A.Weisz1, D.Andrzejewski2, 1Office of Cosmetics and Colors, CFSAN, FDA, Chantilly, VA 20151, 2Office of Scientific Analysis and Support, CFSAN, FDA, College Park, MD 20740
The present work describes (a) the identification and characterization of a newly-found contaminant, 2-bromo-3, 4, 5, 6-tetrachloroaniline (2BTCA), in the color additives D&C Red Nos. 27 and 28 (phloxine B) and (b) the determination of the extent and level of 2BTCA contamination in certified lots of these colors. For these purposes, 2BTCA and its positional isomer 4-bromo-2, 3, 5, 6-tetrachloroaniline (4BTCA) were synthetically prepared. 4BTCA was used as the internal standard for the quantification of 2BTCA in the colors. Test portions from 35 certified lots of D&C Red Nos. 27 and 28 were analyzed for 2BTCA using a solid-phase microextraction/GC-MS method. Those lots were submitted for certification by both domestic (seven) and foreign (four) manufacturers during the past four years. Of the test portions analyzed, 22 (62.9%) contained 2BTCA in amounts ranging from 0.15 ppm to 435.7 ppm with an average value of ~131.7 ppm. The remaining 13 (37.1%) test portions contained no 2BTCA or less than 0.01 ppm, which is the limit of quantification of the present method. The analyses revealed substantial differences in the level of 2BTCA across lots from the same manufacturer as well as among different manufacturers. The wide range of 2BTCA levels found in the analyzed lots suggests that the presence of 2BTCA in D&C Red Nos. 27 and 28 may be avoided or significantly reduced during the manufacturing process. A chemical pathway that could explain the presence of 2BTCA in these color additives is also proposed.
C.Beaudry1, P.Eilers2, S.Hall1, 1Office of Seafood, FDA, Laurel, MD, 2Office of Seafood, FDA, Dauphin Island, AL
The Washington Seafood Lab currently uses a modification of the AOAC method for HPLC analysis of domoic acid (amnesic shellfish poison). With the standard method, tryptophan in seafood extracts often coelutes with domoic acid confounding analysis. Triethylamine, a mobile phase modifier, has been found to help resolve tryptophan and domoic acid (Eilers 1996). In order to better understand this effect and optimize conditions, the current study examined the effects of different triethylamine concentrations and pH levels on domoic acid and tryptophan separation and efficiency. Twenty mobile phases were made at five concentrations of triethylamine and four levels of pH. Test mixtures of domoic acid and tryptophan were analyzed. Triethylamine was found to work by reducing retention time and tailing of the tryptophan peak, while having little effect on domoic acid. Increased pH caused a more marked reduction in tryptophan retention time relative to domoic acid but this benefit was offset by lower efficiency. The observations can be explained by competition of triethylamine with the analytes for free silanol sites on the column, and by the relative pKa values of tryptophan and domoic acid. Ideal routine run conditions for the column used were 1 mM triethylamine at pH 2.5.
J.Brower1, J.C.Reepmeyer1, T.Moore1, L.F.Buhse1, M.M.Nasr1, A.S.Hussain2, 1CDER, Division of Pharmaceutical Analysis, St. Louis, MO, 2CDER, Office of Pharmaceutical Science, Rockville, MD
Government stockpiles of drugs needed for bioterrorism include only adult solid dosage forms. For effective pediatric application, the drugs need to be ground and mixed with appropriate food/drinks. Additionally, the dosage form must have good stability and dose uniformity as well as reasonable taste and palatability. To study doxycycline stability and dose uniformity, tablets were ground, visibly divided into halves and quarters and mixed with low fat chocolate milk. Stability of the ground material over several days was also investigated. The following preparations were used to evaluate 24 hour stability (room temperature and refrigerator): water, apple juice with table sugar, low fat milk, low fat chocolate milk, regular chocolate milk, chocolate pudding, grape jelly, strawberry jelly, yogurt with cherry flavor, and simple syrup with FlavoRx sour apple flavor. Doxycycline was found to be stable as a ground powder for one week or for at least 24 hours in the food/drinks evaluated. Dose uniformity for the half tablet portions was good; the quarter tablet portions, exhibited higher variability.
D.Toler1, J.Brower1, M.M.Nasr1, L.F.Buhse1, N.Sadrieh2, 1CDER, Division of Pharmaceutical Analysis, St. Louis, MO, 2CDER, Office of Pharmaceutical Science, Rockville, MD
The government has a large supply of some drugs including Cipro for use in the event of an emergency for inhalation anthrax. For Cipro, the dosage form recommended for children is a suspension, but the government pharmaceutical stockpile does not include a suspension formulation for this drug. In order to deliver effective dosages to children, the tablets must be disintegrated and mixed with appropriate food/drinks. The prepared dosages must have good stability and dose uniformity as well as reasonable taste and palatability. In this study, suspensions of the tablets in water were mixed with various foods/drinks (water, apple juice, Log Cabin Syrup, Similac, strawberry jelly, chocolate milk, and chocolate syrup). Stability of these preparations was studied over 7 days. Dose uniformity using common dispensing cups was determined. Stability data show that Ciprofloxacin HCl suspended in water and mixed with Log Cabin Syrup, apple juice, strawberry jelly, Similac and chocolate milk can be stored for 24 hours in a refrigerator. The suspension mixed with water or chocolate syrup can be stored for 1 week in the refrigerator.
J.Brower1, L.Revelle1, B.J.Westenberger1, M.M.Nasr1, L.F.Buhse1, N.Sadrieh2, 1CDER, Division of Pharmaceutical Analysis, St. Louis, MO, 2CDER, Office of Pharmaceutical Science, Rockville, MD
Lindane is commonly used to treat scabies and lice. Although the label recommends using non-permeable gloves when applying the lotion or shampoo, the extent to which Lindane can cross the glove barrier, or the relative permeability of various types is unknown. Because Lindane is stored in body fat and adrenal glands, observed toxic effects will be cumulative. Four types of gloves were used in this study (vinyl, latex, latex blend with neoprene, and nitrile). Gloves were inverted and approximately 40mL of shampoo or lotion were evenly distributed in the glove. Gloves were suspended in water at 37C, with stirring, and samples of the water were taken at 5, 30, and 60 min. Samples were extracted by solid phase extraction and analyzed by Gas Chromatography with electron capture detection. All gloves were found to be impermeable for short duration exposure (5 minutes). At one hour, nitrile and latex with neoprene gloves showed no Lindane permeability while vinyl and natural latex gloves showed considerably greater permeability. The latex glove with the greatest permeability, allowed <0.1% permeation of the applied Lindane concentration. At long exposures, greater permeability was observed for the shampoo than for the lotion.
U.R.Cieri, FDA
Reserpine , rescinnamine and deserpidine, alkaloids obtained from the roots of Rauwolfia plants are used as hypotensive agents. Commercial products containing reserpine alone or in combination with a diuretic were very common thirty or forty years ago but their use has dropped dramatically in recent years because other more effective antihypertensive agents were developed and marketed. Today there are still a few products containing reserpine alone or in combination. Rescinnamine and deserpidine never enjoyed great popularity but occasionally a product containing one of these two alkaloids appears in the market. Atropine (d-l Hyoscyamine) sulfate and l-Hyoscyamine Sulfate, alkaloids obtained from Atropa belladonna or similar plants , have identical chemical structure but have different stereo configuration. Commercial products containing a belladonna alkaloid are used as anticholinergic, mydriatic or antidiarrheal agents. A review is presented of methods, both official and non official, for the analysis of commercial products containing a Rauwolfia or a Belladonna alkaloid. An evaluation is also made on the specificity and accuracy of each method; the issue of detecting impurities and degradants is also discussed briefly.
K.K.Cook1, D.Fitelson2, P.Delmonte1, K.D.White1, E.Grundel1, M.P.Yurawecz1, J.I.Rader1, 1CFSAN, FDA, College Park, MD, 2JIFSAN, UMD College Park, MD
While sales of herbal supplements have increased significantly in recent years, information about the composition of botanical raw materials has not kept pace. Pyrrolizidine alkaloids (PAs) are common toxins produced by many species of flowering plants. These alkaloids may be carcinogenic, mutagenic, teratogenic and, with chronic use, hepatotoxic. Plants containing these alkaloids, including comfrey, may pose significant health hazards to those consuming them in certain kinds of teas , tinctures, salves or other traditional remedies. We purchased authenticated comfrey (Symphytum x uplandicum) leaves and roots from Botanical Liaisons, Boulder, CO. Retrorsine, a PA standard, was purchased from Sigma-Aldrich, Inc. (St. Louis, MO) Retrorsine was used to predict the specta and chromatographic behavior of other PAs contained in this species of comfrey. Thin layer chromatography (TLC), mass spectrometry (MS) and authenticated standards symphytine, symlandine and echimidine, generously provided by Research Triangle Institute (Research Triangle Park, NC), have been used to identify and target other PAs for collection and further identification. Various extraction procedures (e.g., extraction in methanol: ammonium hydroxide and straight methanol) have been utilized and the alkaloid compositions of the resulting extracts compared using HPLC and TLC.
L.S.de Jager, G.A.Perfetti, G.W.Diachenko, OFAS, CFSAN, FDA, College Park, MD
There is a growing movement in the United States towards using natural therapies and herbal dietary supplements as alternatives to western drugs. Since the passage of the Dietary Supplement Health and Education Act of 1994, marketing of supplements containing herbal ingredients has increased. Over the past few years, several food products containing botanical ingredients have appeared on the market. These “functional foods” occupy a tenuous position between dietary supplement and food. Most dietary supplement ingredients have not been pre-approved as food additives or determined to be Generally Recognized as Safe (GRAS). Although many methods have been developed to detect marker compounds from botanicals, little work has been done for the detection of these compounds in complex food matrices. In this study, analytical methodology has been developed for the detection of characteristic bioactive compounds of St. John’s wort (Hypericum perforatum L.) and kava (Piper methysticum) in functional food and drink products. Using various sample preparation techniques, in conjunction with LC, detection limits in the low ppb range have been obtained. Using these methods, a variety of food and drink products purporting to contain St. John’s wort and/or kava were analyzed. LC-MS analysis was conducted on sample extracts for confirmation.
A.V.Del Grosso, J.C.May, CBER, FDA, Rockville MD 20852
Plant pollen source materials used to manufacture allergenic extracts are often processed using perchloroethylene (PCE). In this process, pollens are separated from soil and other contaminants based on density differences while suspended in PCE. Since PCE is a known carcinogen, a study was planned to measure PCE content in representative samples of pollen source materials. A solvent extraction and GC/MS SIM procedure for the determination of residual PCE in pollen source materials has been developed. 10 mg of pollen is extracted with 1 mL methanol and 1, 1, 2-trichloroethane added as an internal standard. Chromatography is on a 5% phenyl column with dimensions of 0.25mm x 30m and 0.25 micron film thickness, operated isothermally at 70oC. Sample is introduced using a 1:20 split injection and detection is by selected ion monitoring. Total chromatographic analysis time is 4.5 minutes. Recoveries of > 90% were obtained from pollen samples spiked with quantities that corresponded to 10% to .01% w/w PCE/pollen. PCE content was determined in 41 lots of pollen source materials representing 32 different types of pollen. Measured concentrations ranged from 15.2 % w/w to less than the assessed limit of quantitation (0.01% w/w).
W.H.Doub1, W.P.Adams2, J.A.Spencer1, L.F.Buhse1, M.M.Nasr1, P.J.Treado3, M.P.Nelson3, D.S.Lester4, A.S.Hussain5, 1DPA, OPS, CDER, FDA, St. Louis, MO, 2OPS, CDER, FDA, Rockville, MD, 3ChemImage, Pittsburgh, PA, 4Pharmacia Corp., 5OPS, CDER, FDA, Rockville, MD
A feasibility study was conducted to examine whether Raman imaging is capable of determining the chemical identity, particle size and PSD of micronized drug substance, principally corticosteroids, in aqueous suspension formulations of nasal sprays. Raman spectra were obtained for the active pharmaceutical ingredient (API) and all excipients for a common formulation of aqueous suspension nasal spray. The formulation was sprayed onto a microscope slide, and brightfield as well as API-specific chemical images were obtained of the dried particles. These images were processed using ChemImage’s software to obtain the PSD of the API particles. Calibration of the imaging system was performed using NIST—traceable microparticles.
API particles were clearly distinguishable from those due to excipients. We also observed API particles adhering to excipient particles. This phenomenon may be an artifact of sample preparation and studies are planned to address this issue. The use of Raman-based chemical imaging enables the qualitative identification of corticosteroid in the presence of microcrystalline cellulose as is found in an aqueous suspension nasal inhalation product. Studies are currently underway to investigate the technique for quantitative particle sizing.
This work was presented at AAPS 2002.
W.H.Doub1, A.M.Wokovich1, J.C.Black1, W.P.Adams2, 1DPA, OPS, CDER, FDA, St. Louis, MO, 2OPS, CDER, FDA, Rockville, MD
Several configurations of the Andersen Cascade Impactor (ACI) were evaluated to assess its utility to provide sufficient precision for the measurement of “fines” in aqueous nasal sprays when used for in vitro bioequivalence (BE) studies. Deposition of nasal inhalation pharmaceuticals were assessed with the full (8–stage), standard (ACI28.3) and 90 lpm (ACI90/28.3) configurations, both at a flowrate of 28.3 lpm. These results were compared to those obtained using the ACI28.3 and ACI90/28.3 configurations employing only three stages (top two and final plus filter). Additionally, three different spherical induction chambers (1L, 2L, and 5L) were examined both with and without the Andersen preseparator.
