrogel matrix containing either dispersed oil droplets, low density polyethylene particles, or both. For each TMM the ultrasonic attenuation coefficient, speed of sound, acoustical impedance, and nonlinear parameter B/A were characterized as a function of different phantom constituents and temperatures up to 70ºC. Also, the thermal conductivity and diffusivity were measured. The values obtained for these physical parameters were consistent with those that have been reported for mammalian tissues. Repeatable temperature elevations were produced for typical HIFU exposures (duration of 10 s, peak temperature of 75º C). These TMMs are appropriate for developing standardized dosimetry techniques, validating numerical models, and determining the safety and efficacy of HIFU ablation devices. Note:This research was supported by the Defense Advanced Research Projects Agency (DARPA) through IAG # 224-05-6016.I. K. Ilev, R. W. Faaland, R. J. Landry, D. Calogero, CDRH, FDA, Rockville, MD
Background: Development and use of intraocular lenses (IOLs) has revolutionized refractive cataract surgery in the last 50 years. An estimated 20.5 million Americans over age 40 have cataracts and more than 1.5 million cataract surgeries are performed per year. IOL dioptric power is a fundamental parameter for characterizing the effectiveness and safety of IOLs. The primary objective of this study is to develop a novel confocal fiber-optic laser method (CFOLM) for accurate and objective measuring of the dioptric power of both positive and negative IOLs.
Methods: The CFOLM operating principle (Pending Patent, March 3, 2006) is based on a simple fiber-optic confocal design. The key element is a single-mode fiber coupler that serves simultaneously as a point light source (3-5 µm diameter) used for formation of a collimated Gaussian beam, and as a highly sensitive confocal point receiver.
Results: The CFOLM design provides high accuracy (<1 µm) in spatially locating the IOL focal point and in measuring the focal length of both positive and negative power IOLs. We have tested IOL samples with both positive (+5 to +30 D) and negative (-5 to -20 D) dioptric powers and we have obtained testing repeatability estimated by a standard deviation in the interval of 0.004-0.06 D/0.003-0.013 D and a relative error in the interval of 0.015-0.3 %/0.02-0.16 %, for positive/negative IOL's, respectively.
Conclusions: We have developed a simple, accurate, completely objective, quick and inexpensive method for measuring the focal length of various focusing elements including positive and negative IOLs, objectives, lenses, contact lenses, eyeglasses, and mirrors. FDA is proposing that this novel test method be incorporated into national and international IOL standards.
C. J. Sauder, C. Wolbert, S. Rubin, K. M. Carbone, T. Malik, CBER, FDA, Rockville, MD
*Equal contributors
Neurotoxic wild type mumps viruses were a major cause of viral meningitis and encephalitis prior to the institution of national vaccination programs. Despite the tremendous success of mumps vaccines in nearly eliminating the disease from many countries, some vaccine strains produce neurotoxic adverse events, e.g., aseptic meningitis. Although relatively rare, meningitis occurs in approximately 1 in 10,000 vaccinees, and the impact on international public health has been significant, e.g., withdrawal of mumps vaccines, public resistance to vaccination, and, in some countries, complete cessation of national mumps vaccination programs. Such issues highlight the need to develop improved pre-clinical safety tests for mumps virus vaccines, e.g., using rapid molecular biology based methods. We report here the development of a neurotoxic, wild-type mumps virus infectious clone. Using our rat-based clinical neurotoxicity model, this infectious clone will allow us to identify the causal association of specific mumps virus genes and genetic mutations on neurotoxic outcomes and will, for the first time, provide us with molecular tools to attempt development of a rapid quantitative pre-clinical mumps safety test.
L. Sheng1 , B. Orrison1 , F. Cai1 , Y. Zhu1 , H. Murata1 , A. Pal1 , M. Athanasiou2 , D. Blair2 , S. Hughes2 , J. Coffin2 , A. Lewis1 , K. Peden1 , 1CBER, FDA, Bethesda, MD, 2NCI-FCRF, Frederick, MD
Background: Vaccines and other biological products manufactured in cells contain contaminating residual DNA derived from that production cell substrate. The cell substrates currently used for the manufacture of licensed vaccines in the US are mainly primary cells (of avian or monkey origin) and primate diploid cell strains (WI-38, MRC-5, FRhl-2). One cell line, the Vero cell line, is used for inactivated poliovirus vaccine (IPV) and a rotavirus vaccine in the US, and is used for the live attenuated oral poliovirus vaccine (OPV) in Europe. The urgency to use more cell lines for vaccine production has arisen due to the need to develop vaccines against HIV/AIDS, against emerging agents (such as SARS), against potential agents of bioterrorism, and against a pandemic influenza. The cell lines proposed for use are neoplastic and some are tumorigenic. Since 1954, there has been a proscription on the use of tumor cell lines for vaccine production. One of the reasons for this proscription is the perceived risk of the oncogenic activity of DNA. Because the data on DNA oncogenicity in animals are few, we are attempting to address the risk of DNA by developing quantitative animal models.
Methods: We have generated expression plasmids for the activated human H-ras oncogene and for the murine c-myc proto-oncogene. These genes are driven by the Moloney sarcoma virus long terminal repeat (LTR) and use the bovine growth hormone poly(A) termination sequence. Their oncogenic activity was confirmed in vitro using the focus formation transformation assay in NIH 3T3 cells and Rat-1 cells. To increase the efficiency of tumor induction, plasmids were constructed that expressed both oncogenes. The activity of these plasmids has been assessed in vivo by inoculating them into different strains of mice by the subcutaneous route. Tumors that arose were removed and cell lines were established. PCR and Southern hybridization were used to confirm the presence of the oncogenes, and Western analysis was used to demonstrate oncoprotein expression.
Results: Two strains of adult and newborn mice (NIH Swiss and C57BL/6) were inoculated with different amounts of either H-ras alone or a combination of H-ras plus c-myc. Tumors were only found when both DNAs were inoculated and only at the highest amount of DNA (12.5 micrograms of each). The NIH Swiss mouse was more sensitive than the C57BL/6 mouse, and newborn animals were more sensitive than adults. Using plasmids that expressed both oncogenes, tumors could be induced in newborn NIH Swiss mice with 1 microgram of DNA. PCR and Southern hybridization analyses demonstrated that both the ras and myc genes were present in the cell lines, and Southern analysis suggested that the tumors were clonal. Western analysis demonstrated that both oncoproteins were expressed in the cell lines tested. These results demonstrate that cellular oncogenes can induce tumors by subcutaneous inoculation and indicate that DNA can be oncogenic in normal mice.
Conclusions: Because tumors were only induced when both ras and myc were inoculated, and because the level of DNA required for tumor induction was at least 1 microgram of a plasmid that expressed both oncogenes, the oncogenic risk from cellular DNA in vaccines containing less than or equal to 10 ng of cell-substrate DNA is likely less than 1 in 107. This calculation is based on the fact that an oncogene is present in the human genome at a level about 1 in 105, and 1 microgram is oncogenic and 10 ng of DNA is present in the vaccine. This calculation does not take into account the fact that two oncogenes are required. Nor does it consider any reduction in biological activity obtained by reducing the size of the DNA or chemical treatments, both of which we have shown can reduce activity by several logs.
A. Agrawal, S. A. Miller, S. Matchette, J. Pfefer, CDRH, FDA, Rockville, MD
Background: A non-invasive technique for quantifying sunscreen efficacy would be useful to replace the current standard of dermal UV exposure and biopsy. In this study we explore the potential for using a noninvasive approach based on diffuse reflectance spectroscopy (DRS) to provide critical information on sunscreen efficacy in the UVA (320-400 nm) and UVB (290-320 nm).
Methods: Eight sunscreens were selected to investigate a range of typical filter ingredients including octinoxate, avobenzone and titanium dioxide. Reflectance data was collected with a commercially-available, fiberoptic DRS device using artificial skin tissues with and without sunscreen applied. This data was used to calculate Sun Protection Factor (SPF), UVA protection factor (UVAPF), and critical wavelength (CW). Signals collected by DRS showed very low reflectance levels, particularly in the UVB.
Results: There was a significant amount of variance between SPF calculations for different samples and agreement with advertised SPF values was poor. However, UVAPF and CW data showed much less variance than SPF. Errors in SPF may be due to the lack of signal strength in the UVB and non-uniform application of sunscreens. UVAPF and CW can be calculated with some degree of consistency and show a moderate level of agreement with limited data in the literature.
Conclusions: While our DRS approach appears to be moderately effective for non-invasive evaluation of sunscreen efficacy in the UVA and UVB, modifications to the approach are needed to achieve results that are highly accurate and repeatable. Artificial tissue models show significant promise as a test method for optical spectroscopy-based devices.
A. Prabhakaran, J. DiBella, W. S. Woltosz, M. B. Bolger, Simulations Plus, Inc., Lancaster, CA
Purpose: To simulate the release profiles of Adinazolam Mesylate and observe the influence of various ratios of HPMC K4M and Lactose.
Methods: We used DDDPlus™ simulation software (Simulations Plus, Inc.) to simulate the release profiles of Adinazolam Mesylate, HPMC K4M and Lactose. Different concentrations of HPMC-Lactose (filler) ratios were used to characterize the swelling and release behavior. The HPMC grade K4M (MWt 95000) was used to characterize the polymer chain disentanglement concentration (Cp) which influenced the polymer erosion. The active drug concentration was held constant at 3% (w/w) and HPMC-Lactose ratio % (w/w) was varied as 80:17, 63:32, 50:47, and 35:62. DDDPlus™ simulations for these formulations were compared to the experimental data from the literature ["Gao, 1996"].
Results: DDDPlus™ simulations accurately matched the in-vitro data. It was observed that the polymer content and its molecular weight highly influenced the swelling kinetics and release of Adinazolam Mesylate. The matrix with 80% HPMC showed the slowest drug release. A decrease in HPMC concentration increased the release rate of Adinazolam Mesylate. In terms of absolute mass of HPMC released, the profiles were almost superimposed conforming to the experimental data. This can be explained in terms of Cp, which is dependent on the polymer molecular weight.
Conclusion: The release simulation of a swellable polymer matrix with varying formulation parameters can be accurately predicted and results indicate the high influence of polymer grade on the swelling and release kinetics.