Early experiments showed the 1L chamber to be too small for use with aqueous nasal sprays and it was eliminated from all subsequent comparisons. Using either remaining chamber, the ACI90/28.3 configuration allows as much as 40% more drug to be collected in the CI, although absolute amounts are very small (0.5-1%) with both configurations. A dramatic increase in the measurement precision is observed when the 2L chamber is used. The use of the 3–stage ACI configurations yields additional improvement in the precision of the measurement of “fines”, particularly with the 2L chamber.
This work was presented at AAPS 2002.
R.J.Mobley, J.C.Archer, P.Barnes, V.Litman, S.Shojaee, M.K.Halbert, J.J.Eckert, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
Typical sample preparation for dioxin analysis utilizes an acidified silica gel or acetonitrile cleanup that may be followed by further cleanup using activated carbon. Cleanup of high fat samples with acid silica requires large quantities of reagents. Under these conditions, the recoveries are inconsistent. The traditional cleanup using acetonitrile suffers from low recoveries. An improved method, FDA LIB 4084 uses a silica gel cleanup followed by a reusable carbon column then a micro acid silica/alumina fractionation. Although LIB 4084 generally gives acceptable results for low fat samples, frequent problems have been encountered with high fat matrices. The analysis of samples such as vegetable oils, butter, shortening, fish oil, and Vitamin E frequently result in carryover problems with analyses when reusing carbon columns. Dioxin elution profiles with toluene reveal the elution solvent volume is significantly affected by the matrix. This results in the need to replace the carbon column or use large volumes of toluene to clean the carbon column between analyses. This paper proposes the use of a single-use carbon column with high lipid samples. The benefits of this approach are elimination of re-extractions caused by carryover from previous extractions, lower solvent usage and ability to use an auto-feed technique.
G.Hirsch, M.K.Halbert, R.V.Furth, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
Blue fish sample extracts that have previously been prepared and analyzed for dioxin / furan analyses were re-analyzed for three, co-planar, polychlorinated biphenyls (co-PCBs); PCB 77, PCB 126 and PCB 169. These co-PCBs have been given toxic equivalency factors (TEFs) that when added with the dioxin / furan TEF contributions could result in a total toxic equivalent (TEQ) that is of concern. Currently, the United States has no set TEQ tolerance level, however any consumable part of a fish near 1 pg/g usually indicates a need for follow-up studies. TEQ values, from dioxin / furan contributions, ranged from 0.9 to 2.3 pg/g for five blue fish extracts. The addition of these three co-PCBs elevated the TEQ range to 1.6 -11 pg/g.
S.Shojaee, J.C.Archer, R.Vocque, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
There are several advantages to using the Accelerated Solvent Extractor (ASE-300) over conventional Soxhlet extraction for animal feeds. These include:
For the work reported in this presentation, five grams of various feed samples were spiked with the internal standard 13C12-labeled dioxin and furan congeners, with Ottawa sand serving as the method blank. The ASE-300 extraction conditions were completed at 125°C under 1500 psi in approximately 20 minutes. The solvents used were 1:1 hexane:dichloromethane. The extracts were subjected to a clean-up process of multi-layered silica gel and alumina columns and then analyzed by gas chromatography/high resolution mass spectrometry (GC/HRMS).
C.Earnheart, L.M.Pence, M.K.Halbert, G.Hirsch, J.C.Archer, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
When utilizing and adopting the Chemically Activated LUminescence eXpression (CALUX) bioassay to detect dioxins efficiently, recovery methodologies must provide results without affecting the cells or analyses. Different recovery methods are available for CALUX® analysis. This cellular dependent bioassay system detects compounds that activate the Aryl hydrocarbon Receptor (AhR).
The recovery method recommended by the manufacturer uses a surrogate technique in which one of the duplicate extracts is spiked with a 14C12 dioxin. The recovery is then calculated from a scintillation counter response. Another method calculates percent recoveries from a single extract per sample batch. Using only a batch recovery for calculations assumes no matrix affect and thus is not a true recovery.
Our approach to achieving recoveries is to add 12C12 1, 2, 3, 4-TCDD to each sample and quantitatively determine recoveries by GC/Mass Spectrometry. Advantages to this approach include:
J.C.Archer, L.M.Pence, L.Bluhm, C.Earnheart, J.J.Eckert, FDA/ORA/Arkansas Regional Laboratory, 3900 NCTR Rd., Jefferson, AR 72079
In the Chemically Activated LUminescence eXpression (CALUX®) bioassay, the presence of dioxins and furans in a sample triggers the production of light via a genetically engineered cell line containing the luciferase gene. The CALUX® methodology allows for the simultaneous analysis of a large number of samples, thereby making it a useful screening method for prioritizing sample analyses and reducing the number of samples analyzed by traditional means, namely Gas Chromatography/High Resolution Mass Spectrometry (GC/HRMS). Before the CALUX® method could be used routinely, it was necessary to show that the assay was as sensitive as traditional methods and that there was good agreement between the methods according to established guidelines. Samples representing various types of matrices (feed and feed components; vitamins, minerals, fats, etc.) were analyzed using the CALUX® method. The results from the CALUX® method were then compared to results obtained by traditional methods (extraction, GC/HRMS). Archived samples representing high, moderate and low total Toxic Equivalency (TEQ) values were chosen for the comparison in order to assess the accuracy, precision and sensitivity of the detection method over a wide range.
M.Farahani, CDER, FDA, Rockville MD
The least studied and understood type of DNA damage in cells is the cross-linking between DNA and proteins. Hydroxyl radicals induce cross-linking between phenylanaline (Phe) and 2-deoxyribose (dR) via formation of corresponding free radical intermediates. In this study, aqueous solutions of the mixture of Phe (0.5 x10-3 M, pH 6.3) and dR (0.92 x10-3 M), were saturated with oxygen-free N 2O for 30 min and irradiated in a 60Co gamma source dose range 110-440 Gy, dose rate 110 Gy/min. Samples (10 mg) dried with a rotary evaporator were trimethylsilylated (TMS) in Teflon-capped Hypovials with 0.1 mL each of Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine (1:1) by heating for 30 min at 140°C. The cross-linked products were separated and identified by capillary gas chromatography-mass spectrometry. In the present work, we find that hydroxyl radical induced cross-links between (Phe) and (dR) can take place in model aqueous systems. The newly discovered cross-link between 2-deoxyribose and Phenylalanine may also serve as a model for radiation or free radical induced cross-linking between DNA and proteins and in general between sugar moieties and amino acids.
P.J.Faustino1, A.B.Ciavarella1, E.B.Asafu-Adjaye1, C.R.Brownell1, L.X.Yu2, R.C.Lyon1, 1Division of Product Quality Research, Kensington, MD 20895, 2Office of Generic Drugs, Rockville, MD 20855
A simple, efficient and highly specific reverse phase HPLC method was developed that simultaneously measures plasma concentrations of hydrochlorothiazide (diuretic) and propranolol (B-blocker) in patients given an FDA-approved combination solid oral dosage form tablet. Analytes were extracted from human plasma with silica based (SPE) C-18 cartridges used in combination with unendcapped Cyano SPE cartridges. Hydroflumethiazide and labetalol were added to the plasma samples and standards as internal standards (IS) in the two-phase continuous SPE extraction. Samples were analyzed on an Agilent 1100 series HPLC equipped with a 1046A fluorescent detector and an 1100 series UV detector. Separation was achieved on a 250 X 4.6 mm, 5 micron Phenomenex C-18 (2) Luna column using a 20% ACN/20 mM PO4, pH=3.0 reverse phase isocratic method. Drugs and IS’s were detected simultaneously utilizing fluorescent detection (EX 232 EM 400) for propranolol and labetalol (IS) and ultra-violet detection (272 nm) for hydrochlorothiazide and hydroflumethiazide (IS). The method was validated using FDA’s Guidance for Industry, “Bioanalytical Method Validation”. Hydrochlorothiazide and propranolol met all validation acceptance criteria as defined in the guidance. Hydrochlorothiazide and propranolol had average recoveries ranging from >90-102% and r2 of 0.993 to 0.998 over the analytical range of 2.5-150 ng/mL.
M.L.Gay, R.A.Niemann, S.M.Musser, FDA, College Park, MD
As part of our efforts to develop methods for the quantitation and confirmation of identity of ingredients in dietary supplements, we have extended our previously reported analytical method for the determination of ephedrine-type alkaloids from Ephedra sinica (Ma Huang) in dietary supplements to include synephrine. Synephrine is structurally related to the ephedrine-type alkaloids and occurs naturally in bitter orange (Citrus aurantium). It is often an ingredient in dietary supplements, including those containing Ephedra. We also developed an MS/MS method for the analysis. Results of the analysis of 25 finished products by this method were compared with those obtained by APCI using increased tube lens voltage to achieve adequate fragmentation. A plot of the results obtained by LC/MS/MS vs. those obtained by LC/MS. (i.e. y vs. x) gives excellent agreement, as demonstrated by the slope of 0.986 and the correlation coefficient (R2) of 0.9991. Comparison of the results for synephrine in the eight products found to contain it also demonstrated good agreement.
George.M.Hanna, NERL, FDA, Jamaica NY
1H and 13C NMR methodologies are described for the determination and the characterization of melatonin (N-acetyl-5-methoxytryptamine), a hormone used as supplementary drug in the alleviation of jet-lag and other sleep disorders, utilizing a 400 MHz spectrometer without the need of pure reference standards. The developed methodologies are able to differentiate between melatonin and its precursor neurotransmitter serotonin, assess chemical structure of these compounds, and determine the quantity in the dietary supplement formulation. The NMR methodology was found suitable to monitoring the photo-stability, with the ability to detect degradation products. The quantitative analysis was based on the integrals for the methyl proton of 2-methyl-2-propanol served as an internal standard at 1.24 ppm and the methoxy protons at 3.86 ppm. The precision was established by analyzing synthetic mixtures of the analyte and internal standard. Excellent agreement was verified between the assay results and the quantities of analyte in the mixture.
C.B.Nochetto, D.N.Heller, FDA, Laurel, MD
CVM's Office of Research is developing a novel approach to the higher throughput analysis of food tissues from animals for presence of drug residues. This approach is based liquid chromatography-electrospray tandem mass spectrometry (LC/MS/MS), which has the ability to detect a wide variety of drug classes. Ionization conditions were used that were compatible with a generic, wide-range LC gradient. Acquisition parameters were tailored for compounds that eluted at various times during the gradient. The second multi-class extraction method to be combined with this approach uses solid phase extraction (SPE) of eggs with a silica phase. This method was capable of extracting multiple compounds from two major drug classes: polyether ionophores (e.g., salinomycin, monensin) and macrolides (e.g. erythromycin, tylosin). Sensitivity and selectivity by LC/MS/MS was very good: residues could be detected below10 ng/g (ppb). Residue-incurred eggs, i.e., eggs from hens dosed with these drugs, were analyzed during method development. The method is designed for use in surveillance for potential misuse of drugs in eggs.
P.J.Kijak1, J.D.von Bredow1, C.B.Nochetto1, R.J.Condon2, W.T.Hammack3, A.Brown3, 1CVM, FDA, Laurel, MD, 2RJC Associates, Inc. Myersville, MD, 3Florida Department of Agriculture and Consumer Services, Tallahassee, FL
The US, EU, Canada, and many other countries prohibit the use of Chloramphenicol (CAP) in food producing animals because it has been associated with aplastic anemia in humans. Early in 2002, sub ppb amounts of CAP were discovered by the EU in shrimp and honey imported from China. In order to provide Federal and state laboratories with validated screening methods for CAP, we evaluated two commercial screening tests for CAP, the Charm II test and the RIDASCREEN test in shrimp and honey. A statistically designed protocol was developed to evaluate the selectivity and sensitivity of the screening tests for CAP. Both tests were capable of detecting CAP at 0.3 ppb in shrimp; however, both tests exhibited a positive bias for negative samples. If the tests were used as labeled, this bias would lead to an unacceptably high rate of false positive results. For both kits, we were able to develop alternative protocols for data interpretation that would give a significantly lower percentage of false positive results while maintaining the ability to detect samples containing 0.3 ppb CAP in shrimp. Both test kits were also evaluated for the detection of CAP in honey.
Board A-29 Validation of Methods to Confirm Chloramphenicol at 0.1 ppb in Shrimp, Crabmeat, and Honey: Collaboration between FDA CVM, FDA Office of Regulatory Affairs, Florida Dept. of Agriculture and Consumer Affairs, and the Canadian Food Inspection Agency
M.C.Carson, C.B.Nochetto, D.N.Heller, K.Ferbos, P.J.Kijak, Division of Residue Chemistry, Office of Research, CVM, Laurel MD 20708
Chloramphenicol (CAP) is a potent and cheap antibiotic that is associated with aplastic anemia and other toxic effects in humans. It is banned from use in food-producing animals in the US. Low levels of CAP were detected by analysts in Europe, Canada, and some US states in imported shrimp, honey, and other commodities. Existing FDA methods could only detect 1-2 parts per billion CAP. In July 2002 the Commissioner committed the FDA to begin analyzing imported foods with methods capable of confirming CAP at 0.3 ppb, consistent with enforcement levels used in other countries, and also consistent with the claimed detection limits of marketed screening assays. FDA needed to quickly validate methods to confirm CAP at sub-part per billion concentrations. The Florida Department of Agriculture and Consumer Services and the Canadian Food Inspection Agency had recently developed appropriate LC-MS-MS methods for shrimp and honey, respectively, which they shared with the FDA. We validated Florida’s method to confirm 0.1 ppb CAP in shrimp and crabmeat, and we validated Canada’s method to confirm 0.1 ppb CAP in honey. We adapted FDA’s existing shrimp method (LIB 4284) for analysis on a triple quadrupole, lowering its limit of confirmation from 1 ppb to 0.1 ppb, and validated the modified method. Of three locally purchased imported canned crabmeat samples, two contained CAP. One locally purchased honey sample contained CAP.