C. Ramirez1 , S. M. Madson1 , T. B. Bickell1 , M. B. Buen1 , C. L. Burns1 , J. S. Cholensky1 , P. L. Dexter1 , D. E. Farmer1 , R. L. Farmer1 , E. D. Gonzales1 , M. A. Jackson1 , K. D. Kallander1 , C. R. Kiessling1 , W. M. Kiessling1 , J. A. Kinney1 , E. W. Laster1 , C. M. Lemons1 , M. H. Loftis1 , M. E. Maselli1 , L. T. Michel1 , Z. A. Miller1 , T. L. Morales1 , C. I. Shaw1 , P. D. Stahnke1 , M. Z. Thomas1 , K. A. Watts1 , J. N. Sofos2 , 1FDA, Denver, CO 80225, 2Department of Animal Sciences, Colorado State University, Ft. Collins, CO 80523
Serotyping and Pulsed Field Gel Electrophoresis increase resources needed to resolve potential cross-contamination issues in sample analyses.The current Salmonella analysis control, Salmonella Gaminara ATCC8324, may be present in foods and does not exhibit any uniquely distinguishable characteristics.In this study, we explored the potential use of a green fluorescent protein (GFP)-positive strain (Salmonella Typhimurium DT104 ISSAGFP) to be used as a control. This chromosomally modified, stable culture of S. Typhimurium DT104 ATCC700408 (Noah et al., Journal of Food Protection, 2005, 68:680-686) was evaluated for its fluorescent quality during routine sample analysis as a concurrent control with S. Gaminara.Each control strain was inoculated in pre-enrichment broth, transferred to RV and TT. Then, the designated control strain was streaked to all plates indicated in the BAM, while the GFP strain was streaked to one of XLD, HEK or BS and to TSA or TSAYE plates. Colonies were then examined for fluorescence under ultraviolet light.All 251 plates of S. Gaminara evaluated were negative (100%) for fluorescence. In contrast, of 248 plates with the GFP strain evaluated, 236 were positive (95%). Six analysts did not detect fluorescence in one (mostly HEK) medium, but detected it in the other (TSA and/or XLD), while two analysts observed negative fluorescence in both media used (TSA and XLD) in one sample each.The GFP property could additionally be verified under fluorescent microscopy. A GFP-positive strain could be useful as a control by allowing easy determination of cross-contamination in analyses without the need of additional testing.
S. K. Sharma, R. C. Whiting, CFSAN, Collage Park, MD 20740
Anelectrochemilumninescent immunoassay for Clostridium botulinum neurotoxins was evaluated for its ability to detect toxins in food and in culture filtrates. Examination of 77 type A strains, each grown from a single colony, revealed variable levels of toxin production but all were positive for toxin. The assay was found to be suitable for detecting types A, B, E and F botulinum neurotoxin from a variety of food matrices representing solids, semi-solid and liquids. The sensitivity of the assay for toxin complex in buffer was found to be 200 pg/ml for types A, B and E and1000 pg/ml for type F neurotoxin complex. The assay could readily detect 1 ng/ml of neurotoxin types A, B, E and F from food samples. The assay appears to be an effective tool for high through-put screening of the food supply for botulinum neurotoxin.
S. Trujillo1 , N. J. Fico2 , H. Njapau1 , B. J. Cañas1 , D. L. Park1 , 1CFSAN, FDA, College Park, MD, 2JIFSAN, University of Maryland, 0220 Symons Hall, College Park, MD 20742
Aflatoxin M1 (AFM1) is the hydroxylated metabolite of aflatoxin B1 that can occur in milk. Feeding lactating dairy cows aflatoxin B1-contaminated corn and cottonseed meal in dairy rations have resulted in aflatoxin M1-contaminated milk and milk products, including nonfat dry milk, cheese, and yogurt. Although aflatoxin B1 has long been regulated by the U.S. Food and Drug Administration (FDA), due to the nature of the toxin, lactating animals are still exposed to it. Therefore, aflatoxin M1 may be an unavoidable genotoxic compound that can occur in milk and milk products. Hence, the FDA has set up an action level of 0.5 ng/g in milk and milk products. The need for the milk industry to comply with the established regulations necessitate the use of validated test kits. Reliable and validated rapid test kits are therefore required by industry as well as State and Federal agencies to monitor aflatoxin M1 levels in raw milk prior to processing as well as in the finished products, including pasteurized milk, yogurt, and cheese. Three commercial test kits [Aflatest P® immunoaffinity column (IA) VICAM LP, Watertown, MA; Charm SL™ Raw Milk lateral flow AFM1 test strip (LF), CHARM Sciences, Inc., Lawrence, MA, and an electro-potential biosensor (BI), R-Biopharm, AG Damstadt, Germany], for the detection of aflatoxin M1 in milk are available, but have not been validated at the regulatory level. The performance of each of the three kits was evaluated in two commodities (raw and pasteurized milk) at different concentrations.
M. L. Virata-Theimer1 , S. J. Thorpe2 , M. W. Yu1 , 1CBER, FDA, Rockville, MD, 2NIBSC, Herts, UK
Background: Hemolysis following intravenous immunoglobulin (IGIV) therapy has been linked to not only anti-D (Rh) but also anti-A and anti-B hemagglutinins. In Europe, manufacturers are required to test IGIV products for anti-A and anti-B using an indirect antiglobulin test (IAT) with maximum limits specified at <1:64 for a 3% IgG solution. In the US, manufacturers set their own specifications using the IAT and no maximum limits are imposed by the FDA.Currently, no standardized reagents and methods are available. Recently, Thorpe et al (Biologicals 2005;33:111-6) evaluated the anti-A and anti-B titers in IGIVs distributed in Europe and demonstrated that a direct hemagglutination method gives more consistent results, whereas the IAT is not sensitive.This direct method was recently accepted by the European Pharmacopoeia and FDA as the preferred reference test for measuring anti-D in IGIV.
Methods: 168 lots, representing 11 US-licensed IGIVs produced in 2000-2005, were tested using Thorpe et al's direct method.
Results: The highest antibody titer observed for either anti-A or anti-B was 1:32 when assayed with a starting IgG concentration of 5% per lot.There were 3 lots each having such a titer (3/168; 1.8%). The median titers for anti-A and anti-B were 1:4 (71/168; 42%) and 1:8 (80/168; 48%), respectively.
Conclusions: Current lots of US-licensed IGIVs had anti-A and anti-B titers at < 1:32 for a 5% IgG solution.Using this direct method with reference reagents (soon to be established), we can standardize anti-A and anti-B testing and set maximum specifications to ensure IGIV product safety.
T.O. Woods, CDRH, Rockville, MD
Each year, hundreds of thousands of patients receive new implants. Simultaneously, each year the scope and complexity of interventional MRI (Magnetic Resonance Imaging) procedures continue to expand, requiring an increasing array of patient monitors, support systems and surgical tools that are safe and function correctly in the MR environment. As a result, the issue of the safety of implants in the MR environment is of paramount importance to the medical community and to the millions of patients who receive MRI scans each year.
As the use of MRI spread rapidly in the 1990s, FDA/CDRH recognized the need for standards to address the safety of implants and other medical devices in the MR environment. In 1997, CDRH requested that ASTM develop suitable test methods and guidance to address safety and effectiveness issues for medical devices in the MR environment. The request led to the formation of ASTM task group F04.15.11 on the safety and compatibility of implant materials and medical devices in the MR environment. The group first met in 1998 and decided to develop test methods for evaluating some of the principal safety concerns for implants:magnetically induced displacement force and torque, radio frequency (RF) heating, and image artifact. To date, they have written and published five standards addressing testing and marking medical devices for use in the MR environment. These standards have all been recognized by FDA and are frequently used in medical device submissions. Work continues to expand the scope of the methods to cover electrically active implants.
Y. S. Yang, A. S. Carlin, P. J. Faustino, R. C. Lyon, C. D. Ellison, M. L. Hamad, A. R. Bryant, E. H. Jefferson, M. A. Khan, DPQR, CDER, FDA, Silver Spring, MD
Background: Drug products are often repackaged by pharmacists and repackaging companies from their original FDA approved container closures.This study was conducted in affiliation with the Office of Compliance to address regulatory concerns and to identify stability issues associated with repackaging.
Methods: We investigated the stability of 50 mg metoprolol tablets packaged in their original HDPE containers and repackaged in USP Type A unit dose blister packs. Controlled stability studies were conducted at 25oC/60% RH (1-12 months) and 40oC/75% RH (1-3 months). Products were analyzed for potency, dissolution, water content and hardness. Tablets were also analyzed noninvasively by near-infrared (NIR) spectroscopy and NIR chemical imaging.
Results: Neither stability condition significantly affected the attributes measured for tablets in the original containers. 25oC/60% RH had no significant affect on the repackaged tablets. However, the repackaged tablets gained significant amount of water (3.5% to 8.5%) in 4 weeks at 40oC/75% RH. The increase in water content changed the tablet performance as measured by a decrease in hardness (8 kp to ~0 kp) and an increase in tablet dissolution (51% to 92 % at 5 min). The increase in water was clearly detected by NIR methods.
Conclusions: Although, USP Type A repackaging provides the highest level of protection from moisture (<0.5 mg water permeation per day at 23oC/75% RH), certain moisture sensitive drug product formulations may not be suitable for repackaging. The stability of drug products can be monitored by "at-line" NIR techniques for rapid assessment and correction of storage conditions.
L. Graham, C. Serrano, I. Reischl, K. DeBell, E. Bonvini, B. Rellahan, Division of Monoclonal Antibodies, OBP, OPS, CDER
Background: The Division of Monoclonal Antibodies regulates products aimed at modulating lymphocyte activity for the treatment/prevention of graft rejection, autoimmune diseases and cancer. The development and validation of potency assays that accurately reflect the mechanism of activation of such therapeutics is problematic due to the inherent variability of cell based assay systems. The recent development of phospho-specific antibodies raises the possibility that these reagents could be used to quantitate the activity of immuno-modulatory therapeutics in a sensitive and robust assay.
Methods: Site-specific mutations, transient and stable transfections, calcium-mobilization, reporter gene assays, immunoblotting, and fluorescence-activated cell sorter (FACS) techniques were used to identify and characterize phospholipase Cγ1 (PLCγ1) phosphorylation.
Results: The recent use of phospho-specific antibodies combined with flow cytometry analysis has opened new applications that include characterization of scarce cell populations, activation pathways, drug screening, disease phenotyping and biomarker identification. Our laboratory uses Phospholipase C gamma-1, a model protein that integrates downstream signaling events from multiple receptors, to investigate and understand common and unique signaling cascades that are utilized by selective immuno-modulatory antibodies. Utilizing antibodies that specifically recognize phosphorylated tyrosines 775 and 783 of PLCγ1, we sought to determine if the degree of lymphocyte activation could be determined by assessing the levels of Y775 and Y783 phosphorylation in a Phospho-flow based assay. Our results suggests that the Phospho-flow assay is highly reproducible and robust, but it is subject to false-positives due to cross-reactivity of the phospho-specific antibodies and to secondary reagents that may be used during cellular activation and intracellular staining.
Conclusion:Phospho-flow assays could be developed and validated as potency assays for immuno-modulatory therapeutics.
W. K. Lee1 , H. Kim1 , K. Y. Song2 , K. H. Seo3 , 1SNU, Seoul, South Korea, 2JIFSAN, UMD, College Park, MD, 3CFSAN, FDA, College Park, MD
We investigated the effect of soy bean extracted chiro-inositol and myo-inositol on the delay of the pathogenesis of diabetic vascular disease in vitro. Cataractogenesis was induced by incubating the rat lenses in medium 199 containing 20mM xylose. 5.6mM concentration of chiro-inositol (CI), myo-inositol (MI), and chiro-inositol and myo-inosiol mix (CM) that were added to each of 20mM xylose medium were measured the effect on the opacity, IC50 of aldose reductase, the oxidative stress, the polyolpathway, lactate dehydrogenase leakage and sugar influx for 48 hours. CI, MI and CM decreased significantly the opacity of lenses measured the Image Analyzer when compared with the lenses cultured in 20mM xylose medium only. We observed the reversion of the cataract in lenses cultured in CI and CM added to the medium after 36 hours. Inhibition concentration by 50% of aldose reductase activities were evaluated as 2.37 g/ml (CI), 2.86 g/ml (MI), and 3.05 g/ml (CM) in vitro using IC50 value and percent inhibition of chemiluminescence, respectively. We expect CI, MI, and CM to be more effective aldose reductase inhibitor,for IC50 of them is stronger than other inhibitors. In the amounts of GSH and the activities of glutathione peroxidase,CM increased by 200%. Increased GSH contents and glutathione peroxidase activities may contributethe greater anti-oxidative system can protect lens cell from cataract-induced oxidative stress. The studies describedin this report were undertaken to examine the effects on the polyol pathway by measuring sorbitol and xyliotol contents, the activities of aldose reductase,and the rate of fructose production. The sorbitol contents and the rate of fructose production of the cataract lenses in CI added to medium were found to be significantly reduced by 80% and 58%. Indicating is CI plays an important role in reduction of polyols that cause osmotic stress. The effect of CI in reducing lactate dehydrogenase (LDH) leakage were correlated with cell damage associated with cataract formation was also examined. Leakage of LDH into the culture medium was significantly reduced by 45% in MI added group, by 46% in CM added group, and by 60% in CI added group. This result means these inhibited cataractous damage. Lastly, we demonstrated that the rate of sugar influx into the lenses cultured in CI added medium was significantly decreased by 73%. We can found the effect of CI oninhibition of influx of sugar into rat lenses. CI reduced catractogenesis through blocking sugar entrance into lens. In conclusion, the effect of CI, MI, and CM is significantly good for the delayof diabetic cataract that is multi-factorial, even till 48 hours. Especially, CI has the best effect among them. We need to considerate the role of CI in cataract in relation to the reduction of conversion of MI to CI in diabetes and require the further study for the mechanism of CI and MI in cataractous lens.
K. Y. Song1 , Y. C. Yoon2 , D. K. Jeong3 , K. H. Seo4 , 1JIFSAN, UMD, College Park, MD, 2Konkuk University, Seoul, South Korea, 3Kosin University, Busan, South Korea, 4CFSAN, FDA, College Park, MD
The objectives of this study were to determine the hydrolysis of lactose among probiotic bifidobacteria by high performance liquid chromatography (HPLC) and to select bifidobacteria with high extra- and intra-beta-galactosidase activity. Five commercial probiotic bifidobacteria were used for this test. Lactose and its byproducts in culture suspensions by beta-galactosidase activity of bifidobacteria were efficiently separate by HPLC. The beta-galactosidase activity which was detected by hydrolysis of lactose were different among five Bifidobacterium species. In tested samples, Bifidobacterium bifidum (BBB) had the greatest beta-galactosidase activities, Bifidobacterium longum (BBL) 3, Bifidobacterium infantis (BBI) and BBL 1 had the middle, and BBL2 had the least. The beta-galactosidase activities of the sonicated samples were higher than those of the unsonicated samples in all bifidobacteria after 4.5 hours of incubation (P<0.01). After 4.5 hours incubation, the amounts of lactose in the sonicated samples were decreased to 79% by the beta-galactosidase of BBB, to 67% by that of BBL 3, to 52% by that of BBI, to that of BBL 1, and to 16% by that of BBL 2 (P<0.01).
S. O. Choi1 , Y. C. Yoon1 , K. Y. Song2 , K. H. Seo3 , 1Konkuk University, Seoul, South Korea, 2JIFSAN, FDA, College Park, MD, 3CFSAN, FDA, College Park, MD
This study was conducted to evaluate the quality of yoghurt manufactured from different concentration factors (CF1.00 as control, CF 1.67, CF1.82, CF 2.00, and CF 2.22) using by ultrafiltration and to determine its quality changes during storage at 5 C for 0, 3, 6, 9, 12, and 15 days. All samples were analyzed for general composition, solids-not-fat (SNF), total solids (TS), non-protein-nitrogen (NPN), viscosity, whey separation, lactic acid bacteria (LAB) count, sensory evaluation. As the concentration factor of raw milk increased, (1) concentration of fat, protein, and SNF steadily increased. However content of NPN and LAB count were relatively unchanged. (2) With increase in concentration factor, the incubation time to reach pH 4.6 increased (CF1.00 as control- 4 hr 30 min, CF 1.67- 5 hr, CF1.82- 5 hr 15 min, CF 2.00- 5 hr 30 min, and CF 2.22- 6 hr) and changes in titratable acidity become slow during incubation. (3) As concentration factor increased, the viscosity also increased but the volume of whey separation decreased. The higher the concentration factor was, the higher the values of sensory evaluation was. Thus, control sample received the lowest value. (4) During the storage of yoghurt samples, content of fat, protein, SNF, TS, NPN, and volume of whey separation were relatively remained same. However, lactose concentration and sensory evaluation value slightly decreased while viscosity somewhat increased. (5) When comparing control sample to treated samples, protein, SNF, and TS concentrations were slightly decreased in control. However, lactose concentration was remarkably decreased in control. (6) Based on all of these results in this study, yoghurt manufactured using by ultrafiltration improved quality compared to conventional yoghurt by adding skim milk because of higher concentration in fat, protein and viscosity, and lower amounts of lactose and whey separation. Furthermore, quality of yoghurt was improved with a higher number of concentration factor.
J. U. Kim1 , Y. C. Yoon1 , K. Y. Song2 , K. H. Seo3 , 1Konkuk University, Seoul, South Korea, 2JIFSAN, UMD, College Park, MD, 3CFSAN, FDA, College Park, MD
This study was performed to investigate the quality and shelf life of Tofu added dairy byproducts as WPC and WP. The Tofu manufactured contained the amount of 0%, 2%, 4%, and 6% WPC under the moulding weight of 13.3 g/cm2, and of 0%, 2%, and 4% WP under the moulding weight of 13.3 g/cm2, 17.8 g/cm2, and 22.2 g/cm2. The mixture ratio of CaCl2 and glucono-delta-lactone (GDL) used for Tofu coagulant was 9:1, 8:2, 7:3, and 6:4. When the higher addition of WPC and WP to soymilk, the content of moisture was decreased, but content of total solids, protein, fat, carbohydrate, and ash were increased in premix. The yield was increased in Tofu with addition of WPC to compare to traditional Tofu (P<0.05), while Tofu with WP showed a little increase, but there was no significant between Tofu with WP and traditional Tofu in yield (P<0.05). The general composition of all Tofu manufactured in this study was affected by different addition of WPC and WP, by the different condition of moulding weight, and by different mixture ratio of CaCl2 and GDL as Tofu coagulant to compare to traditional Tofu. In the rheology, Tofu with WPC showed high score than tofu with WP and traditional Tofu. In sensory evaluation, Tofu with WPC and WP showed the better than traditional Tofu. But, after the storage of 60 hours (2.5 days), the Tofu with WPC and WP began to spoil, showed discolor on the surface, and germinated mould. The traditional Tofu showed the good shelf life than Tofu with WPC and WP. Further study is going to improve shelf life of Tofu with addition of WPC and WP to keep up with the condition of the traditional Tofu.
S. M. Shin1 , Y. C. Yoon1 , K. Y. Song2 , K. H. Seo3 , 1Konkuk University, Seoul, South Korea, 2JIFSAN, UMD, College Park, MD, 3CFSAN, FDA, College Park, MD
This study was conducted for analyzing general composition and change in quality of soymilk yoghurts manufactured by adding skim milk or whey powder to soy milk treated by heat of 95 C for 5 min and 120 C for 10 min, respectively. (1) During the storage of soymilk yoghurt, content of total solids, protein, fat, and lactose slightly decreased, whereas viscosity, content of ash, NPN, and the number of lactic acid bacteria were nearly not changed. (2) pH and titratable acidity were rapidly changed in all soymilk yoghurt for 3 hours of incubation. (3) The number of lactic acid bacteria in control sample was 7.8 x 108, in soymilk yoghurt added skim milk, 4.97 x 108, and 5.02 x 108, respectively. In soymilk yoghurt added whey powder, 5.9 x 108, and 5.5 x 108, respectively, according to degree of heat treatment with 95 C for 5 min and 120 C for 10 min.(4) Viscosity of yoghurt samples became lower, as heat treatment was high in temperature and long in time. (5) The value of sensory evaluation was relatively high in soymilk yoghurt added skimmilk which was treated by heat at 95 C for 5 min, however, the value was significantly lower than that of control sample. (6) Lactose, glucose and galactose were detected in all samples using by HPLC, it was because lactose could be degraded into glucose and galactose for 3 hours on incubation.