L.P.Lue1, A.Vancura2, 1NRL, ORA, FDA, Jamaica, NY 11433, 2St. John's University, Jamaica, NY 11439
Cephalosporins (CEF) are b-lactam antibiotics with cephalosporinic nucleus, produced together with penicillins (PNC) by a fungus Cephalosporium acremonium. Chemical synthesis involves conversion of penicillin nucleus into a cephalosporinic nucleus. Therefore, it is very common to find the contaminant of penicillin in active pharmaceutical ingredient (API) and dosage forms of cephalosporins. However, this contamination phenomenon of penicillins in cephalosporins is not acceptable in medicinal practice. The HPLC on C18 reversed phase columns with UV or fluorescence(FD) detection has been utilized to detect penicillin in API’s of cefadroxil and cefoperazone. FD enhanced the detection level ten times. The low levels of penicillins in cephalosporine (ppb) after derivatization with HgCl2 or 4-fluoro-7-nitro-benzofuran were detected by UV detector or FD , respectively.
T.P.McNeal1, C.Olivo2, T.H.Begley1, 1OFAS, FDA, College Park, MD, 2Virginia Tech, Blacksburg, VA
Vinyl chloride is the building block for polyvinyl chloride (PVC) polymers. Polyvinyl chloride production is second only to polyethylene in the US. Polymers of PVC are used in building construction, housewares, water pipes and food packaging. Vinyl Chloride monomer (VCM) is a human carcinogen. Thus the levels of residual VCM in food packaging are of interest to the Food and Drug Administration (FDA). In 1986, FDA determined that thick walled PVC food packaging, i.e., bottles and blister packages, were safe provided the polymer contained < 10 parts-per-billion (ppb) residual VCM. Since then, no data are available that show residual VCM levels in these food packages are < 10 ppb. To determine whether the residual VCM levels in PVC containing food packages in current use are < 10 ppb, a survey and analysis of PVC containing food packages was conducted.
The analytical procedure employed for these determinations utilizes automated static headspace sampling and capillary GC with a PLOT capillary with mass selective detection. Polyvinyl chloride and Saran™ films, PVC bottles and blister packages were analyzed for residual VCM. Residual VCM levels found in the packages ranged from none detected (< 1 ppb) to ca. 275 ppb. The package containing 275 ppb residual VCM was a not a food contact material. The Repeatability for VCM in PVC polymers at the 5 parts-per-billion level had a coefficient of variation of < 1%. Recovery of VCM at the 5 ppb level was essentially 100%. Data on residual VDCl levels in Sarans ™ also will be presented.
M.Miyahara1, T.Mashimizu2, Y.Kobayashi3, T.Maitani1, 1National Institute of Health Sciences (NIHS), Setagaya, Tokyo, 2Japan Electron Optics Laboratory (JEOL), Akishima, Tokyo, 3Japan Atomic Energy Research Institute (JAERI), Takasaki, Gunma
After extensive studies on irradiated food for years, food irradiation technology has been utilized for food safety recently. Detection methods for irradiated foods are still required by leveling regulation. In this study we discuss detection method for irradiated foods using ESR. Bones and shells consist of hydroxyapatite Ca10(PO4)6OH2 or of calcium carbonate. Those compounds retain radicals in the crystal lattices, which are induced by irradiation. ESR is a radical monitor. Samples were prepared by modified procedures suggested by EN. Bone and shell samples were washed to remove muscles and dried over phosphorus oxide (V) in vacuum. ESR can detect those radicals in bones (fish, frog legs, wing sticks) and shells of mussels and shrimps. Spectra of irradiated bones of fish, frog legs, wing sticks, and shells of mussels were same as these of hydroxyapatite. The signals were detectable at 0.5kGy about for one year. Those results were comparable with the results of EN method. Irradiated shells of shrimps and prawns were different from those of bones. Thus preliminary results showed new scope of the method.
P.J.Nyman, G.W.Diachenko, G.A.Perfetti, CFSAN/OFAS, FDA, College Park, MD
A survey of soy sauces and related products available in the U.S. was conducted to determine the levels of 3-monochloropropane-1, 2-diol (3-MCPD) and 1, 3-dichloro-2-propanol (1, 3-DCP) in these products. Fifty-five retail samples were purchased and analyzed for 3-MCPD. 3-MCPD determinations were made according to a gas chromatography/mass spectrometry method that was validated by a collaborative trial. Eighty-five percent of the samples analyzed were found to contain greater than the detection limit of 0.005 ppm (g/g) for 3-MCPD. Thirty-three percent were found to contain greater than 1 ppm; the highest level was 876 ppm 3-MCPD. Thirty-nine of the samples analyzed for 3-MCPD also were analyzed for 1, 3-DCP by using a modified method developed and validated in-house. Fifty-six percent of the samples analyzed for 1, 3-DCP were found to contain greater than the detection limit of 0.055 ppb (ng/g) for 1, 3-DCP; the highest level was 9.8 ppm 1, 3-DCP. Products manufactured in Asia contained the highest chloropropanol levels.
P.J.Nyman, G.W.Diachenko, G.A.Perfetti, CFSAN/OFAS, FDA, College Park, MD
A GC/MS method for 3-MCPD in foods and food ingredients was modified for the determination of 1, 3-DCP in soy and related sauces. The method was validated by using a blank soy sauce. The detection limit, quantitation limit, and recoveries were determined and identities were confirmed by MS on the basis of analyses of test portions spiked with 1, 3-DCP at 10, 25, 50, and 100 ng/g. The spiked test portions were quantitated by using an internal standard calibration curve. For the spiked test portions, the mean internal standard-corrected recovery for 1, 3-DCP was 100 percent with a relative standard deviation of 1.32 percent. The limits of detection and quantitation were determined to be 0.055 and 0.185 ng/g, respectively. The method also was compared with a headspace GC/MS method that was recently developed by the U.K.’s Central Science Laboratory. Results from the method comparison showed that the recoveries for the spiked test portions, as well as the amounts of 1, 3-DCP found in the retail products, were comparable.
J.C.Reepmeyer, CDER, FDA, St. Louis, MO 63101
Fraudulent over-the-counter topical products, sold for the treatment of psoriasis, have been found to contain undeclared designer corticosteroids, which possess modified ester substituents. Corticosteroids, extracted from topical products, were analyzed by HPLC on a Waters Symmetry C-18 column with a mobile phase gradient consisting of either acetonitrile-water or methanol-water using diode-array detection and positive ion atmospheric pressure chemical ionization liquid chromatography mass spectrometry (APCI LC-MS). Betamethasone-17-propionate-21-butyrate (BMPB), -21-isobutyrate (BMPI), and -21-stearate (BMPS), potential designer corticosteroid candidates, were synthesized as standards. Known corticosteroid pharmaceuticals, such as betamethasone-17, 21-dipropionate (BMD), were analyzed for comparison. All corticosteroids studied gave prominent molecular ions [MH]+ by positive ion APCI LC-MS. BMD gave fragments corresponding to [MH - HF]+, [MH - CH3CH2COOH]+, [MH - HF - CH3CH2COOH]+, and [MH - HF - CH3CH2COOH - H2O]+. Interestingly, BMPB gave fragments with the same losses as BMD, and thus gave atomic mass units that were 14 units above those for BMD. Fragments due to loss of butyrate were absent or negligible, which demonstrated selective fragmentation of the 17-propionate ester over the 21-butyrate ester. A similar phenomenon was observed with BMPI and BMPS. BMPB and BMPS were identified as compounds present in fraudulent topical products.
J.C.Reepmeyer, CDER, FDA, St. Louis, MO 63101
Ivermectin and clorsulon are antiparasitic agents used in veterinary medicine, sometimes used in combination. The purpose of this work was to develop a method for the simultaneous analysis of ivermectin and clorsulon in injection solutions by high-performance liquid chromatography (HPLC), to differentiate between two ivermectin analogs, and to confirm structures by ultraviolet (UV) spectroscopy and mass spectrometry. Clorsulon (CSL), ivermectin methyl analog (IVM-B1b), and ivermectin ethyl analog (IVM-B1a) were separated by reversed-phase HPLC on a short (4.6 mm x 50 mm) C-18 column using a mobile phase gradient consisting of acetonitrile-methanol-water, and detected by UV at 244 nm and by negative ion atmospheric pressure chemical ionization liquid chromatography mass spectrometry (APCI LC-MS). UV area measurements were found to be linear for all three compounds. Repeatability measurements (n = 6) for CSL, IVM-B1b, and IVM-B1a gave 0.13, 0.29, and 0.08% relative standard deviations, respectively. The two ivermectin analogs gave molecular ions [M-H]- as their base peaks by negative ion APCI LC-MS. Clorsulon also gave a molecular ion with prominent isotope peaks due primarily to the presence of three chlorine and two sulfur atoms, but the base peak was due to loss of hydrogen chloride [M-H-HCl] -.
F.J.Schenck1, J.E.Hobbs1, S.J.Lehotay2, 1ORA, FDA, Atlanta, GA, 2ARS, USDA, Wyndmoor, PA
A fast and inexpensive method for the analysis of pesticide residues in fruits and vegetables (produce), named QuEChERS, developed at a USDA-ARS Laboratory, was evaluated. The procedure entails vortexing 10 g of produce sample with 10 mL of acetonitrile, and effecting a phase separation by salting out with MgSO4 (4 g) and NaCl (1 g). Cleanup of the acetonitrile extract was performed using dispersive SPE, which entails vortexing the extract with a small quantity of SPE sorbent and MgSO4. Gas chromatography or liquid chromatography was then utilized to perform quantitative analysis of the pesticides. Produce samples containing various incurred organochlorine, organophosphorus and N-methyl carbamate pesticide residues were analyzed using the QuEChERS method, a published acetonitrile extraction method used by the Canadian Pest Management Regulatory Agency and the acetone extraction (Luke) method used by the FDA. Similar results were obtained using all three methods.
L.V.Podhorniak1, F.J.Schenck2, A.J.Krynitsky3, F.Griffith1, 1OPP, EPA, Fort Meade, MD, 2ORA, FDA, Atlanta, GA, 3CFSAN, DPIC, College Park, MD
A reverse phase liquid chromatographic method with both fluorescence and mass spectrometric detection is presented for the analysis of 13 parent N-methyl carbamate pesticides and their metabolites, as well as piperonyl butoxide, for a total of 24 compounds found in selected fruits and vegetables. The commodities chosen were of special concern to EPA as these commodities had the least amount of monitoring data for dietary exposure estimates used in risk analysis. The method uses an acetone extraction, followed by an aminopropyl SPE cleanup. Determination of residues is by reverse phase liquid chromatographic column fitted with either fluorescence or a mass spectrometric detector. A set of six samples can be prepared in one working day. Recovery data are presented from selected commodities for some of EPA's fruit and vegetable crop groupings.
V.A.Vega, F.J.Schenck, ORA, FDA, Atlanta, GA
An extraction technique is described for aflatoxins B1, B2, G1, and G2 in peanut butter using matrix solid phase dispersion (MSPD) in conjunction with immunoaffinity column (IAC) clean up. 2.0 grams of peanut butter sample is blended with 3 grams of C-18 SPE sorbent. A column prepared with the C-18-peanut butter matrix is washed with 5 mL water, and then the aflatoxins are eluted with 15 mL methanol. The eluate is diluted with water and subjected to an IAC clean up. The aflatoxins are then quantitated by reversed phase liquid chromatography with fluorescence detection. Recoveries will be determined using a negative control sample which will be fortified from 0.5 ppb to 20.0 ppb. The method provides a rapid, specific and easily controlled assay for the analysis of aflatoxins in peanut butter, with minimal solvent usage. The MSPD method reduces organic solvent consumption by 85% and hazardous waste by 80% compared with the AOAC method.
S.L.Snellings1, S.C.Harris2, M.A.Jackson2, D.W.Milller1, 1NCTR, FDA, Jefferson AR 72079, 2Northrop Grumman, NCTR, FDA, Jefferson AR 72079
The Scientific Imaging and Analysis System (SIAS) was developed at the National Center for Toxicological Research (NCTR) in the Division of Chemistry as a way to perform colorimetry and pseudo-spectrophotometry in a cost-effective, rapid manner. The potential benefits to the FDA are numerous, including the documentation of gels, animals, tumors, and microorganisms. Other applications include explosive detection, food analysis, and organic chemistry. SIAS uses a digital camera to image objects in color or grayscale. Images can be collected individually or as a time series, which allows real-time color analysis. Each image is automatically corrected for lighting and camera effects using custom color standards. Using SIAS custom software, specific pixels in an image can be analyzed, and calibration curves, concentration, diffusion kinetics, thickness, and spectral data can be calculated. Data analysis can be performed on a single image or a template can be applied to a series of images using auto-analysis. SIAS can be a benchtop or handheld unit, which is perfect for field analysis. One use of SIAS is the colorimetric analysis of tuna fillets that may have undergone carbon monoxide treatment. SIAS quantitates the level of carbon monoxide in the tuna based exclusively on the color of the meat.