C. H. Song1 , Y. C. Yoon1 , K. Y. Song2 , K. H. Seo3 , 1Konkuk University, Seoul, South Korea, 2JIFSAN, UMD, College Park, MD, 3CFSAN, FDA. College Park, MD
This study was carried out to compare the composition (fat, protein lactose, and total solids) of Mozzarella cheese whey with retentate's and permeate's one by ultrafiltation, and also to investigate seasonal change of the composition. Glycomacropeptide and beta-lactoglobulin of whey protein, which was concentrated by ultrafiltatrion and heating whey, were isolated by DEAE-sepharose fast flow as ion-exchange chromatography. As a result of the experiment for nine months, the average level of fat was 0.77%, protein 1.46%, lactose 4.13% and TS 7.13%. When Mozzarella cheese whey was concentrated by reduction 10 times, the average level of fat at retentate was 5.76%, protein 6.47%, lactose 3.98%, and TS 17.2%. At permeate, the average level of fat was 0.22%, protein 0.47%, lactose 4.24% and TS 5.77%. To compare with the level of composition between concentrated and untreated whey, the fat was increased 7.5 times, protein 5.6 times, and total solids 2.5 times, but lactose was decreased 0.96 times. On seasonal changes of whey composition, the fat level was low in summer and winter, but high in spring and autumn. The level of protein, lactose and total solids was high in autumn and winter, but low in spring and summer. Approximately 1g GMP/1 L was isolated from Mozzarella cheese whey. GMP was identified using by SDS-PAGE and HPLC amino acid analysis. Also approximately 1.6 g beta-LG/1 L was isolated from Mozzarella cheese whey. beta-LG was identified using by SDS-PAGE and HPLC amino acid analysis, and it's molecular weight was 18,000 Da.
S. H. Yoo1 , Y. C. Yoon1 , K. Y. Song2 , K. H. Seo3 , 1Konkuk University, Seoul, South Korea, 2JIFSAN, UMD, College Park, MD, 3CFSAN, FDA, College Park, MD
This study was carried out to compare the composition (fat, protein lactose, and total solids) of Cheddar cheese whey with retentate's and permeate's one by ultrafiltation, Glycomacropeptide and beta-lactoglobulin of whey protein, which was concentrated by ultrafiltatrion and heating whey, were isolated by ion-exchange chromatography. As a result of the experiment for nine months, the average level of fat was 0.68%, protein 1.17%, lactose 4.53% and TS 7.03%. When cheese whey was concentrated by reduction 10 times, the average level of fat at retentate was 5.06%, protein 5.97%, lactose 4.08%, and TS 15.2%. At permeate, the average level of fat was 0.21%, protein 0.48%, lactose 4.30% and TS 5.72%. To compare with the level of composition between concentrated and untreated whey, the fat was increased 7.4 times, protein 5.1 times, and total solids 2.1 times, but lactose was decreased 0.9 times. About 1g GMP and less than 1.6 g beta-LGper 1 L were isolated from Cheddar cheese whey, and were identified using by SDS-PAGE. To compare with GMP and beta-LG of Mozzarella cheese whey, there was no significant difference between Mozzarella cheese whey and Cheddar cheese way (P<0.05).
J. H. Lee1 , Y. C. Yoon1 , K. Y. Song2 , K. H. Seo3 , 1Konkuk University, Seoul, South Korea, 2JIFSAN, UMD, College Park, MD, 3CFSAN, FDA, College Park, MD
This study was carried out to evaluate the quality and utilization possibility of Mozzarella cheese analogs manufactured by mixtures of soy milk and raw milk (1:1, 1:2, 1:3, 2:1, and 3:1) in comparison with Mozzarella cheese analogue (soymilk) and Mozzarella cheese (raw milk) manufactured by traditional method. The more addition of soy milk to raw milk in premix, the content of protein increased but there was no increased in the content of lactose and SNF. During the storage of 30 days at 4 C, total solids, and NPN of Mozzarella cheese analogue were increased, but fat, lactose, and protein were decreased. Ash did not change. According to the results of physio-chemical components of Mozzarella cheese analogue, the more soy milk addition to raw milk, the lower pH showed. The changes of pH decreased a little but titratable acidity increased. The traditional Mozzarella cheese showed much higher in actual yield and predicted yield than Mozzarella cheese analogue. At a result of electrophoresis analysis, Mozzarella cheese analogue showed more other bands than traditional Mozzarella cheese showed alpha-casein and beta-casein bands. Also on the result of physical characteristics analysis, hardness was affected by addition of soy milk, cohesiveness and brittleness was affected by addition of raw milk, and elasticity was not almost affected. Hardness hardly changed but elasticity, cohesiveness and brittleness decreased a little. The traditional Mozzarella cheese showed higher in meltability than Mozzarella cheese analogue. But there was no difference in browning, oiling-off between traditional Mozzarella cheese and Mozzarella cheese analogue (P<0.05), but Mozzarella cheese analogue showed the decreased in stretching. In the sensory evaluation, traditional Mozzarella cheese showed the better in body texture, appearance and flavour than Mozzarella cheese analogue. Further study is going to improve the sensory of Mozzarella cheese analogue.
S. H. Lee1 , Y. C. Yoon1 , K. Y. Song2 , K. H. Seo3 , 1Konkuk University, Seoul, South Korea, 2JIFSAN, UMD, College Park, MD, 3CFSAN, FDA, College Park, MD
This study was carried out to evaluate the quality and utilization possibility of Mozzarella cheese analogs manufactured by mixtures of soy milk and concentrated raw milk (1:1) in comparison with Mozzarella cheese manufactured by traditional method. Raw milk was concentrated by ultrafiltration as CF 1, CF 2, CF 3, CF4, and CF 5. According to the result of the analysis of components in Mozzarella cheese Analogue, after the storage of 30 days, total solids, water soluble nitrogen and non protein nitrogen of Mozzarella cheese analogue were increased, but fat, lactose, and protein were decreased. Ash did not change. At a result of electrophoresis analysis, it was classified as alpha-, beta- and kappa-casein in Mozzarella cheese analogue. In Mozzarella cheese analogue made from premix, the higher concentration rate of raw milk add to soy milk, the lower the hardness, elasticity, cohesion, and brittleness. And when the concentration factor of raw milk in premix was increased, meltability increased in accordance with the concentration rate. But the degree of meltability was lower than traditional Mozzarella cheese.In Sensory evaluation results, Mozzarella cheese analogue showed the better than traditional Mozzarella cheese in formation, appearance, flavour. Further study is going to manufacture the various products of Mozzarella cheese analogue to meet the various demands of consumers.
W. S. Jin1 , Y. C. Yoon1 , R. L. Song2 , K. H. Seo3 , 1Konkuk University, Seoul, South Korea, 2JIFSAN, UMD, College Park, MD, 3CFSAN, FDA, College Park, MD
This study was carried out to develop Mozzarella cheese analogue by utilizing some of the dairy-products in the form of WPC 34, WPC 80, whey protein (WP), demineralized whey powder (DWP) and lactose powder (LP) along with soy milk. Soy milk was separated blended with 5% WPC 34 (A), WPC 80(B), DWP(C), WP(D) and LP(F) and similarly with 10% WPC 34 (F), WPC 80(G), DWP(H), WP(I) and LP(J). These admixtures were used in the preparation of Mozzarella cheese. Mozzarella cheese analogue prepared by blending, soy milk with above whey-products were subjected to various physico-chemical and sensory attribute studies in comparison with the Mozzarella cheese made with raw milk (R) by traditional method. The Mozzarella cheese and analogue were stored for 30 days at 4 C. The physicochemical and sensory quality changes during storage were monitored and the results are summarized below. The physico-chemical and sensory qualities of Mozzarella cheese and analogues and their storage stability. (1) The pH of all the treated samples except the cheese prepared from group G was lower than the control Mozzarella cheese. (2) Electrophoresis study has shown more bands of protein of high molecular weight in the Mozzarella cheese analogues than in the control Mozzarella cheese. (3) The rheological studies as studied by rheometer revealed that, the hardness of Mozzarella cheese analogues are lesser than the control Mozzarella. But the elasticity, cohesiveness and brittleness of the analogues were higher than the control samples. (4) With regard to meltability, the control sample has shown higher meltability than any of the Mozzarella analogue. During storage for 30 days at 4 C, there was continuous increase in meltability of the Mozzarella cheese but on the contrary there was increase in meltabilty up to 10th day of storage and later on there was decrease in the meltability with respect to all the analogues samples (5) Mozzarella cheese prepared by the traditional method has shown a higher browning, and stretching as compared to all the types of analogues but a higher oiling off was observed in Mozzarella cheese analogues than the control. (6) From the sensory evaluation studies as represented by sensory scores, it is apparent that the Mozzarella cheese prepared by traditional method is superior with respect body texture, appearance and flavour as compared to Mozzarella analogues. However, the analogues were also highly acceptable. The storage study revealed that there was improvement in body texture, appearance and flavour of control Mozzarella cheese during 30 days of storage at 4 C. But, in case of Mozzarella analogues, there was slight decrease in scores with respect to these attributes with the progress of storage.
K. Y. Song1 , Y. C. Yoon2 , S. S. Yoon3 , K. H. Seo4 , 1JIFSAN, FDA, College Park, MD, 2Konkuk University, Seoul, South Korea, 3IFBB, Yonsei University, Seoul, South Korea, 4CFSAN, FDA, College Park, MD
The objective of this study was to evaluate the quality of Mozzarella cheese as new type cheese. The milk used for producing Mozzarella cheese was classified by fat amounts (0.5% and 3%), by bacterial count (<30,000 CFU/ml and <100,000 CFU/ml), and by concentration factor (CF 2 and CF 3). On making Mozzarella cheese in accordance with the combination of several factors as described above, the addition ratio of rennet and starter was classified by 50%, 65% and 80% to compare to that of traditional Mozzarella cheese. The general components of raw milk, retentate, permeate and whey were examined. Retentate concentrated by ultrafiltration was higher than raw milk in fat, protein, and solid-not-fat contents, but was lower in lactose. The content of protein, lactose and SNF was higher than raw milk in whey. The general components of Mozzarella cheese made form the combination of several factors were affected in total solids, ash, fat, lactose, protein, water soluble nitrogen, non protein nitrogen, and NaCl during the storage of 3 months. Also, the physicochemical property of Mozzarella cheese was affected by fat, bacterial count and addition ratio of starter and rennet. Actual yield and predicted yield, adjusted to 50% TS, were affected by concentration factor, fat content, and addition ratio of starter and rennet. At results of UREA-PAGE analysis, the content of alpha-s-casein decreased but beta-casein was constant during the storage of 3 months 4 C. Hardness, elasticity, cohesiveness, and brittleness were affected by fat content, addition of starter and rennet. Meltability and fat leakage were affected by bacterial count and fat content. Browning, oiling-off and stretching were affected by bacterial count, fat content, addition of starter and rennet, and temperature. Flavor, body texture, appearance, color and firmness decreased by bacterial count, fat content, addition of starter and rennet.