J.A.Spencer1, L.F.Buhse1, M.M.Nasr1, N.J.Westenberger1, J.P.Jasper2, 1Div. Pharmaceutical Analysis, 2Molecular Isotope Technologies
We wished to learn whether Isotope Ratio Mass Spectrometry (IRMS) could distinguish between API materials made by different manufacturers and between different lots of material by the same manufacturer. Four sample sets, each representing a particular drug substance, were created from API materials. Each sample set for IRMS consisted of five identical vials to be analyzed. Analysts were given the name of each of the four pharmaceutical samples for safety reasons but no other details. The sample sets were distinguished as follows:
Bivariate plots (d15N vs. d13C; dD vs. d18O) showed a high degree of clustering for Set A and less clustering for Set B. Set C showed tight clustering for each of the two manufacturers, but strong differentiation between the two sources. Set D showed widely separated points in all the plots. For the API samples examined, distinctions were readily made between different manufacturers of the same drug substance. IRMS instrumental precision was high enough to discern different lots of a pure active pharmaceutical made by a single manufacturer.
J.A.Spencer1, L.F.Buhse1, M.M.Nasr1, N.J.Westenberger1, A.S.Hussain2, E.H.Jefferson2, 1Div. Product Quality Research
‘Process Signature’ denotes measurable variances in chemical and physical properties of a material that are process dependent. This was a study to learn whether NIR reflectance spectra of powdered APIs can establish sameness of samples from the same lot and, conversely, can distinguish between APIs from different lots or sources. Samples consisted of five individual vials of four different API materials as follows: Set A — five samples, same manufacturer, same lot; Set B — same manufacturer, five lots; Set C — two manufacturers, one lot from each; and Set D — five manufacturers. NIR reflectance scans were made of each of the API samples through the bottom of 1ml glass vials. The direct NIR reflectance spectra for Set A show a very low standard deviation as a function of wavelength, whereas the five spectra for Set D differed substantially in slope and offset. Set B exhibited about four times and Set D ~ 20 times the standard deviation of Set A. For these API samples, NIR reflectance spectral variations demonstrate sameness of multiple samples from a single lot. Meaningful spectral differences can be observed between the lots of API and even larger variations could be seen between different manufacturers.
J.A.Spencer1, L.F.Buhse1, M.M.Nasr1, A.S.Hussain2, 1Div. Pharmaceutical Quality Research
In 2001 the FDA initiated a program to stimulate the development of Process Analytical Technology (PAT) in the manufacture of pharmaceuticals. This calls for substantial changes in manufacturing technology, the philosophy of product quality control, the drug approval process and, ultimately, the regulatory atmosphere. There are a variety of fast, non-destructive instruments and sensors such as Near Infrared, Raman and particle sizing. These are now commonplace in non-regulated industries and will play an important role in PAT-based pharmaceutical manufacture. Measurements from such combinations of devices will inevitably be joined into multidimensional process control models. Central to the implementation of PAT is the use of chemometric tools such as PCA and PLS. In pharmaceutical manufacturing these will need to be implemented with detailed validation protocols that can be properly managed by the manufacturer and understood by the FDA.
Two popular techniques, NIR and Raman, were used to measure spectra of a series of Active Pharmaceutical Ingredients (APIs). The sample sets came from different manufacturers and lots. We show that data preprocessing such as derivativization or MSC which is typically done for chemical analysis, can dramatically reduce the amount of process dependent information (process signature) contained in the raw NIR spectra.
P.R.Sundaresan, K.D.White, J.I.Rader, CFSAN, FDA, College Park, MD
Teucrium species are rich in neoclerodane diterpenoids. Teucrium species known as germander have been used in traditional folk medicine for their stimulant, diuretic, antipyretic and antiseptic properties. However, the furano-neoclerodane diterpenoids present in germander have been implicated in the in vivo hepatotoxicity of this botanical. The lack of commercially available reference standards prompted us to develop in-house standards for Teucrin A and other diterpenoids. Authenticated germander was used as the source material. Acetone extracts of powdered plant material were prepared. They were analyzed by HPLC using Synergi Max-RP columns with monitoring at 220 nm. Limited amounts of Teucrin A and other diterpenoid standards were analyzed on a Synergi Max-RP column to determine their retention times and correlation coefficients. The same standards of Teucrin A and other diterpenoids were subjected to concurrent mass spectral analysis. Teucrin A and diterpenoids such as dihydroteugin, teuflin, teucrin H1 and teucvidin were identified by LC/MS. Isolation and characterization of germander standards on a preparative scale are under way. The chemical identities of these potential reference standards will be confirmed by NMR spectroscopy.
S.Wang, Lab E, FBC, Northeast Regional Laboratory, ORA, FDA, Jamaica, NY 11433
The ion trap has an important limitation: its maximum ion storage capacity is small, approximately 107ions. When the number of ions exceeds 105, space-charge repulsion occurs, thus, its mass resolution deteriorates. To overcome the limitation, the AGC is applied. The AGC is implemented by automatic control of the ionization time for EI or CI, or the injection time for ESI or APCI/AAPI. The AGC software sets up to maintain the optimum quantity of ions into the ion trap according to a linear equation: the number of ions stored in the ion trap is proportional to the ionization time or ion injection time. The linear equation is based on a linear approximation of first order kinetics. The linear approximation is based on the assumption that only a small proportion of ions initially present (less than 20%) is consumed, but the assumption is not true very often. The linear approximation of first order kinetics applied by AGC only is a special case of a more general and accurate kinetics. In order to better quantitatively understand the MS data calculated by the AGC, the more accurate and general kinetics must be developed for ion trap MS. Accurate and general kinetics are presented.
G.M.Ware, FDA
Hypoglycin A (HG-A) is a water-soluble natural toxin found in ackee fruit. It is similar to leucine and isoleucine in chemical structure, chromatographic and photometric properties. This paper describes a liquid chromatographic (LC) method for the resolution of HG-A, leucine and isoleucine. Chromatographic stationary phases C18, C8 and C2 showed little selectivity for the separation of HG-A from leucine and isoleucine. Resolution was obtained on phenyl, diol and polar group end capped C18 stationary phases using a mobile phase of acetonitrile/ methanol/water. Leucine, isoleucine and HG-A were detected at 210 nm. Method validation included the determination of the selectivity, (a), chromatographic resolution, (Rs) detection and quantitation limits for leucine, isoleucine and HG-A. The purity of HG-A standard containing 1% leucine and isoleucine was characterized. Low milligrams of pure HG-A were isolated by analytical column. Chromatographic conditions used for the analytical separation were scaled up to a semi-preparative method capable of isolating 25 to 50 mg HG-A per injection. UV and IR spectra for pure HG-A were used to confirm purity. Hypoglycin A standard was further characterized by GC/MS.
L.Xu, T.Heinze, A.Pogge, W.Slikker, Jr., L.C.Schmued, NCTR, FDA, Jefferson AR
Fluoro-Jade B contains a number of structural analogues of Fluoro-Jade, a well-known and useful dye for detection of degenerating neurons in tissue sections. It exhibited a higher affinity for degenerating neurons than Fluoro-Jade. The present research focused on isolation, purification and identification of Fluoro-Jade B components and will ultimately distinguish the most biologically active compound(s). The reaction mechanism suggests about nine possible reaction products corresponding to a 1:1, 2:1, 3:1 and 4:1 ratio of the starting material resorcinol and benzenetetracarboxylic dianhydride. The molecular weights of the predicted structures based on these ratios could be 346 (two isomers), 438 (two isomers), 420, 512 (two isomers) and 586 (two isomers), respectively. The analytical HPLC method consisted of a reversed phase C18 column and a mobile phase with a pH gradient. The LC/MS data supported the theoretical predictions. The quantitative separations were performed by silica gel column chromatography, TLC and preparative reversed phase HPLC. For each fraction collected, the negative-ion electrospray mass spectra showed the expected [M-H-] ions and reasonable fragmentation with in-source collision-induced dissociation (CID).
A.X.Yang, B.Bhattacharya, J.Mejido, J.Han, R.K.Puri, Microarray Program, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, CBER, FDA, Bethesda, MD 20892
CBER has joined forces with the National Cancer Institute and developed a state-of-the-art microarray program. In this program, not only do we provide hands-on-training to our researchers/reviewers, we have been optimizing the production of high quality arrays for use in scientific experiments. Recently, we purchased a human oligo set consisting of 16, 659 70-mer oligos. Each oligo was selected from the 3’ end of the gene and optimized using BLAST search. The gene array list (GAL) file was generated in collaboration with John Powell of Center for Information Technology (CIT), National Institutes of Health (NIH). We created the human oligo nucleotide ‘chip’ by using GeneMachine spot arrayer. Oligos were deposited on the surfaces of poly-L-lysine coated glass slides and arranged in 32 subarrays with 23 x 23 spots. After printing, oligo chips were cross-linked and slides were blocked with succinic anhydride to reduce background hybridization with fluorochrome dyes. The quality controls of these chips included scanning of autofluorescence of SSC, and hybridization with 9-mer Cy3-labeled oligo nucleotide probe. Chips were only acceptable if >95% spots were uniformly printed. The quality of these chips was further confirmed by hybridization with total standard RNA obtained from human brain tumor cells. GenePix 4000B and ScanArray Express HT were used for image acquisition and data collection. Data were further analyzed using different softwares, primarily the mAdb database, CIT, NIH. Current efforts are focused on quality control and standards development. These activities are expected to enhance knowledge base and ability to evaluate results of microarray experiments in product development or clinical trial design.
G..C.Ziobro, CFSAN, FDA, College Park, MD 20740
Concerned about the presence of filth contaminants such as rat, mouse, and other animal hairs and excreta; whole insects, insect parts, and excreta; and other extraneous materials which, because of their repulsiveness, would not knowingly be eaten or used, Dr. Wiley hired B.J. Howard to develop microscopic methods to isolate, identify, and quantify these contaminants in foods, drugs, and cosmetics. Over the past 100 years Howard and his successors have analyzed over 48,000 regulatory samples and have published several hundred papers on the detection of these materials. The most recent series of papers detailed the regulatory action criteria for filth and other extraneous materials that are the scientific basis of Compliance Policy Guide Sec. 555.600. This poster will outline the history of filth method development how it influenced America's health.
F.A.Bencsath, L.Quinonez, H.Valentin-Perez, J.D.Barnett
Diesel oil is one of the largest volume petroleum products spilled at sea by ship accidents. The spills may contaminate seafood and result in the closure of the affected fisheries. Taint-free seafood is required for reopening. We used instrumental analyses to quantify contamination as a complement to the qualitative assessment of the human sensory panels. We used an electronic nose instrument and dynamic headspace gas chromatography-mass spectrometry (GCMS). Oysters were tainted by exposing them for 48hours to seawater contaminated with 10ppm diesel oil, then were depurated in clean seawater for six weeks. After the exposure, the oysters were found tainted by sensory assessment. GCMS analysis identified C10-C17 alkanes and 60 aromatic compounds: benzene, indane, tetralin and naphthalene homologues. The total hydrocarbon concentration decreased exponentially during depuration, from the initial 26ppm to 0.5ppm (the threshold of human olfaction) in 24 days. The electronic nose, calibrated by a mixture of six representative aromatic hydrocarbon compounds, provided very similar quantitative data for the oyster samples with total hydrocarbon concentration of 2ppm and above.
F.J.Schenck1, D.J.Donoghue2, J.E.Hobbs1, 1ORA, FDA, Atlanta GA, 2Univ. of Arkansas, Fayetteville AR
A method for the analysis of N-methyl carbamate pesticide residues in eggs is presented. The purpose of the study is to provide residue data for exposure estimates used in risk analysis. The method uses an acetonitrile extraction, a graphitized carbon and aminopropyl SPE column cleanup and determination by reverse phase liquid chromatography with fluorescence detection. The average recoveries of 11 fortified (1.0 — 10.0 ppb) carbamates from eggs ranged from 80-100%. Single Comb White Leghorn hens were treated with carbaryl. Hens dusted once with a 10% carbaryl powder produced eggs containing 76, 54, and 42 ppb carbaryl and 69, 33 and 34 ppb of the carbaryl metabolite 1-napthol on days 1, 2 and 6 post-dosing, respectively. Hens sprayed with a 1% carbaryl solution produced eggs containing 14, 15, 23, 14, and 19 ppb carbaryl and 5, 6, 6, 6 and 15 ppb 1-napthol on days 2, 3, 4, 5 and 7 post-dosing, respectively.
T.Liu, J.Muller, Z.Ye, CBER/FDA/Rockville MD
The matrix protein (M1) of influenza virus plays a central role in viral replication. In relation to viral growth and morphology, we studied the RNP-binding activity of M1s from high-growth strain A/Puerto Rico/8/34 (A/PR8/34) and the relatively low-growth wild-type strain A/Nanchang/933/95. The RNP-binding strength of M1 was studied by disruption of M1 from M1/RNP complexes with salt and acidic condition. Our results indicated that binding of M1 of high-growth A/PR8/34 was more difficult to break than the binding of M1 of low-growth A/Nanchang/933/95. Consistent with the presence of M1 in A/PR8/34, binding of M1 of Resvir-9, a reassortant containing P, M and NS genes from A/PR8/34 and the rest of genes from A/Nanchang/933/95 and retaining relative high-growth characteristic, was relatively difficult to break than the binding of M1 of A/Nanchang/933/95. Physical properties of morphological features of these viruses were studied by velocity sucrose gradient centrifugation and transmission electron microscopy of purified viral particles, and by immunofluorescence staining of hemagglutinin expressed on the surface of infected cells. The results demonstrated that high-growth strains, A/PR8/34 and a relative high-growth reassortant, Resvir-9, had characteristics associated predominantly with spherical particles, while the low-growth strain, A/Nanchang/933/95, had characteristics of filamentous particles. These studies indicate that the binding between M1 and RNP complex might determine viral growth and morphology.
Board B-01 Microarray Detection of Antibiotic Drug Resistant Strains of Mycobacterium tuberculosis (MTB)
X.Tang1, S.L.Morris2, J.J.Langone1, S.A.Khan3, P.M.Regan4, L.E.Bockstahler1, 1CDRH, FDA, Rockville, MD 20857, 2CBER, FDA, Rockville, MD 20857, 3NCTR, FDA, Jefferson, AR 72079, 4WEAC, ORA, FDA, Winchester, MA 01890
Global public health is threatened through the emergence of potentially dangerous antibiotic drug-resistant strains of MTB. We are developing a model MTB system to establish a protocol utilizing microarray technology for rapid and accurate detection of clinically relevant point mutations in MTB genes associated with drug resistance. DNA templates of MTB rpoB and katG genes containing clinically relevant point mutations were generated by recombinant PCR techniques. Template sequences were confirmed by DNA sequencing. We prepared Cy5 dye-labeled single-stranded DNA target fragments from the templates by a modified asymmetric PCR technique. Wild type and mutant MTB gene-specific oligonucleotide probes (15-25 bases) were spotted onto glass slides. Following hybridization of the DNA target fragments with the array bound oligo probes, the results were determined using a laser scanner. We have used this technique successfully to detect and identify (<24 hrs) on a single microarray slide a number of wild type and clinically relevant mutant rpoB and katG gene target sequences. We plan to expand our microarray system to the detection of mutations in the other MTB genes associated with drug resistance. Validation and further refinement of our microarray protocol will be required for its application to clinical diagnoses.
W.H.Doub1, A.M.Wokovich1, G.J.Singh2, W.P.Adams3, 1DPA, OPS, CDER, FDA, St. Louis, MO, 2DPE, OPS, CDER, FDA, Rockville, MD, 3OPS, CDER, FDA, Rockville, MD
Nasal spray drug products are characterized in part via measurement of plume geometry and spray pattern as suggested in the U.S. Food and Drug Administration (FDA) June 1999 Draft Guidance for Industry, Bioavailability and Bioequivalence Studies for Nasal Aerosols and Nasal Sprays for Local Action. At the present time, plume geometry is typically measured using high-speed photography often with manual actuation. Spray pattern is assessed by collecting the spray on a glass or plastic sheet followed by manual measurement.
We have evaluated the SprayVIEW NSP System (Image Therm Engineering, Sudbury, MA) with respect to its ability to characterize both plume geometry and spray pattern. The SprayVIEW NSPis a nonimpaction system using a sheet laser and high speed digital camera to image the plume geometry and spray pattern. This system provides intensity profiles that increase objectivity for the determination of the spray pattern perimeter and shape.
We also see areas where improvements might be made. We find a need for the user to assess the homogeneity of the laser beam as this factor has a profound effect on the image of the spray plume. Additionally, there is still much subjectivity involved in measuring parameters which describe plume geometry.
T.J.Flynn, P.W.Wiesenfeld, S.C.Sahu, CFSAN/OSCI/OARSA, FDA, Laurel, MD
Hepatotoxicity is the leading cause of post-market withdrawal of drugs and of warning notices for dietary supplements. The present study evaluated the effects of model compounds, some with known hepatotoxicity, on a human (HepG2/C3A) hepatocyte cell line. Cells were cultured on 96-well plates and exposed to 9 concentration levels of test agent for 48 hr. Specific endpoint assays, which could all be conducted in a plate reading fluorometer and which reflect known mechanisms of hepatotoxicity, included: generation of reactive oxygen species; depolarization of mitochondria; steatosis; and induction or inhibition of cytochrome P450 activities. All parameters were normalized for total DNA content per well as a measure of number of viable cells. Each model compound generated a unique response profile based on which endpoints showed a concentration-related increase, decrease, or no change. Only 3 of 12 compounds tested showed cytotoxicity as measured by a significant decrease in total DNA. For some model compounds that are human drugs and have known hepatotoxicity (e.g., valproic acid), some endpoints responded at concentrations comparable to therapeutic blood levels. These findings suggest that this test system may serve as a high throughput screening assay for hepatotoxicity of food- or dietary supplement-related compounds.
L.Zhang1, E.F.Lizzio1, E.Gubina1, T.Chen1, H.Mostowski2, S.Kozlowski1, 1DMA OTRR, 2OCGT
There are two distinct phenotypes of T-cell cytokine responses that lead to different effector functions and to different outcomes in disease processes. Although evidence suggests a possible role of the local microenvironment in the differentiation or localization of T-cells with these phenotypes, there are no examples of divergent T-cell cytokine phenotypes with the same antigen specificity concurrently existing in different tissue compartments. Utilizing a CD8+ T-cell adoptive transfer model for GVHD, we demonstrate a potent type 2 cytokine response develops in the spleen while a potent type 1 cytokine response simultaneously develops in the testis. These experiments demonstrate for the first time that cytokine production can be oppositely polarized in different organs of the same individual. This may have important implications for organ specific pathology in infection or autoimmunity: infections or autoimmune diseases that affect multiple organs may have heterogeneity in tissue cytokine responses that are not revealed in systemic lymphocyte cytokine responses. Therefore, attempts to modulate the immune response phenotype may ameliorate pathology in one organ while exacerbating pathology in another.
D.B.Lyle, J.A.Barrett, J.C.Shallcross, S.C.Wood, J.J.Langone, CDRH, FDA, Rockville MD
Damage to endothelial cells, which line the interior of blood vessels, can occur in balloon angioplasty and/or the placement of stents within blood vessels. A key aspect of minimizing injury and subsequent restenosis of the vessel is the proliferation of endothelial cells which re-line and cover the denuded, exposed extra-cellular matrix. We have adapted a fluorescent plate reader methodology which uses the DNA-binding dye Hoechst 33342, for easily and quickly quantifying proliferation of the porcine aorta endothelial cell line PECSV.46 in 96-well culture plates during and following exposure to a variety of potential proliferation-altering drugs or biomaterials. PECSV.46 was optimally labeled with 50 mg/ml Hoechst 33342 for 1 hr. Binding of the dye to DNA resulted in a dose-dependent increase in fluorescence intensity within the wells. A linear response was observed for cell numbers between 2, 000 to 20, 000 cells/well, corresponding to 10% and 100% confluency. Detection did not require washing the dye from the wells, thus minimizing possible disturbance or loss of cells. This model system will facilitate the study of contrast agents, drugs used on drug-eluting stents, or device biomaterials for their potential to cause or modulate vascular toxicity.
M.E.Bishop1, S.Dertinger2, J.McNamee3, M.M.Moore1, A.Aidoo1, S.Harper4, R.Frobish5, J.L.Weaver6, W.Witt1, W.Slikker, Jr.1, C.Hotchkiss1, M.Hayashi7, J.T.MacGregor8, 1NCTR, FDA, Jefferson, AR, 2Litron Laboratories, Rochester, NY, 3Health Canada, Ottawa, Ontario, 4CFSAN, FDA, Laurel, MD, 5CVM, FDA, Laurel, MD, 6CDER, FDA, Laurel, MD, 7NIHS, Tokyo, Japan, 8NCTR, FDA, Rockville, MD
Pre-market testing of regulated products includes assessment of chromosomal damage in vivo. The traditional assay is assessment of the induction of micronuclei (cellular DNA fragments) in reticulocytes from mouse bone marrow using microscopic scoring. We are evaluating a flow cytometric assay that may permit routine monitoring of micronucleus frequency using peripheral blood from the rat, dog, non-human primate, and human, using mechanistically characterized agents known to induce micronuclei. The use of peripheral blood in these species has previously been limited by splenic activity that selects against micronucleated cells. The proposed method, which uses anti-CD71 labeling to identify young reticulocytes and a malaria standard that allows cross-laboratory standardization, may overcome this splenic selection problem. We have compared the new method with established methods in rats, and have identified suitable antibodies for flow cytometric assay in the beagle dog. Flow cytometric scoring gave superior counting statistics and lower variability than manual scoring. With appropriate validation, it may be possible to integrate the flow cytometric assessment into routine testing and eliminate the need for a separate mouse assay.
O.A.Maximova, R.E.Tafts, K.L.Pomeroy, D.McMahon, D.M.Asher, Laboratory of Bacterial, Parasitic & Unconventional Agents, DETTD, OBRR, CBER, FDA, Rockville MD 20852
Abnormal prion protein (PrP) associated with transmissible spongiform encephalopathies (TSEs), often called “scrapie-type PrP” (PrPSc), can be detected in tissues using immunohistochemistry (IHC). Evaluation of PrPScby IHC is often subjective–based on recognition by an experienced evaluator of specific PrPSc staining in tissue sections, sometimes reported semi-quantitatively using a four-point scale (0 to 4+). Evaluations are often unambiguous but may be inconsistent between evaluators or by the same evaluator on different days. FDA has sometimes had to base decisions regarding safety of products on information that includes results of PrPSc IHC to diagnose TSE in donors of blood or tissue or in patients who underwent invasive procedures with reusable FDA-regulated instruments. Quantitative IHC criteria would aid FDA in making such decisions. We sought to develop a system facilitating objective and consistent decisions about PrPSc IHC using computer image analysis to reduce variability and inconsistency in evaluations of tissue specimens. Using commercially available computer software (MetaMorph® Universal Imaging), we optimized capture and analysis of tissue images from sections of scrapie-infected hamster brain to quantify positive-signal intensity and percentage of selected tissue areas occupied by deposits of PrPSc. We believe that this approach may be useful for TSE research and for improved immunodiagnostics.
O.A.Maximova1, R.E.Taffs1, K.L.Pomeroy1, J.Ridge1, D.McMahon1, V.Ogryzko2, G.Amexis3, E.M.Dragunsky3, K.M.Chumakov3, D.M.Asher1, 1Laboratory of Bacterial, Parasitic & Unconventional Agents, DETTD, OBRR, CBER, FDA, Rockville MD 20852, 2NICHD/NIH, Bethesda MD 20895, 3Laboratory of Methods Development, DVP, OVRR, CBER, FDA, Rockville MD 20852
Detection of abnormal “scrapie-type” prion protein (PrPSc) in tissue is the gold standard for histopathological diagnosis of transmissible spongiform encephalopathies (TSEs). Immunohistochemistry (IHC) most often uses formalin-fixed paraffin-embedded (FFPE) tissue to identify PrPSc accumulations and characterize distribution patterns. Considerable disparity in IHC protocols can cause substantial variation in results. To reduce variation, we developed and optimized IHC for two types of specimens–necropsy tissues and cell cultures. We found a strong correlation between results of infectivity assays and PrPSc immunoreactivity in sections of scrapie-infected hamster brain. We developed a novel technique using “touch” prints of freshly cut brain tissue directly onto glass slides, facilitating rapid IHC detection of PrPSc. We modified the basic IHC protocol to detect the normal “cellular” prion protein (PrPC) and mutant forms of PrPC in fixed transfected SY5Y human neuroblastoma cell cultures expressing multiple copies of mutant PrP-encoding (PRNP) genes associated with familial Creutzfeldt-Jakob disease and similar TSEs. PrPC was detected in transfected cultured SY5Y cells but not in normal progenitor cells. The methods may help to improve early diagnosis of TSEs in potential donors of human tissues and in animals used as sources of raw materials and in-process reagents in FDA-regulated biological products.
S.L.Snellings1, L.A.Groves1, T.C.Schmitt1, D.W.Miller1, W.B.Parsons2, 1National Center for Toxicological Research, 2Arkansas Regional Laboratory, Jefferson, AR 72079
Ammonia, dimethylamine, and trimethylamine are volatile bases produced during seafood decomposition. Human sensory analysis is the method most commonly used to characterize seafood in terms of freshness or quality. An ion chromatography analytical method with conductance detection has been described for measuring the formation of these constituents as decomposition products in shrimp. The salient elements of the method begin with the extraction of the volatile bases with brine containing 0.02M sulfuric acid. Next, the acid salts are rapidly transformed to their respective free volatile bases by the addition of 40% sodium hydroxide. Lastly, the basic solution is purged with argon and the volatile gases are trapped in 5 mM methanesulfonic acid solution. Subsequent analysis with ion chromatography allows the quantification of ammonia, dimethylamine, and trimethylamine in the 5 mM methanesulfonic acid solution. We will describe a colorimetric method for determining total volatile bases that is based on the sample preparation for the IC method. The IC, colorimetric and human sensory method will be scrutinized for consistency.
V.J.Tomazic-Jezic, A.D.Lucas, B.A.Sanchez
Adverse reactions to NRL proteins are caused by topical, parenteral or respiratory exposures. NRL proteins bound to glove powder can cause serious reactions in NRL glove users. We evaluated protein extracts from medical gloves with and without accompanying powder. The ratios did not correlate with either the total amount of protein or powder on the glove, or glove weight, indicating that other factors may affect the protein binding. To investigate this possibility, we exposed clean powder to NRL antigens under various conditions and evaluated protein levels on starches and respective protein solutions. We found that protein binding was dose-dependent; with the saturation point at the concentrations above 200 µg/ml. Marked differences were observed among the individual starch preparations. Two cross-linked corn starch powders showed a strong affinity to attach proteins, while the protein binding to cooking corn starch and oat starch was much lower. Those differences were stronger with proteins in water than in PBS. The data indicate that physico-chemical properties of glove powder and some procedural factors play a role in the protein binding affinity.