Y. A. Kim1 , H. Kim1 , K. Y. Song2 , K. H. Seo3 , 1SNU, Seoul, South Korea, 2JIFSAN, UMD, College Park, MD, 3CFSAN, FDA, College Park, MD
This study was performed how soybean-derived pinitol (3-O-methyl-D-chiro inositol) works the cataractogenesis of organ cultured-rat lenses using 20mM D-xylose induced cataract of the lens. Rat lenses were cultured in 20mM xylose medium with or without pinitol. The opacity of the lens was measured at intervals of 12 hours with CCD camera and analyzed with Image Analyzer. Pinitol was added in increasing amount from 1.25 mM to 7.4mM to the xylose medium. The opacity has decreased the most in the lens group of 2.5mM and 4.9mM concentration of pinitol. After cultivating of 24 hours, 2.5mM pinitol and 4.9mM pinitol adding reducedthe opacity of cataract group by almost 34%, 25% compared to those cultured in xylose only medium.After cultivating of 36 hours,2.5mM pinitol and 4.9mM pinitol adding reducedthe opacity of cataract group by almost 50%, 45% compared to those cultured in xylose only medium.This preventive effect of pinitol on the cataractogenesis lasted for 60hrs. It is general that mechanisms of cataractogenesis is explained by oxidative stress and polyol pathway. Thus, We investigated the biochemical change of rat lenses in sugar cataract and the effect of 2.5mM pinitol and 4.9mM pinitol on the changes of polyol pathway and oxidative stress after 24 hours of cultivating. As for polyol pathway, the glucose influx into the lenses cultured 2.5mM pinitol and 4.9mM pinitoladded medium was reduced by 65.7%, 65.8% and xylose influx into lenses was decreased by 48.2%, 69.1% compared to those cultured in xylose only medium. Compared to lenses cultured in xylose only medium, sorbitol and xylitol contents of the lenses cultured in 2.5mM pinitol and 4.9mM pinitoladded medium were significantly decreased by 89.8%, 90.7%. Aldose reductase activities of the lenses cultured in 2.5mM pinitol and 4.9mM pinitoladded medium were significantly decreased by 34.1%, 45.2% compared to lenses cultured in xylose only medium. As to oxidative stress, GSH contents of the lenses cultured in 2.5mM pinitoladded medium were 3.5 times as much as GSH contents of lenses cultured in xylose only medium. and GSH contents of the lenses cultured in 4.9mM pinitoladded medium were 3.7 times as much as GSH contents of lenses cultured in xylose only medium. GPX activities of the lenses cultured in 2.5mM pinitoladded medium were 1.9 times as much as GPX activities of lenses cultured in xylose only medium. And GPX activities of the lenses cultured in 4.9mM pinitoladded medium were 3.1 times as much as GPX activities of lenses cultured in xylose only medium. Lactate dehydrogenase activities of medium 2.5mM pinitol and 4.9mM pinitol added group were decreased significantly. In conclusion, soybean-derived pinitol inhibited the developing cataract. This delay may have brought about by the inhibition of polyol pathway and the reduction of oxidative stress caused by polyol pathway increasing when sugar cataract was developing.
CATEGORY M: ENGINEERING AND PHYSICSW. Kainz1 , A. Christ2 , T. Kellom1 , S. Seidman1 , N. Nikoloski2 , B. Beard1 , N. Kuster2 , 1CDRH, FDA, Rockville, MD, 2IT'IS - The Foundation for Research on Information Technologies in Society, Zeughausstrasse 43, 8004 Zurich, Switzerland
We present new definitions for obtaining reproducible results in numerical phone dosimetry. Numerous numerical dosimetric studies have been published about the exposure of mobile phone users which concluded with conflicting results. However, many of these studies lack reproducibility due to shortcomings in the description of the phone positioning. The new approach was tested by two groups applying two different numerical program packages to compare the specific anthropomorphic mannequin (SAM) to 14 anatomically correct head models. A novel definition for the positioning of mobile phones next to anatomically correct head models is given along with there essential parameters to be reported. The definition is solely based on anatomical characteristics of the head. A simple up-to-date phone model was used to determine the peak spatial specific absorption rate (SAR) of mobile phones in SAM and in the anatomically correct head models. The results were validated by measurements. The study clearly shows that SAM gives a conservative estimate of the exposure in anatomically correct head models for head only tissue. Depending on frequency, phone position and head size the numerically calculated 10 g averaged SAR in the pinna can be up to 2.1 times greater than the peak spatial SAR in SAM. Measurements in small structures, such as the pinna, will significantly increase the uncertainty; therefore SAM as designed for SAR assessment in the head only. Whether SAM will provide conservative value for the pinna depends on the pinna SAR limit of the safety standard considered.
The opinions and conclusions stated in this paper are those of the authors and do not represent the official position of the Department of Health and Human Services. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.
W. Kainz1 , S. Seidman1 , R. Qiang2 , J. Chen2 , 1CDRH, FDA, Rockville, MD, 2The University of Houston
Currently, a variety of anatomical models are available for electromagnetic numerical dosimetry. Up to now all available anatomical models are voxel-based. The body, or parts of the body, is represented by small cuboids, usually cubes. Voxel-based models have several disadvantages. A proposed solution to overcome these disadvantages is to use CAD (Computer Aided Design) based anatomical models. Instead of representing anatomical structures as a composition of voxels, CAD objects can define individual organs. The main advantages of anatomical CAD models are: 1. every organ or the whole model can easily be scaled, 2. boundaries of the organs are well defined by a smooth surface, 3. the model can be meshed in any resolution as needed, 4. because the model will be meshed for each simulation graded meshes need no re-meshing, 5. organs with the same electrical properties can be combined in groups, 6. data formats are standardized, and 7. the posture of the model can be changed using existing CAD software. We developed several CAD models based on commercially available CAD data, laser scanned humans and MRI images of humans. We also developed CAD models of pregnant women based on the laser scans of a woman in her 34th week of pregnancy and MRI data. Fetus, bladder, uterus, placenta and bones are based on MRI data of a woman in the 35th gestational week. The easy scaling and realignment of internal parts was only possible because of CAD-based anatomical data.
The opinions and conclusions stated in this paper are those of the authors and do not represent the official position of the Department of Health and Human Services. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.
S. Paquerault1 , N. Petrick1 , B. Sahiner2 , K. J. Myers1 , H. Chan2 , 1CDRH, FDA, Rockville, MD, 2University of Michigan, Ann Arbor, MI
We are developing a bilateral pairing technique to help reduce false-positives identified by a single-view computer-aided detection (CAD) system for breast masses.In this study, we compare the performance of the proposed bilateral CAD to a single-view CAD.A database of 172 right/left breast pairs containing 205 biopsy-proven masses was used.Single-view CAD was run on each image using a lax selection threshold so that 5 objects per image were retained.The automated bilateral pairing algorithm identified all objects in a left breast mammogram "matching" a CAD detected object in the corresponding right breast image and visa-versa.Bilateral pairing was based on geometrical correspondence between objects with a matching score derived from a paired right/left object feature set and a linear discriminant analysis classifier.Leave-one out resampling was used to train/test the technique.We compared the FROC performances of the single-view CAD, the proposed bilateral technique and a modified CAD using manual pairing of bilateral structures.At a per-lesion detection sensitivity of 0.7, there were 3.8 FPs/image for the original CAD, 3.3 for the proposed technique, and 2.2 for the modified CAD using manual matching, a 12.6% and 42.1% reduction, respectively.At an FP rate of 1.0 per image, the sensitivities for the original CAD, the proposed technique and the modified CAD using manual matching were 0.47, 0.51 and 0.60, respectively.Preliminary results show that CAD with bilateral pairing did not achieve a significant FP-reduction.
J. Pfefer, A. Agrawal, S. Matchette, CDRH, FDA, Rockville, MD
Background: Fluorescence- and reflectance- based imaging techniques have a strong potential to improve clinical detection of pathologies such as cervical neoplasia. However, quantitative understanding of data collected by these approaches necessitates information on tissue optical properties in vivo. At present, there is minimal in vivo data on the optical properties of many tissues in the range that is most relevant - the ultraviolet A to visible.
Methods: We report here on the development and evaluation of a second-generation diffuse reflectance system for measurement of tissue optical properties using a multi-fiber optical probe with maximum separation distance of 2.5 mm. Improvements over the prior system include the implementation of an imaging spectrograph, high sensitivity CCD camera and in-line neutral density filters to maximize dynamic range and signal to noise ratio. Absolute measurements of tissue reflectance were enabled though calibration of the reflectance system. Spatially-resolved reflectance data sets were generated for absorption coefficients from 1 to 25 cm-1 and reduced scattering coefficients from 5 to 25 cm-1 through direct measurement of well-characterized tissue phantoms at 405 nm and Monte Carlo simulations. Multivariate calibration models were generated using a neural network algorithm and simulated reflectance distributions.
Results: These models were used to estimate the optical properties of tissue phantoms from reflectance measurements. By comparing predicted and known optical properties, system accuracy was found to be + 0.3 cm-1 for absorption coefficient and + 0.7 cm-1 for reduced scattering coefficient.
Conclusions: Advances in system instrumentation have produced a significant improvement in the accuracy of our optical property measurement system.
C. S. Kim, D. M. Saylor, B. J. Dair, CDRH, Rockville, MD 20852
Y.G. Tirat-Gefen, DESE-OSEL-CDRH-FDA
Background: Development and validation of medical devices
are expensive and time consuming tasks.
Methods: We reviewed state-of-the-art computing technologies of the interest of designers of new medical devices with emphasis to high detailed simulation of human physiology.
Results: Software technologies are becoming essential tools in simulation of biomedical systems. In silico pre-evaluation of the effects of a new device may become the standard practice in the industry sometime in our lifetime. It is expected that large-scale/ high detail computational simulation of cells, tissues and organs of the human body will involve both open source and proprietary code of universities, research centers, and commercial providers. Grid computing and semantic web technologies are converging at a fast speed to address the issues of interoperability, data security and high computational cost of bioinformatics software. We propose a software architecture to allow large scale distributed simulation of integrated human physiological systems.