V.J.Tomazic-Jezic, B.A.Sanchez
Allergenicity of NRL glove powders is well known, but the quantitative relationship to the allergenicity of the entire glove is not established. This study evaluated binding of individual NRL allergens to several glove powders. Two cross-linked corn starch powders, cooking corn starch and oat starch were exposed to ammoniated (AL) or non-ammoniated (NAL) raw NRL proteins. The amounts of starch-bound allergens were determined using the FIT Biotech allergen kit. The ratios of four individual allergens were similar for all starches, although the total level was different. However, the allergen ratio on starches was significantly different from the ratios in either AL or NAL extracts. While Hev b 6.02 comprised about 60% of total allergen in NAL extract, only 12-30% Hev b 6.02 was bound to starches. In contrast, there was only 3-7% of Hev b 1 allergen in NAL extract, but on powders, 35-45% of total allergen was Hey b 1. All samples demonstrated significantly higher binding of Hev b 1, and lower propensity for Hev b 6.02 allergen. These indicate that allergenic properties of NRL glove and glove powder may be different.
P.W. Wiesenfeld, T.J. Flynn and S.C Sahu, CFSAN/OSCI/OARSA, FDA, Laurel, MD
Hepatotoxicity is one of the most frequently reported adverse findings in safety assessment of food additives, drugs and dietary supplements. It is important, therefore, to develop in vitro screening assays and end-points that can quickly and accurately identify potential liver toxicants. Human and rodent hepatocyte cell lines (HepG2/C3A, WRL-68, CL9) were used to evaluate endpoints for apoptosis and cytotoxicity. Among the endpoints used were: cell viability, cytochrome P450 activity, programmed cell death (apoptosis), liver enzyme activity in the medium, and an indicator of steatosis. Caspase 3 activity was compared with classical markers of cytotoxicity such as, release of lactate dehydrogenase (LDH) into the medium. Caspase 3 and cytotoxicity end-points were dose, time, and cell-line dependent. C3A cells exposed to progesterone for 4-hours, did not produce any change in DNA concentration or Caspase 3 activity, but increased LDH release by 25%. Caspase 3 activity decreased with increasing dose and time (2, 4 and 6 hr) when C3A cells were exposed to androstendione (1.5 - 200 µg/ml). Under these conditions, there was no change in LDH activity and only a small decrease in total DNA content. In CL9 cells, 48-hour exposure to different concentrations of galactosamine increased release of LDH, decreased DNA content and Caspase 3 activity. Multiple cell lines, and end-points are needed to predict hepatotoxicty of model hepatotoxic compounds.
H.Golding1, L.R.King2, R.E.Taffs3, M.Merchlinsky2, N.Eller4, D.E.Scott4, J.Manischewitz2, 1DVP, CBER, FDA, Rockville, MD, 2DVP, CBER, FDA, 3DETTD, CBER, FDA, 4DH, CBER, FDA
In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, are under way.
There is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the Plaque Reduction Neutralization Test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia neutralization assay based on the expression of a reporter gene, b-galactosidase. Using a previously constructed vaccinia-b-galactosidase recombinant virus vSC56, we developed a neutralization assay, which is rapid, sensitive and reproducible. The read-out is automated. We show that the neutralizing titers (50% inhibitory dose) for several vaccinia immune globulin products (VIG) measured in our assay were very similar to those obtained in classical PRNT assays. A new FDA VIG standard was established for distribution to other laboratories. The new assay is currently used in several collaborative studies in primates and mice, and to evaluate clinical samples from human vaccine trials. The new assay will serve as an important tool for pre-clinical and clinical trials of new smallpox vaccines, and evaluation of therapeutic agents to treat vaccine associated adverse reactions.
D.N.Busick, S.A.Brown, K.Merritt, Office of Science and Technology, CDRH, FDA, Rockville, MD
To prevent patient-to-patient transmission of Creutzfeldt-Jakob Disease, the World Health Organization (WHO) recommends destroying instruments used on tissues possibly contaminated with CJD. Because it is impractical and costly to destroy instruments that are designed to be reusable, an alternative is to thoroughly decontaminate before reusing instruments that have contacted high-infectivity tissues (e.g., brain, spinal cord) of patients with confirmed or suspected CJD. The WHO and the US Centers for Disease Control and Prevention (CDC) have suggested that one of the following CJD-decontamination methods be used prior to routine cleaning and sterilization of surgical instruments:
- Autoclave in 1 N sodium hydroxide at 121°C for 30 minutes; OR
- Soak in either 1 N sodium hydroxide or 5% bleach for 60 minutes, then autoclave (dry or in water) for 60 minutes.
We subjected a variety of surgical instruments to the WHO-recommended decontamination methods to determine the effects on the instruments. Some instruments tolerated the decontamination procedures well; others did not (for example, gold-plated forceps handles corroded after soaking in bleach). We also subjected identical instruments to two standard methods for testing corrosion resistance, to determine whether the methods could predict the instruments’ response to decontamination.
M.Farahani, CDER, FDA, Rockville MD
Dental clinics are faced with increasingly stringent requirements for the treatment of both inorganic and organic wastes. Electron beam was used to reduce heavy metal ions such as lead and mercury with high efficiency. Radiolytically produced e-aq and H react with the heavy metal ions to reduce them to lower oxidation states, leading to formation of the atomic metal. Radiolysis was performed using an electron accelerator (7 MeV) with a dose rate of 0.4-o.6 kGy/3s pulse. Lead chloride (1x10-3M), mercury chloride (1x10-3M) were prepared for irradiation. All solutions were saturated with either air or N2. Sand was added to the solutions to nucleate precipitation. Following irradiation all solutions were filtered; syringe filters were used in the absence of oxygen for lead-containing samples, and Whatman 42 filter paper for mercury-containing samples. The soluble lead ions after the radiation treatment were measured with Atomic Absorption Spectrometer. The absorbed dose to achieve 99.9% removal of Hg ion from an aqueous solution is as low as 3 kGy. These results demonstrate that that electron beam technique can be used to convert water-soluble heavy metal ions to insoluble particles.
I.K.Ilev1, R.W.Waynant1, K.R.Byrnes2, E.Gorman1, J.J.Anders2, 1CDRH, FDA, Rockville, MD 20857, 2USUHS, Bethesda, MD 20814
A fundamental in vivo/ex vivo study of optical properties and light transmission characteristics of single and multiple tissue layers and blood in a Sprague Dawley rat model is presented. In our experiments, we utilize either coherent laser sources with various energy and spectral characteristics, or incoherent light sources in a broadband spectral range covering the visible and near-infrared. The measurement techniques are based on a minimally invasive fiber-optic approach that includes the use of dual-fiber sensor probes with input fibers for delivery of the illuminating light emission and output fibers for collection and delivery of the detected optical signals. The sensor probes are placed into different tissue layers (skin, sub-cutaneous connective and deep connective tissue, back muscle, bone and spinal cord) or blood, and broadband spectral transmission characteristics of these media are measured in vivo and ex vivo. The transmission spectra are analyzed in order to determine the specificity of interaction of different tissues with light. The main goal is to determine the most effective coherent or incoherent light sources to be used for specific minimally invasive photobiomodulation therapeutic and optical diagnostics techniques.
L.N.Kerr, L.D.Cash, M.P.Chaput, L.G.O'Malley, E.M.Sarhrani, J.C.Teixeira, W.S.Boivin, S.A.Mailhot, WEAC, FDA, Winchester, MA 01890
Current test methods evaluate the presence of defects in finished product, not the dynamics of glove use. A method to assess the durability of medical examination gloves made of various materials was evaluated. For each glove type, three sets of 100 gloves were tested. The first set of gloves was “challenged” using the durability method. The second set was manually donned and conditioned through a series of tasks simulating clinical use. The third set was unstressed control gloves. The gloves from each set were water-leak tested and the results recorded. The durability method created defects in all areas of the gloves. The durability method produced defects at similar rates to the simulated clinical method for all the glove types tested. Defect rates correlated well with target defect rates established from the literature. Durability varied among glove types with powder-free vinyl being the least durable (42% defect rate) and powder-free, textured chloroprene the most durable (3% defect rate). The durability method was reproducible with a coefficient of variation of 2.4%. This method can be used to establish the durability of medical examination gloves of a variety of materials.
W.Limm1, T.H.Begley2, T.Lickly3, S.G.Hentges4, 1OFAS, FDA, College Park. MD, 2OFAS, FDA, College Park, MD, 3Dow Chemical, Midland, MI, 4American Plastics Council, Arlington, VA
Diffusion coefficients of limonene in various linear low density polyethylene (LLDPE) and low density polyethylene (LDPE) resins have been determined from sorption data using a thermogravimetric method. From these data, one can determine whether polymer synthesis parameters such as choice of catalytic process or comonomer result in substantial differences in polymer barrier properties.
For example, LLDPE is currently manufactured using either one of two distinct catalytic processes: Ziegler-Natta (ZN) and metallocene, a single site catalyst. ZN catalysis is a heterogeneous process that has proven itself over the last half-century. It involves a transition metal compound containing a metal-carbon bond that can handle repeated insertion of olefin units. In contrast, metallocene catalysis has fewer than 20 years of history, but has generated much interest due to its ability to produce highly stereospecific polymers in very high yield. In addition to high stereospecificity, metallocene-catalyzed polymers are significantly lower in polydispersity than traditional ZN counterparts.
Absorption and desorption testing of heat-pressed films made from LLDPE and LDPE resins of varying processing parameters indicates that diffusion coefficients of limonene in these resins do not change substantially enough to warrant a change in the way FDA accesses LLDPE barrier properties.
T.O.Woods1, S.A.Brown1, S.G.McNamee1, K.Merritt2, A.D.Lucas2, V.M.Hitchins2, 1Division of Mechanics & Materials Science, CDRH, FDA, Rockville, MD 20850, 2Division of Life Sciences, CDRH, FDA, Rockville, MD 20850
This study addresses two immediate concerns for CDRH reviewers: the effects of reprocessing on OBNU devices and the effects of loading on degradation of absorbable materials. OBNU sutures are commonly reprocessed for reuse, though they are labeled for single use. In addition, the use of absorbable materials in loaded applications in tissue engineering and fracture repair continues to expand, and it is important to establish methods to determine the effects of static and cyclic loading on resorbtion rate. We previously showed that repeated ethylene oxide sterilization (reEtO) can affect the strength of absorbable sutures out of package. Here we continue our investigations to examine the effects of loading on the retained strength after immersion in saline of new and 5x reEtO suture composed of a variety of absorbable materials. A test apparatus was constructed which, in 37ºC saline, simultaneously subjects 5 suture loops to static or cyclic loads without constraining the specimen length. Material degradation was assessed by measuring tensile strength after static or cyclic loading and after immersion with no load. Results indicate that both load and resterilization decrease suture strength after immersion and that resterilization has an increasing effect on degradation with longer immersion times.
L.A.ZarembaCDRH, Rockville MD
MRI examinations of patients with implanted medical devices can produce significant heating of these devices, which is a potential hazard to patients. However, the MRI itself can be used to measure the temperature increase of the implant. The proton resonant frequency changes by 0.01 ppm/0C. This can be used to estimate the temperature distribution using a technique called phase mapping in which a map of the phase of the image signal, rather than its magnitude, is obtained. Phase mapping tests have been conducted in a phantom on the 2 tesla MRI at MOD I. Maps were acquired using a gradient echo sequence with an echo time of 12 msec, producing a phase change of about .064 rad/0C at 2 tesla. The technique was used to examine the temperature distribution surrounding wire configurations simulating implanted medical devices. Results were verified with a Luxtron fiberoptic temperature probe. Phase mapping has the advantage that it produces an image of the temperature distribution, while current methods using probes measure temperature at only a few points. The technique could be used as an alternative to temperature probes in the ASTM standard test method for measuring the heating of implanted medical devices during MRI.
S.Lute1, L.Norling2, J.Brown3, S.Herzer4, M.Voisard4, Y.Xu2, K.Brorson1, 1DMA/OTRR Bethesda MD, 2Genentech, Inc, 3ONDC/CDER Rockville MD, 4Amersham, Inc.
A potential safety concern in biotechnology purification schemes that employ re-use of column media is loss of the virus removal capacity of the chromatographic purification operation. One approach to assure safe reuse of resins is to define chromatography performance attributes that predict retrovirus clearance during extended re-use. In previous small-scale experiments, protein A columns were cycled 150 to 460 times using concentrates of murine hybridoma harvests, standard low pH elution buffers and different cleaning solutions. These studies identified Ab step yield and breakthrough as performance attributes that decay prior to retrovirus log10 reduction value (LRV) when protein A media is multiple-cycled. In these follow-up studies, we are evaluating viral clearance in multiple-cycled anion exchange media. Weak and strong anion exchange media from multiple vendors are being subjected to 250 or more cycles using a variety of cleaning buffers (NaOH, NaCl, and HCl-based). The resins are being challenged with virus-spiked model proteins in both flow-through and bind & elute mode. A variety of performance attributes are being measured including retrovirus clearance using two independent Q-PCR based assays, DNA clearance, and step yield. Our preliminary results emphasize the importance of proper cleaning of resins for chromatographic performance, including viral clearance.