Conclusion: We discussed, in this presentation, the state-of-the-art in grid computing software technologies relevant to the design and validation processes for medical devices, and our current work in deploying these technologies to implement a large scale distributed platform for physiological simulation that will allow the use of both open source and proprietary software components.
D. L. Walsh, R. J. Schroeder, S. F. Stewart, CDRH, FDA, Rockville, MD
Background
Inflammatory bowel disease (IBD) affects nearly 1 million people in the U.S., causing intestinal ulcerations and bowel injury. The anti-inflammatory drug mesalamine, administered rectally, is one effective treatment for IBD. However, an IBD patient discovered that a generic mesalamine enema drug product recently approved by FDA was subjectively more difficult (and painful) to administer than the proprietary version. A study was initiated to determine whether the observed differences were measurable.
Methods
Differences in the proprietary vs. generic mesalamine bottles were quantified by mechanical testing. The force required to squeeze the drug out of the bottle was measured by compression testing, performed in a universal testing machine. Compressive force and displacement were measured for 5 bottles each of the proprietary and two generic versions. Other methods included tensile testing of the bottle wall and bend testing of the nozzles.
Results
The maximum force required to compress each type of generic bottle (48.9N±5.4N and 87.2N±4.6N) (mean±sd) was significantly higher than for the proprietary bottle (30.3N±3.7N) (p< 0.0001 for each comparison). Nozzle stiffness for the generics was also measurably higher than for the proprietary.
Conclusions
Measurable differences in compressive force and bottle nozzle stiffness were observed between the generic and proprietary enema bottles. Such differences in the performance of a drug delivery system may adversely affect patient compliance with and subsequent effectiveness of the treatment. Collaboration between CDER and CDRH laboratories should be established to investigate standard specifications and/or test methods for this drug delivery system. Further studies of currently marketed enema products are also needed to better define the range of acceptable performance.
M. A. Gavrielides, N. Petrick, K. J. Myers, NIBIB/CDRH Laboratory for the Assessment of Medical Imaging Systems, Division of Imaging and Applied Mathematics, Office of Science and Engineering laboratories, Center for Devices and Radiological Health, Food and Drug Administration, Rockville, MD 20852
Introduction/Background: Diagnostic thoracic procedures using computed tomography (CT) often include comparisons of scans acquired with different slice thicknesses. In this manuscript, we investigated the potential for alignment of different CT scans from the same patient using skeletal knowledge of the thoracic region. Skeletal matching was selected because it is expected to be less susceptible to differences associated with patient breath hold, positioning and cardiac motion.
Methods: Our method utilized the positioning of the ribs relative to the vertebra for matching. It also included matching the scapula when visible in the scans. Rib positioning was described by the angles formed between the vertebra centroid and combinations of pairs of rib centroids visible on each CT slice; this was used as the primary matching mechanism. Scapula morphology was described using a feature based on the local maxima of the distance transform. Since the scapula is not visible in all slices of a full scan, its description was limited to only defining the potential range of slices. A cost function incorporating the difference of features from rib positioning and scapula morphology between two slices was derived and used to match slices.
Results: The method was evaluated on an independent set of 10 pairs of full and partial CT scans. Assessment was based on whether or not slices containing known nodules between each pair of scans were overlapping after the alignment procedure. Results showed that the proposed metric correctly aligned 9 out of 10 scans.
Conclusions: The preliminary results are encouraging for using this method as a first step towards temporal analysis of lung nodules.
CATEGORY N: BIOENGINEERED TISSUES, BIOMATERIALS, STEM CELL AND OTHER CELLULAR PRODUCTSM. K. Dewanjee1 , M. Lizak2 , S. R. Husain1 , R. K. Puri1 , 1FDA, Bethesda, MD, 2NINDS, Bethesda, MD
Background: Current methods of cellular tracking using optical imaging are not suitable for patients due to toxicity of the reagents and/or low tissue penetration. In addition, the currently used agent of magnetic iron oxide nanoparticles is not suitable for optimal MRI due to susceptibility artifact.
Methods: We developed charge neutral gadolinium complexes (CNGC) and characterized them for cell loading, cell retention and cell viability of Gd-loaded cells using Gd-153 tracer. We used the Gd-(pyrithionate)3 complex as a model CNGC for optimizing the labeling of neutrophils and lymphocytes. We evaluated their viability after Gd-loading, circulation in blood and migration after intra-arterial (IA) and intravenous (IV) administration in nude mice (n = 6) implanted subcutaneously with human glioblastoma. The mice anaesthetized with 5% Isoflurane and maintained at a breathing rate of ~45 breaths/min and body temperature of 37ºC were imaged at 5 minutes, 24 and 48 hours after IA or IV injection. All images of thorax, abdomen and pelvis, were taken on a 7 Tesla Pharmascan (Bruker-Biospin, Billerica, MA). T1-weighted images were taken using a gradient echo sequence. The MRI-parameters were: TR/TE = 200 ms/2.2 ms, flip angle = 90 degree and matrix = 256x256. Twelve slices (1 mm thick) were acquired in different orientations for each image.
Results: Lymphocytes were loaded to a level of 20-100 pg/cell maintaining a viability of more than 90%. Immediately after injection, the cardiac chambers, major blood vessels and stomach wall could be imaged with MRI. One day post Gd-lymphocyte injection, the spleen could be imaged perhaps due to lymphocyte pooling.
Conclusions: These studies indicate that cells could be labeled with CNGC and cell fate could be effectively monitored noninvasively using high resolution MRI.These studies provide a model of safety assessment of cellular therapy by monitoring trafficking of cells in vivo.D. B. Lyle, J. C. Shallcross, J. C. Breger, V. M. Hitchins, J. J. Langone, CDRH, FDA, Silver Spring, MD 20903
Background: FDA is receiving increasing numbers of applications that contain tissue-engineered natural products, raising new regulatory/review questions. Of particular concern is determining acceptable endotoxin levels. We examined the inflammatory potential of alginates which are being used to encapsulate cells for clinical purposes.
Methods: Alginate micro-spheres were produced by calcium cross-linking with an electrostatic encapsulator system. Cultured RAW 264.7 murine macrophages were treated with micro-spheres made from 20 mg/ml "crude" or "ultra-pure" alginates in presence or absence of interferon-gamma. Micro-spheres were made from ultra-pure alginate spiked with known amounts of purified endotoxin. Culture medium supernatants were assayed for nitric oxide (NO), an indicator of macrophage activation/inflammation, via nitrite-induced increase in fluorescence of 2,3-diaminonapththalene. Endotoxin concentrations were assayed using a Limulus amebocyte lysate chromogenic kit.
Results: There was 18.5 EU/ml of "natural" endotoxin in the crude and <0.1 EU/ml in the ultra-pure alginate. Neither alginate directly stimulated RAW 264.7 cells to produce NO. However, adding interferon-gamma to the media revealed a NO response to crude but not ultra-pure alginate. Equivalent NO responses were observed when cells were exposed to either purified endotoxin in media or three times that amount incorporated into ultra-pure alginate micro-spheres.
Conclusions: These data demonstrate that purified endotoxin added to ultra-pure alignate simulates the effect of natural endotoxin present in crude alginate. Reduction of NO production by RAW cells was observed when endotoxin was incorporated into micro-spheres rather than present in the culture media, suggesting that endotoxin may be less inflammatory in solid biomaterials than those that readily dissolve.
C. L. Zimliki1 , D. Mears2 , V. M. Chenault1 , 1CDRH, FDA, Rockville, MD, 2ICBM, University of Chile, Santiago, Chile
Background: Psammomys Obesus is a Type II diabetic animal model exhibiting a nutritionally induced progression similar to humans. This rat also exhibits diabetic complications, making this animal a useful tool to evaluate device/drug combination products, drugs that improve the glucose-sensing system at the cellular level (e.g., sulphonylureas) and devices to treat diabetic complications (e.g., diabetic ulcer dressings). However, conversion of rats into the diabetic state can take days or weeks, is variable between rats and requires constant monitoring of blood glucose and serum insulin to ascertain the diabetic state. The diabetic state at the cellular level results in a loss of glucose sensing. Currently, these pathways are not fully understood and need to be identified to better evaluate the safety and effectiveness of the next generation of glucose sensing devices.
Methods: A static insulin secretion system, islet electrical activity, and calcium imaging were used to detect the stimulus-secretion coupling pathways for glucose and other insulin potentiators/inhibitors.
Results: In the evaluation of the insulin response to glucose and known potentiators/inhibitors of insulin release, we found that healthy control sand rats have similar glucose sensing to known murine models. Surprisingly, we found that incubation of isolated islets from healthy animals overnight in our media resulted in glucose-insensitivity and no response to potentiators of insulin release.
Conclusions: These results suggest that P. Obesus glucose-sensing mechanisms are similar to murine models and that incubation of these islets in our media may be a reliable and quick method for converting these cells into the diabetic state. CATEGORY O: OBESITY AND NUTRITIONM. Calvo1 , L. H. Garthoff1 , R. B. Raybourne1 , U. S. Babu1 , C. Kelly2 , S. Lodder2 , M. J. Feeney2 , B. Minor2 , D. Beyer3 , R. Beelman3 , J. Pecchia3 , K. Paley3 , N. Chikthimmah3 , P. Mattila4 , 1CFSAN, Laurel, MD 20708, 2Mushroom Council, Dublin, CA 94568, 3The Mushroom Research Center, Penn State University, University Park, PA 16802, 4MTT Agrifood Research, Finland
Background: In response to the Mushroom Council's 2004 call for proposals examining the ability of edible mushrooms to boost innate immunity, CFSAN scientists proposed to optimize vitamin D content ofedible mushrooms throughexposure to sunlight (UVB) and to test their ability to increase disease resistance, regulation of immune response and mammary tumor development in rodents. Vitamin D's emerging role in the prevention of chronic diseases such as cancer and in the regulation of immune response has incited the need for more vitamin D rich foods. Funding this proposal created the need for a pilot study to demonstrate feasibility of exposing mushrooms to short periods of UVB to produce an optimal level of vitamin D in a normal serving size of mushrooms (84 g).
Method: We examined the influence of UVB light on mushrooms during growing, harvesting and post-harvesting phases. Dr. Pirjo Mattila of MTT Agrifood Research in Finland analyzed the amount of vitamin D2 in samples with different light exposure times using straight-phase HPLC followed with vitamin D2 quantification by reverse-phase HPLC.