C.R.Clavet1, A.B.Margolin2, P.M.Regan1, 1University of New Hamphsire
Monoclonal antibodies H1206 (anti-HSV-2 gG-2) and H1379 (anti-HSV-1 gG-1) bound to tosylactivated paramagnetic dynabeads® (Dynal®) have been used to isolate herpes simplex virus type-specific glycoproteins G-1 and G-2 from solubilized HSV infected cell extracts. Monoclonal antibody H1206 was unreactive to the immunomagnetically purified gG-1 and, conversely, monoclonal H1379 was unreactive to immunopurified gG-2. The immunoaffinity purified native glycoproteins were used as markers in conjunction with full Western blot profiles to HSV-1 and HSV-2 to determine the respective serological reactivity to the viruses. Immunoblotting a number of human and rabbit HSV positive antisera obtained from at the Centers for Disease Control (CDC), Atlanta, GA., demonstrated the effectiveness of this methodology. Sera characterized by Western blot and serological reactivity to the immunopurified gG-1 and gG-2 could be used to test the performance characteristics of HSV-1 and HSV-2 clinical serological immunoassays.
Board D-03 Evaluating the Safety and Effectiveness of "Aberration-Free" Ophthalmic Refractive Surgery
B.Drum, M.Eydelman, G.Hilmantel, CDRH, FDA, Rockville, MD
FDA has well-established methods for evaluating spherical and astigmatic refractive surgery. However, new techniques for measuring the optical performance of the eye are now being used with refractive surgical lasers to treat “higher order” aberrations (e.g., coma and spherical aberration) in addition to defocus and astigmatism. Even though many of the effects of higher order aberrations on visual function are still poorly understood, FDA must evaluate the safety and effectiveness of these new treatments. Some specific difficulties include:
1) the potential visual improvement is small and dependent on type of aberration; 2) to date, virtually all refractive surgical treatments have made higher order aberrations worse; 3) treatment outcomes are influenced by uncontrollable factors such as healing, aging, accommodation, pupil size and ocular biomechanical effects; 4) small errors in laser alignment can produce large errors in outcome; and 5) some aberrations may be necessary for optimal visual performance.
FDA’s need to evaluate aberrometry-guided refractive surgery is fueling major research efforts in the vision, ophthalmic and optometric research communities. The results of this research together with outcomes from FDA-regulated trials contribute to a continuing evolution in the types of data and analyses needed for pre-market approval of refractive surgical devices.
Y.Yang1, P.S.Pine2, H.Davis2, P.J.Faustino1, R.C.Lyon1, L.X.Yu3, J.P.Hanig2, 1Division of Product Quality Research, Kensington, MD 20895, 2Division of Applied Pharmacology Research, Laurel, MD 20708, 3Office of Generic Drugs, Rockville, MD 20855
An isocratic reversed-phase HPLC method was developed and validated according to FDA’s Guidance for Industry, “Bioanalytical Method Validation”, for the determination of isotretinoin in plasma and brain tissue from mice following oral single and multiple doses. Plasma sample preparation included deproteination with acetonitrile-perchloric acid followed by centrifugation. Brain tissue was homogenized at 4oC in a solution containing 0.5 mg/mL each of EDTA and ascorbic acid. Homogenates were immediately extracted with acetonitrile-perchloric acid followed by centrifugation. The supernatants were analyzed by HPLC. Benz[a]anthrancene-7, 12-dione was used as the internal standard. Chromatographic separation was achieved on a C18 column (Phenomenex) using an acetonitrile-aqueous 0.5% acetic acid (85:15, v/v) elution. The average extraction efficiency was >95% for plasma and >82% for brain. The lower limit of quantification was 30 ng/mL for plasma and 3.0 ng/0.1g for brain tissue, respectively. The linear range for plasma was 30 to 600 ng/mL, and for brain was 15 to 80 ng/0.1g. The R2 ranged from 0.9891 to 0.998. The peak concentrations (Cmax) of isotretinoin are 2.36 and 2.99 ug/mL in plasma, 34 and 39 ng/0.1mg in brain following a single and multiple doses, respectively. In both cases, Tmax was 1 hr.
T.K.Lee1, E.L.Koo1, A.C.Chang1, D.Sands2, C.Whitton2, C.Longstaff2, 1CBER, FDA, Rockville, MD 20852, 2NIBSC, S. Mimms, UK
An international collaborative study was organized to identify and calibrate a replacement standard for the current US Standard Thrombin, Lot J, and the 1st International Standard (IS) for alpha-Thrombin (89/588). The study involved 25 laboratories from 15 countries. Laboratories were asked to assay two candidate replacement standards, C and D, relative to both the US Standard and the IS using either fibrinogen clotting or plasma clotting methods, and/or chromogenic assays. Data analysis indicated that candidate D is the more suitable replacement for the US Standard and IS. From clotting assays, the potency of candidate D was 106.4 US units/ampoule against the US Standard, and 114.5 IU/ampoule against the IS. Averaging provided 110 Units/ampoule (both US units and IU). The ratios between the potencies derived from the chromogenic and clotting assays indicated that candidate D had a higher proportion of alpha-Thrombin than candidate C. Initial stability studies after 6 months of storage at elevated temperatures showed that both candidates were equally stable with a projected loss of activity of <0.1% per year at -20 °C. It is proposed that candidate D be adopted as the 2nd International Standard for Thrombin with a potency of 110 IU/ampoule.
J.Mauldin1, K.Carbone1, R.Yolken2, S.Rubin1, 1CBER, FDA, Bethesda, Maryland 20892, 2Johns Hopkins University, Baltimore, Maryland 21218
Currently either plaque reduction neutralization assay (PRN) or hemagluttination inhibition assay (HIA) are acceptable measures of protective serological immune responses to mumps. However, there is debate as to whether rapid and quantitative serological assays (e.g., ELISA) can be used as indicators of mumps serological responses. Thus, we compared serological responses to wild type mumps in a group of 75 human sera using two commercially available Mumps IgG ELISAs, Wampole Laboratories (Cranbury, New Jersey, USA) and IBL (Hamburg, Germany), to those obtained using an in-house plaque reduction neutralization (PRN) assay. Because the induction of a neutralizing antibody response is a reasonable index of immunity against mumps virus, results of the PRN assay were used as the standard against which ELISA results were compared. The results indicate that the specificity of the two ELISAs ranged from 83%-91% and the sensitivity ranged from 72%-76%. These results suggest that assays that detect functional antibody, e.g., virus neutralization assays, are of greater utility than ELISAs for assessing mumps immunity.
M.L.Pombo1, I.G.Berthold2, E.Gingrich2, M.T.Jaramillo3, M.Leef2, J.L.Arciniega2, 1FDA, Bethesda MD. National Institute of Hygiene "Rafael Rangel", Venezuela, 2FDA, Bethesda MD, 3FDA, Bethesda MD. National Public Health Laboratory, Mexico
Potency testing of the only anthrax vaccine licensed for human use in the US involves protection of actively immunized guinea pigs from a lethal challenge with a virulent strain of Bacillus anthracis. The objective of this work was to validate an anti-PA (protective antigen) ELISA to measure antibody response in mice. Validation of the assay according to the guidelines of the International Conference on Harmonization (ICH) rendered the following characteristics: linearity (working range: 40-1, 159 EU/ml; r2 = 0.9983), accuracy (91-105% recovery), precision (3-20%CV, repeatability; 1-9%CV, intermediate precision per day and per analyst), detection limit (4 EU/ml), and quantification limit (40 EU/ml). Findings support the use of this ELISA in an alternative immunogenicity (potency) test of PA-containing vaccines in mice and for the standardization of reference materials.
M.W.Trucksess1, V.A.Brewer1, K.M.Williams1, C.D.Westphal1, J.Heeres2, 1CFSAN, FDA, College Park, MD, 2JIFAN/University of Maryland student
Peanuts are one of the eight most common allergenic foods and a large proportion of peanut-allergic individuals have severe reactions, some to minimal exposure. Specific protein constituents in the peanuts are the cause of the allergic reactions in the sensitized individuals who ingest the peanuts. To avoid accidental ingestion of peanut contaminated food, methods of analysis for the determination of the allergenic proteins in foods are important tools. Such methods could help identify foods inadvertently contaminated with peanuts thereby reducing the incidence of allergic reactions to peanuts. Commercial immunoassay kits are available but need study for method performance which requires reference material for within and between laboratory validation. In this study NIST peanut butter, SRM 2387, was used. A polytron homogenizer was employed to prepare an homogenous aqueous peanut butter suspension for the evaluation of method performance of some of the commercially available immunoassay kits such as Veratox Peanut Allergen Test (Neogen Corp.), Ridascreen Peanut (R-Biopharm GmbH), and Bio-Kit Peanut Protein Assay Kit (Tepnel). Each gram of the aqueous peanut butter suspension contained 643 g peanut, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. The suspension was found to be homogenous, stable, reproducible and applicable for adding into ice cream, cookies, breakfast cereals and chocolate for recovery studies at spiked level ranged from 12 mg/kg to 90 mg/g.
H.Wang, A.V.Del Grosso, J.C.May, OVRR
Formaldehyde is often used in bacterial vaccines either as a stabilizer or inactivating agent. Vaccines against anthrax, diphtheria, hepatitis A, influenza, Japanese encephalitis, and tetanus contain residual amounts of free formaldehyde. Less than 0.02% formaldehyde is permitted in vaccine products by FDA based on four decades of safety research. However, the chemical analysis methods used in determining the formaldehyde content of vaccines rely mainly on cumbersome colorimetric procedures (e.g. Hantzsch reaction, Schiff reaction). The aim of this research is to develop a faster and better HPLC method using pre-column chemical derivatization. The selected derivatization reactions such as 2, 4-Dinitrophenylhydrazine, 4-Nitrobenzylhydroxylamine, 1, 3-Cyclohexanedione and Dansyl Hydrazine were compared based on chemical properties of the derivatization reagents, Conditions required for derivatization reaction, derivative stability and HPLC detection sensitivity. HPLC analytical results for several Anthrax Vaccine Absorbed samples were compared to those obtained by traditional colorimetric methods. The recovery of formaldehyde was tested by spiking the vaccine samples since the yields of the derivatization reactions were found either less than quantitative or immeasurable.
P.H.Duffy1, S.M.Lewis2, M.A.Mayhugh2, R.W.Trotter3, 1NCTR, FDA, Jefferson AR, 2Bionetics Corp., Jefferson AR, 3Charles River Corp., Jefferson AR
Ad libitum-fed (AL) obese rats are typically control groups for biomedical studies. Concomitant to weight gain, age- and obesity-related pathologies develop which are modified by dietary controls. A chronic 110-week study was undertaken to determine the effects of 10, 25 and 40% dietary restriction (DR) in male Sprague-Dawley rats. The survival rate for DR groups exceeded that for the AL group; the largest increase in survival occurred between AL and 10% DR. The incidence and severity of cardiomyopathy and nephropathy was significantly lower in all DR vs AL rats. Compared to AL rats, 10% and 40% DR rats had fewer primary neoplasms; 10% DR rats had fewer malignant lesions than 25% and 40% DR rats, and age at which first benign, primary, and malignant tumors occurred was greater in all DR groups. The largest reduction in tumor number occurred between AL and 10% DR groups. Additionally, the percentage of rats with tumors and number of fatal tumors was significantly lower in all DR vs AL groups. This study proved that low-level DR (10%) is effective in increasing survival rate and in preventing or slowing the progression of neoplastic and non-neoplastic obesity-related diseases.
S.Satchithanandam, C.Oles, M.P.Yurawecz, J.I.Rader, ONPLDS, CFSAN, FDA, College Park, MD
The U. S. Food and Drug Administration is finalizing a regulation to include trans fatty acids in nutrition labeling. The purpose of this study was to determine the trans fat contents of currently available foods. Food products were identified on the basis of market share and were purchased from local super markets. AOAC Official Method 996.01 (Fat Analysis in Cereal Products) was modified as needed for the analysis of trans fat. Food samples were weighed, hydrolyzed and converted to fatty acid methyl esters (FAME) and analyzed by gas chromatography. Trans fat content (g/100g fat) ranged from 0.0-2.43 in salad dressings and mayonnaise, from 0.0-13.23 in vegetable oils and shortenings, from 0.0-25.23 in cake and related products, from 0.0-29.15 in potato chips and corn chips, from 15.95-23.42 in margarine products, from 25.83 to 38.52 in fried potatoes, and from 25.0 to 33.61 in cookies and crackers. The variability of trans fat content is due in part to differences in the type of fats and oils used in manufacturing processes.
J.J.Yin1, M.M.Mossoba1, M.P.Yurawecz1, Y.Ku1, J.K.Kramer2, 1FDA, College Park, MD., 2Southern Crop ProtectiFood Research Center, Ontario, Canadaon,
Conjugated linoleic acid (CLA) isomers are found in dairy products and meats from ruminants and reportedly exhibited anticarcinogenic and other beneficial physiological effects in animal studies. The effect of CLA on the relation between structure and function of membranes is described in this work. Electron spin resonance (ESR) spin-label oximetry was used to evaluate if oxygen transport and oxygen depletion was affected by incorporation of CLA instead of linoleic acid (LA) into membrane phospholipids. Specifically, 1-stearoyl-2- (9cis, 11trans-octadecadienoyl)-phosphatidylcholine (C9T11 CLA) and 1-stearoyl-2-(10trans, 12cis-octadecadienoyl)-phosphatidylcholine (T10C12 CLA) were incorporated into soy plant phosphatidylcholine (Soy PC), egg yolk PC (EYPC), Heart PC and Brain PC bilayers. Moreover, the use of spin labels attached to different carbons along the fatty acid chain makes it possible to carry out structural and oximetric determinations with the same test sample. In our model system, free radical generation was initiated by extended incubation of the liposomes, induction by AAPH, and AMVN. The effects on the oxygen consumption rate of 5 mol% CLA in most PC was significantly larger compared to the 5 mol% LA. However, there is no significant difference in homogenous solutions of those lipids. The perturbation of membrane structure and the increase of the relative oxygen diffusion-concentration products provided a potential mechanism by which CLA incorporated into membrane lipids could affect oxidative stress.