Results: A standard serving size of white button mushrooms exposed post-harvest to UVB for 5 minutes contained 86.9 µg ( 3476 IU) vitamin D2 or869% of the Daily Value (10µg ).
Conclusions: Exposure of white button mushrooms post-harvest to as little as 5 minutes of UVB light produce a significant quantity of vitamin D2. This inexpensive process could provide a significant, unique plant source of vitamin D for vegetarians and individuals who do not drink milk, the major fortified food source.
A.G. Feight, CDER, FDA, Silver Spring, MD
Background: The rising rate of obesity in the US population prompted evaluation of duration of use for the amphetamine congeners. Current product labeling recommends these products not be used for periods longer than 'a few weeks'.
Methods: A longitudinal, patient-level claims database (Caremark Dimension Rx™) was utilized to determine the length of an 'episode' of amphetamine congener therapy for each individual in the database. A patient-level analysis was then conducted to characterize patterns of use.
Results: The final dataset contained 100,992 claims for 21,758 individuals receiving one or more of the amphetamine congeners, including benzphetamine, diethylpropion, mazindol, phendimetrazine, and phentermine. The mean number of episodes per individual was 2.2. Across all individuals, the mean duration of the longest episode was 88.2 days (median 58.0, range 1-1120). Subset analyses were performed by separating individuals receiving only phentermine products (80%); only 'non-phentermine' products (14%); and products from both groups (6%). Although there were small differences in certain key measures between the 'phentermine' and the 'non-phentermine' subsets of data, as compared to the full dataset, the most striking differences were seen among the small subset of individuals who utilized products from both groups. These individuals were more likely to be female, had a greater mean number of prescriptions, and more episodes of therapy. Most importantly, they had a much longer duration of use: a mean of 136 days.
Conclusions: Almost without exception, the amphetamine congeners are currently being used for periods far longer than the labeled duration of a 'few weeks'.
J. W. Parry, L. Su, K. Zhou, L. Yu, University of Maryland, College Park
Herbs, spices, and other plant based natural remedies have long been used for health benefits in traditional medicine, and the seed oils from those plants may also contain many active beneficial components. Seed oils are known to contain fat soluble vitamins and antioxidants, which may protect important biological molecules from oxidative stress. Cold-pressed onion, parsley, cardamom, mullein, roasted pumpkin, and milk thistle seed oils were evaluated for their radical scavenging capacities against the stable DPPH radical and the oxygen radical absorbance capacity (ORAC) test. The total phenolic content (TPC), tocopherols (alpha, gamma, and delta), and carotenoids (beta-carotene, lutein, cryptoxanthin, and zeaxanthin) were also determined. Significant DPPH radical scavenging activities were detected in all seed oil extracts and the activities were found to be time and dose dependent. ORAC values of the cold-pressed seed oil extracts ranged from 1.1 to 1097.5 micromoles trolox equiv/ g oil from the roasted pumpkin seed oil to the parsley seed oil, respectively. The TPC was found to be highest in the onion seed oil (3.4 mg gallic acid equivalents/ g oil) which also contained a significantly higher concentration of alpha-tocopherol than all tested oils. Zeaxanthin was the predominating carotenoid in all seed oils, and roasted pumpkin contained a significantly higher amount of all carotenoids than the other seed oils. The results from this study suggest the possible food application of these cold-pressed seed oils in health promotion and disease prevention through improving human nutrition.
CATEGORY P: SCIENCE AT THE CENTENNIAL: HISTORY AND PERSPECTIVEJ. N. Barrows1 , B. R. Meyers1 , N. Richfield-Fratz1 , C. J. Bailey1 , A. L. Lipman2 , 1Office of Cosmetics and Colors, CFSAN, FDA, College Park, MD, 2Office of Food Additive Safety, CFSAN, FDA, College Park, MD
Since ancient times, naturally occurring coloring agents from vegetable, mineral, and even insect sources have been used to color foods, drugs, and cosmetics. In the mid-1800s, synthetic organic dyes came into use, called "coal-tar colors" because they were first created from by-products of coal processing. Federal oversight of color additives began in the 1880s with authorization of artificial coloring for butter and cheese. Oversight increased with passage of two important laws. The Food and Drugs Act of 1906, commonly called the "Pure Food and Drugs Act," established requirements for seven artificial colors used in foods. The Federal Food, Drug, and Cosmetic Act of 1938 mandated the listing of "harmless and suitable" coal-tar colors (other than coal-tar hair dyes) and made mandatory the previously voluntary certification program. New colors could be added through an application process that established safety. The Color Additive Amendments of 1960 defined "color additive," established a provisional list of color additives in use at that time, and established the petition process for permanently listing color additives (other than coal-tar hair dyes) as "suitable and safe" for a given use in foods, drugs, and cosmetics. The 1960 Amendments contained the so-called "Delaney Clause" which prohibited the listing of a color additive shown to be a carcinogen "in man or animal." Federal oversight of color additives in medical devices began in 1976. Today color additives permitted for use in foods, drugs, cosmetics, and medical devices are listed in 21 CFR Parts 73, 74, and 82. A summary of listed color additives can be found at http://www.cfsan.fda.gov/~dms/col-toc.html.
G. Coody, D. Vasbinder, ORA, Rockville, MD
Widespread promotions of fraudulent patent medicines and nostrums, many with misidentified, secret or hazardous ingredients, were a significant impetus for enactment of the Pure Food and Drugs Act of 1906. Despite predictions that the Act would deal a "death-blow" to harmful nostrums, one hundred years later, American consumers spend billions of dollars on fraudulent health products to prevent or treat incurable and/or serious diseases such as arthritis, cancer, HIV/AIDS, Alzheimer's, diabetes and to lose weight.
Today's fraudulent unapproved drugs, devices, biologics and dietary supplement products are promoted with similar techniques to those used in 19th century America. These techniques include: exaggerated claims to treat incurable and emerging diseases and/or promising quick cures; widespread advertising; reliance on personal testimonials, pseudo medical jargon, dubious science and experts; and claims of revolutionary, ancient or exotic ingredients.
FDA defines health fraud as the deceptive promotion, advertisement, distribution or sale of articles that are represented as being effective to diagnose, prevent, cure, treat, or mitigate disease but which have not been scientifically proven safe and effective for such purposes.
To combat health fraud, FDA conducts surveillance, educates consumers, ;and pursues compliance through communication (e.g. warning letters) and use of enforcement tools (e.g. seizure, injunction, criminal prosecutions).
FDA believes that consumers, through their own choices, can avoid fraudulent health products if they have access to accurate information. FDA will continue to ensure consumers have truthful, scientifically supportable information that will enable them to make prudent decisions regarding their health and the use of FDA-regulated products.
M. D. Ditto, S. Choudhuri, CFSAN, FDA, College Park, MD
In the 100 years since FDA's inception, progress in science has led to the development of new technologies and new products. FDA has often faced the challenge of ensuring that products of new technology are safe and that those products reach the public expeditiously.
Biotechnology, which utilizes all the tools of modern DNA technology or recombinant DNA technology, has yielded many products in both the medical and agricultural arenas. Since the late 1970's, FDA has been engaged in the oversight and regulation of products developed through biotechnology. In 1983, FDA approved the first bioengineered product, recombinant insulin for use as a drug in humans. Since that initial approval many products, including new plant varieties used for food and feed, have been introduced to the marketplace.
In this poster, we describe the development of FDA's policy for bioengineered food and feed plants beginning with the science that made such products a reality. A synopsis of the legal basis as well as the history of the public dialogue that helped to shape FDA's policy is described. Finally, an overview of FDA's Consultation program in which FDA has responded to more than 60 notifications about a new bioengineered plant variety is also presented.
P. M. Gaynor, R. Bonnette, E. Garcia, L. S. Kahl, L. G. Valerio, Jr., CFSAN, FDA, College Park, MD
Under the 1958 Food Additives Amendment to the Federal Food, Drug, and Cosmetic Act, any substance intentionally added to food is a food additive and is subject to pre-market approval by FDA unless the use of the substance is generally recognized as safe (GRAS; the GRAS provision) (or otherwise excepted from the definition of food additive - e.g., color additive). By 1961, FDA had amended its regulations to include a list of food substances that are GRAS under certain conditions of use ("the GRAS list"). During the 1960's, many manufacturers requested FDA's opinion on whether their conclusions of GRAS status were justified and received "opinion letters. "In 1969, FDA removed cyclamate salts from its GRAS list as a result of safety questions, and then-President Nixon directed FDA to reexamine the safety of GRAS substances. In the 1970's, FDA announced that it was conducting a "comprehensive review" of presumed GRAS substances and established rulemaking procedures to affirm the GRAS status of substances that were either on the GRAS list or the subject of a petition ("GRAS affirmation"). To eliminate the resource-intensive rulemaking procedures, in 1997, FDA proposed to replace the GRAS affirmation petition process with a notification procedure ("GRAS notification").
B. R. Meyers, J. N. Barrows, Office of Cosmetics and Colors, CFSAN, FDA, College Park, MD
When the Federal Food, Drug, and Cosmetic Act of 1938 superseded the Food and Drugs Act of 1906, the new law's name announced a significant advance. For the first time, cosmetics became subject to Federal authority. How did we go from cosmetics being completely unregulated—and the use of makeup even morally suspect—to establishing legal requirements for their safety? The new law marked the culmination of years of consumer activism and shifting perceptions, as the use of cosmetics such as makeup and hair dyes became not only socially acceptable but commonplace. These developments paralleled other social changes, such as the entrance of women into the workplace, the growth of the entertainment industry with its attendant glamour, and the wider availability of commercially marketed products. Consumer injuries, most notoriously cases of blindness resulting from "Lash Lure" eyelash dye, also helped propel cosmetic safety requirements into the new law. Since 1938, FDA's regulatory responses have included requiring warning statements, prohibiting or restricting certain ingredients, and pursuing enforcement actions (e.g., warning letters, seizures, injunctions, and import refusals). The Fair Packaging and Labeling Act of 1966 authorized FDA to issue regulations requiring ingredient declarations for cosmetics sold at retail to consumers. In 1972, FDA established the Voluntary Cosmetic Registration Program. FDA's Cosmetics website became available in the 1990s and immediately became an important communications tool. Today FDA continues to monitor emerging cosmetics issues and to inform the public of our findings and conclusions on cosmetic safety.
A. G. Long, J. R. Payette, United States Pharmacopeia
Collaboration between FDA and USP officially began on June 30, 1906. Section 7 of the Federal Food and Drugs Act of 1906 cited the standards of the Pharmacopeia of the United States and the National Formulary as those that would be used by the Bureau of Chemistry to measure standards of strength, quality, and purity of medicines. USP VIII was reprinted in 1907 to honor this new citation in the law. The FDA-USP relationship was reinforced in 1938 with the passage of the Federal Food, Drug, and Cosmetic Act that continued to cite USP standards and added those for packaging and labeling.
In addition to legal recognition, collaboration between these two important public health organizations has existed throughout the century in many other ways, including scientific cooperation and shared leadership. Hundreds of FDA employees have worked with USP to establish standards by participating as members of USP's Expert Committees and its ad hoc Reviewer and Observer Programs.In addition, many former FDA officials have served as USP volunteer leaders, including FDA founder Harvey Wiley, who served as USP President from 1910 to 1920. Former FDA Commissioners Jere Goyan, Arthur Hull Hayes, and Jane Henney also served as USP volunteers. Shared staff leadership has included Joseph Valentino, Jerome Halperin, and Roger L. Williams.
This poster explores the collaborations between FDA and USP and honors the scientists and leaders who have worked together to advance the public health for 100 years.
T. L. Cecil, S. Schuber, United States Pharmacopeia
During the past century USP and FDA have forged a very effective model of scientific collaboration. Following the Kefauver amendments (1962), FDA was charged with ensuring that sponsors of new drug applications fulfill minimal requirements for efficacy as well as safety, typically by completing extensive clinical trials. Ultimately FDA, in partnership with USP, established quality standards as surrogates for certain types of studies and manufacturing controls. Many of the analytical tests and procedures drug sponsors apply to meet FDA's requirements come from USP-NF general chapters and GMP/validation concepts found therein. A private specification concluded between a sponsor and FDA can become a public specification. Once a monograph becomes official, it is enforceable by FDA and other regulatory bodies that can apply its specifications to the control of articles in domestic and international commerce. These specifications establish the identity of the article, help detect counterfeits, and enable the evaluation of adulterated and misbranded articles. Because the general chapter and monograph development and revision process is public and science-based, stakeholders (including the regulated industry) and regulators (FDA and others) can participate in ensuring that the compendial tools at their disposal are of the highest quality and utility. This poster will review the history of collaborations between USP and FDA and will highlight continuing opportunities for cooperative activities to advance public health.
S. A. Anderson, L. T. Beker, E. S. Cho, G. L. Robert-Baldo, B. M. Silverman, S. K. Suggs-Anderson, L. H. Tonucci, ONPLDS, CFSAN, FDA
In the United States prior to the 1906 Pure Food and Drug Act, there was no regulation of human milk substitutes, predecessors of today's infant formula that included evaporated milk and cow's milk. The regulatory history after the 1906 Act is highlighted in a timeline that includes events, discoveries, and expert opinions that precipitated legislation and regulations. Initially regulated with other foods under the 1906 Act, with the authority of the 1938 Federal Food, Drug, and Cosmetic Act, regulation of these products moved forward in 1941 and 1966 with regulations that required label statements to inform about nutrient quality and content, and also to specify when levels were not met for several nutrients. In 1971, minimum requirements were expanded from 4 to 17 nutrients and label statements were required if these minimum levels were not met. The recognition and regulation of infant formula as a separate food supplying minimum levels of 29 nutrients and maximum levels of nine nutrients came with the Infant Formula Act of 1980, along with the authority to establish requirements for quality factors and quality control procedures. The 1986 amendments included requirements for finished product testing, and additions to requirements for good manufacturing procedures, quality control procedures, and product recall. Greater understanding of infant nutritional needs for healthy growth and development has been and continues to be a key element for development of science-based regulations. Moving into the next century, challenges for infant formula regulation continue to evolve with increasingly complex new ingredients and advances in technology.
L. R. Dusold1 , F. S. Fry2 , M. O. Walderhaug2 , D. M. Schmit3 , E. A. Reinhold3 , M. E. Chen1 , 1OIT-CFSAN, FDA, 2CFSAN, FDA, 3Computer Technology Services, Inc. (CTS), Rockville, MD
The Centennial of the Pure Food & Drug Act is an occasion for both looking back and looking forward at the evolution of the tools used by FDA and CFSAN in the promotion and achievement of public health goals. For the past 20 years, an important evolving tool of public health specialists has been the Internet and its predecessor networks (BITNET and NSFNet). Public health specialists have used the predecessor networks to communicate with email and exchange files. In time, as the network evolved, CFSAN used the Internet to conduct research, to improve communication, and to collaborate with industry and academics on public health projects. CFSAN has embraced this technology by connecting to BITNET in 1987, followed by World Wide Web technology in 1993 with FDA's first web site, www.cfsan.fda.gov. This pioneered public health data exchange mechanisms for scientists, the communication of public health messages for consumers with outreach material, and the development of an electronic collaboration environment for the many public health specialists within the Center. CFSAN continues to exploit and to innovate new Internet technologies and seeks to explore the possibilities available in the Internet-2 initiative.G. A. Brindza1 , R. L. Byle1 , L. R. Dusold1 , C. E. Exley2 , D. M. Schmit3 , S. G. Yin4 , 1OIT-CFSAN, FDA, 2OIT-CFSAN, FDA, Retired, 3Computer Technology Services, Inc. (CTS), Rockville, MD, 4OITSS, FDA
History of Computing at CFSAN The Centennial of the Pure Food & Drug Act is an occasion for looking at the historical role of computers and information technology in FDA and the Center for Food Safety and Applied Nutrition (CFSAN). As early as the 1960's, mainframe computers were used at the Bureau of Foods (former name for CFSAN). Over the past 40-plus years, computer use and information technology has become ubiquitous, supporting every aspect of work at FDA/CFSAN from science, research, and CFSAN's core business missions, to administrative tasks. Laboratory Automation in CFSAN has enabled the research scientist to go from manually analyzing a few samples per week to potentially hundreds of samples per week. Advances in computing have enabled what we are capable of doing by providing powerful tools and a mechanism to increase our ability further FDA/CFSAN's Mission to protect the public health and advance science and food research.M. V. Waleski1 , L. K. Parker2 , D. C. Havery2 , 1OFFICE OF COSMETICS AND COLORS, 2COSMETICS AND COLORS
FDA established the Voluntary Cosmetics Registration Program (VCRP) at the request of the cosmetics industry in the early 1970s. The purpose was to create a means for companies to report to FDA post-market data on cosmetic products being sold to consumers in the United States. This information was intended to help FDA fulfill its mission to assure the safety of cosmetic products that are truthfully labeled. Originally the program had three separate parts and used standard paper forms for reporting. In Part I of the original program, a cosmetics manufacturer or packer could register the location of the establishment by submitting form FDA 2511. In Part II of the program, a manufacturer, packer, or distributor could file the ingredient formulation of a cosmetic product already on the market in the United States using a two-part form FDA 2512/2512a. Part II also provided for the filing of an individual raw material using form FDA 2513. In Part III of the program, a cosmetics company could report yearly the number and type of adverse reaction complaints from consumers or physicians using forms FDA 2704 and 2706. Through the years FDA has modified and reduced the program to make it less burdensome for companies to participate. Part I remains unchanged. In 1992 FDA terminated the raw material filing in Part II, and in 1997 FDA terminated Part III completely. Recently the program became fully electronic, giving participants the option of reporting their information directly into a web-based computer system maintained by the agency. The VCRP represents a unique and enduring partnership between the federal government and the cosmetics industry.
G.C. Ziobro, CFSAN, FDA, College Park, MD
B.J. (Burton J.) Howard is known through out the Agency for the development of the Howard Mold Count, which is used to ascertain the adulteration of tomato and fruit products with mold. His career began at the turn of the last century and he retired in 1943. From 1904 until his retirement he was the chief of the Microchemical/Microanalytical Laboratory. During his 40+ years he studied much more than moldy tomatoes. In his early career he developed methodology and collected evidence to support the passage of the Pure Food Act. In 1902, at Dr. Wiley's instruction, he testified before Congress on the mold content in commercial jams. Before the Act was passed in 1906, he published "Some Forms of Food Adulteration and Simple Methods for Their Detection. "After the Act was passed he continued to develop many of the methods used to detect the adulteration of foods, drugs, and cosmetics.
During his career, he published over 20 scientific papers and Bureau of Chemistry Circulars and was awarded one patent. The following reviews his long career and his contributions to food safety and quality.
G.C. Ziobro, CFSAN, FDA, College Park, MD
In 1904, the Microchemical Laboratory was formed in the Bureau of Chemistry. Its early work was to document and report on adulteration in foods. Evidence generated by the laboratory was used to convince Congress of the need of the Pure Food Act that was eventually passed in 1906. Upon passage of the Act, the lab developed a wide variety of methods to isolate, identify, and quantitate contaminants in foods, drugs, and cosmetics. The most famous of the methods, still in use today, is the Howard Mold Count and the Wildman Trap Flask.
In 1928, the Microchemical Laboratory was renamed the Microanalytical Laboratory and subsequently the Microanalytical Branch or Division of Microanalytical Evaluations. In 2004, the laboratory was absorbed into the Biological Constituents Branch of the Division of Natural Products.
In its 100+ year history, over 70 people have worked at its Washington, DC locations. These scientists and support personnel analyzed over 54,000 samples, published over 200 articles and authored or edited 10 books.
L. Robinson, Howard University
Background: The program objective was to develop an innovative method of sharing rich histories and information with library users on Howard University health sciences luminaries, past and present. The history and accomplishments of many of these phenomenal scientists reside inside books, journals and articles on shelves, collecting dust. A method was sought to bring some of these figures back to life in order to enhance academic studies and historical awareness.
Methods: Research was done to identify outstanding health sciences professionals who have made significant contributions to their disciplines. This research included reviewing biographical materials, special collection materials, reference requests, as well as recommendations from faculty and staff. The presentation format selected was the development of a series of 24" x 36" posters. One individual is selected as a subject per poster.
Results: These accomplished scientists are often unknown outside of their respective disciplines. The population served is students, faculty, and health practitioners. University staff, campus and community organizations also view and study the displays during meetings and social events held at the library. The series currently consists of five posters reflecting the fields of medicine, pharmacy, nursing, and dentistry. The poster developer has been invited to speak at professional meetings to share information on specific individuals in the poster series.
Conclusions:In addition to being displayed around the LSHSL library, the posters are available on loan, to organizations. The poster development series has been so well received that it will be an annual program.