S.G.Edelson-Mammel1, R.L.Buchanan2, 1OPDFB, FDA, College Park MD, 2OSCI, FDA, College Park MD
The presence of low levels of Enterobacter sakazakii in dried infant formula have been linked to outbreaks of meningitis and necrotizing enterocolitis in neonates, particularly those who are premature or immunocompromised. In the current study the ability of twelve strains of E. sakazakii to survive heating in rehydrated infant formula was determined at 58°C using a submerged coil apparatus. The observed D58-values ranged from 30.5 to 591.9 seconds with heat resistance appearing to be phenotypically bimodal. The z-value of the most heat resistant strain was 5.6°C. When dried infant formula containing this strain was rehydrated with water pre-equilibrated to various temperatures, a greater than or equal to 4-log reduction in E. sakazakii levels was achieved by preparing the formula with water at greater than or equal to 70°C.
S.M.Etheridge1, G.C.Pitcher2, C.S.Roesler3, 1CFSAN, FDA, Laurel, MD, 2Marine and Coastal Management, Cape Town, South Africa, 3Bigelow Laboratory, W. Boothbay Harbor, ME
Dinoflagellates of the genus Alexandrium are the most common sources of paralytic shellfish poisoning (PSP) toxins, and suspension-feeding bivalves are the traditional vectors. However, in 1999 PSP was detected in the abalone Haliotis midae off the coast of South Africa. The purpose of our research was to identify the source of abalone toxicity and assess toxin retention and biotransformation. Abalone were incubated with a range of toxic media and food sources and were then analyzed for toxin content using high performance liquid chromatography. The abalone were toxic at the start of each treatment (160.15 37.57 g STX eq 100 g-1 tissue). When abalone were fed artificial feed only, they became less toxic. There was no significant difference in toxicity between the other treatments and initial conditions, indicating that cells of Alexandrium catenella, the filtrate of cultured A. catenella, and the kelp Eklonia maxima may all serve as potential toxin sources, or alternatively, abalone will not depurate in the absence of feeding. Maintaining abalone on a diet of artificial feed prior to marketing may serve as a strategy to ensure the safety of this product. The need for including this non-traditional vector in routine PSP monitoring programs is clearly evident.
S.L.Foley1, R.Singh1, S.Gaines2, S.L.Ayers1, K.Bischoff3, J.J.Mauer4, D.G.White1, 1Division of Animal and Food Microbiology, CVM, FDA, Laurel, MD, 2Division of Animal Research, CVM, FDA, Laurel, MD, 3USDA, College Station, TX, 4University of Georgia, Athens, GA
Escherichia coli cause a vast array of illnesses in many animal species, including humans. This organism is believed to have evolved with its animal host, accounting for its host specificity. Since E. coli can cause foodborne illnesses in humans it is important to be able to determine the animal species from which a pathogen originates. The ability to determine the source of that pathogen will help facilitate an understanding of how E. coli disseminates in the food supply. The objective of this study was to evaluate different phenotypic and genotypic discriminatory methods for their ability to distinguish the origin of E. coli isolated from different food animal sources. Two hundred E. coli isolates from cattle (n=54), swine (n=90), and chickens (n=56) were differentiated using serotyping, antimicrobial susceptibility profiling, and ribotyping. The results indicated that there were 34 different serotypes, 76 susceptibility profiles, and 48 ribotype groupings for the isolates. Twenty-five of the 48 ribotype clusters contained more than a single isolate. Nineteen of these groups contained isolates originating from a single animal species. Of the 76 antimicrobial susceptibility profiles characterized, there were 37 groups that contain more than one isolate, and 35 of those groupings contain isolates from a single animal host species. Additionally, E. coli isolates from a number of the serotypes appeared to be more commonly isolated from one host species and tended to cluster in specific ribotype groups and share similar antimicrobial susceptibility profiles. This suggests that there may be some host species-specific differences that can be detected using ribotyping, serotyping, and/or antimicrobial susceptibility profiling.
S.M.Jackson1, Y.Hu2, 1FDA, ORA, NRL, Jamaica , NY, 2FDA, ORA, NRL, Jamaica, NY
Shigella spp are common food borne pathogens, however they are considered to be difficult organism to recover from foods. Regulatory PCR-based assays have been used for the detection of Shigella spp from produce in recent years, and it has been found that sample preparation and DNA extraction steps are crucial for a successful Shigella spp PCR. In order to address the maximum recovery the organism from samples, we analyzed all the technical details related to every step of sample preparation, DNA extraction and PCR detection. In these studies, an optimizing sample preparation method with Polyvinylpyrrolidone (PVP), pathogens concentration with high speed centrifugation, DNA extraction with PrepMan technology, and PCR with chemical hot start were developed and evaluated as a tool for an improved method to recover and detect Shigella spp from produce with sensitivity higher than that of current method. This improved method could be of future importance as an additional regulatory technique for the detection of Shigella spp and other pathogens in produce.
M.M.Mossoba, P.Whittaker, S.F.Al Khaldi, B.D.Tall, F.S.Fry, M.P.Yurawecz, V.C.Dunkel, CFSAN, FDA, College Park, MD
The analysis of fatty acids using gas chromatography is widely accepted as a reproducible method for identifying bacteria. Bacterial fatty acids can also be characterized by infrared spectroscopy (IR), a faster and potentially a more differentiating approach. We have developed a rapid method that applies IR technology to the analysis of bacterial fatty acids for identifying microbial pathogens. Fourteen different bacteria, representing 8 genera and 11 species, were cultured on brain heart infusion agar at 35°C for 24 hours, harvested and then subjected to saponification, methylation and extraction into hexane:methyl tert-butyl ether (1:1). The bacterial fatty acid methyl esters (FAMEs) were measured by heated diamond Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy. A dataset for the bacterial agents was prepared using spectra from replicates prepared on 4-6 different days. The results showed that the spectra obtained were unique for each of the 11 species and that they could be used as a fingerprint for the organisms. In summary, this analytical method provides a rapid procedure for the identification of bacteria based on their unique fatty acid methyl ester spectra.
D.L.Park, H.Njapau, S.Trujillo, OPDFB, CFSAN, FDA, College Park, MD 20740
The significance of mycotoxins to public health and the increase in global trans-shipment of food commodities has led the Food and Drug Administration to initiate a global effort to reduce mycotoxin contamination of agricultural commodities, hence improve the safety and quality of the world’s food supply. In conjunction with the Joint Institute for Food Safety and Applied Nutrition, an international workshop on mycotoxins for training trainers was held July 22-26, 2002, at the FDA facility in College Park, Maryland. Over 100 individuals from 48 countries participated. The participants returned to their respective countries and are in the process of conducting similar workshops. Workshops have been held in Turkey and Mexico, and Nigeria and Tanzania have submitted proposals for similar events. Efforts are also underway to assist Central America and other Southeastern African countries. A global approach is in conformity with science-based efforts to harmonize international food safety standards and regulations, and has the potential for improving the safety and quality of food in economically challenged nations, enhancing the health and productivity of the populace and ultimately increasing economic growth and development. Richer economies in the developing regions will have increased purchasing and choice capabilities to participate in world trade.
H.Njapau1, D.L.Park1, S.Trujillo1, P.J.Cotty2, L.Antilla3, R.Webb3, W.Snuffer4, W.D.Price5, 1OPDFB, CFSAN, FDA, College Park, MD 20740, 2USDA-ARS-SRRC, New Orleans, LA 70124, 3ACRPC, Phoenix, AZ 85040, 4SBTI, Phoenix, AZ 85034, 5OSC/DAF, CVM, Rockville, MD 20855
Milk is widely consumed by infants in the U.S. Cotton, a desirable feed ingredient for dairy cows, can be contaminated with aflatoxin B1. Milk from animals fed aflatoxin B1-contaminated feed can contain aflatoxin M1, a mutagenic substance for which FDA has set an action level. A high pressure/high temperature ammoniation process substantially reduces aflatoxin B1 levels in dairy-ration cottonseed, and the mutagenic potential in milk. The objective of this study is to determine the efficacy of the ambient temperature/ atmospheric pressure (AT/AP) ammoniation process to decontaminate aflatoxin-contaminated cottonseed and determine the decrease in the mutagenic potential of milk from dairy cows fed rations containing decontaminated cottonseed. Preliminary results indicate that the AT/AP tent process reduces aflatoxin levels in highly contaminated (1, 700 ppb) cottonseed by 94% in 74 days in winter in Arizona. At ambient temperatures of 17-26°C, internal process temperatures variably increase from 31- 42°C to 43-85°C in 10 days and gradually decrease to near ambient temperature by day 74. Ammoniated cottonseed moisture declines from 21 to 15% (w/w) in the same period. The study is in progress and will, once completed, provide important information on the efficacy and safety of the AT/AP ammoniation process.
I.A.Ross, P.P.Sapienza, D.E.Hanes, W.Johnson, C.S.Kim
Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, FDA, Laurel, MD, USA.
To determine the release of endotoxin, lipopolysaccharide (LPS), from Salmonella typhimurium in gastrointestinal and plasma conditions, LPS was biosynthetically labeled with 3H on the fatty acyl-chains of S. typhimurium grown in proteose peptone beef extract (PPBE) medium. An aliquot of bacterial cell culture was incubated in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and PPBE medium. At the end of the incubation period the samples were centrifuged and the supernatant measured for radioactivity. The time course for the incorporation of the radiolabel into the cells indicated the uptake increased linearly for the first 60 min and reached steady-state level by 120 min. The LPS released at 120 min was 0.5, 0.8, and 1.9% of the total bacterial LPS into plasma, SIF, and SGF, respectively. The results were significant at p <0.5 level for SIF and p <0.001 for SGF as compared with plasma pH values.
K.H.Seo, I.E.Valentin-Bon, R.E.Brackett, G.R.Henderson, B.S.Eblen, A.J.De-Jesus, CFSAN, FDA, College Park, MD
We developed a real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of SE and applied the technique to naturally contaminated ice cream samples. In this assay, a segment (97-bp) of the gene sefA specific to Salmonella group D strains, such as Salmonella Enteritidis and Salmonella Dublin, was amplified by polymerase chain reaction (PCR). The amplification of the target gene products was monitored in real-time by incorporating fluorescent dye-labeled gene-specific probes in the PCR reaction. When applied to direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection, but populations of SE derived from real-time quantitative PCR were one to three logs higher than provided by most probable numbers and colony-forming units obtained by conventional culture methods. However, the detection and enumeration of SE in naturally contaminated ice cream can be completed in 4 h using the developed real-time PCR method while cultural enrichment method require 5 to 7 days. The real-time PCR assay proved to be a valuable tool that may be useful for the food industry in monitoring its processes to improve product quality and safety.
K.H.Seo, G.Thammasuvimol, R.E.Brackett, S.G.Edelson-Mammel, CFSAN, FDA, College Park, MD
Enterobacter sakazakii (E. sakazakii), a gram-negative, straight rod belonging to the Enterobacteriaciae family and qualifies as a coliform bacterium, is a rare cause of invasive infection with high death rates in neonates. Final confirmation of E. sakazakii colonies can only be done via several systems such as API 20 E™, API ZYM™, or Vitek™ assays, but no DNA or serological based technologies are available. A real time PCR assay for specifically detecting E. sakazakii was developed. A set of primers and probes was designed using E. sakazakii partial macromolecular synthesis (MMS) operon; rpsU gene, 3' end, and primase (dnaG) gene, 5' end. The specificity of the assay was evaluated using nearly 50 other Enterobacteriacea including Enterobacter cloacae. The newly developed assay enables us to detect 102 CFU/ml in pure culture and infant formula in 50 cycles of PCR reaction without enrichment procedure. The assay also was very specific to discriminate E. sakazakii from all other Enterobacteracea strains. The developed real time PCR assay could save up to 5 days and eliminate the need for plating samples on selective/diagnostic agars, and biochemical confirmation steps. The real time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.
Board F-15 Microbial Source Tracking (MST) of Foodborne Salmonella & Campylobacter
R.Singh, S.L.Foley, D.G.White, S.Zhao, S.Simjee, P.F.McDermott, R.D.Walker, Center for Veterinary Medicine, FDA, Laurel, MD
Approximately 76 million people suffer from foodborne illnesses in the U.S. annually at an average cost to the U.S. economy of $15 billion. Many pathogenic bacterial species have been implicated in foodborne diseases, with infections caused by Salmonella and Campylobacter species occurring at higher frequencies than those caused by other species. In this study we present MST as a means of determining the food animal of origin of Campylobacter and Salmonella. We have investigated serotyping, antimicrobial susceptibility, pulse-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for their capacities to distinguish Campylobacter and Salmonella isolates from pigs, cattle, turkey and chickens. For Campylobacter, preliminary analysis of the data suggests that PFGE and MLST provide better discriminatory power than biochemical profiles or serotyping. For Salmonella, serotyping appears to be the best method for certain strains, namely S. Dublin and S. Choleraesuis isolates, in which over 99% are from cattle and swine, respectively. The results from this study could aid in determining the food animal species from which Salmonella or Campylobacter may have originated. Further, the methods can be used as an example for future MST studies of other foodborne pathogens. Data from these studies will aid the FDA’s ability in making science-based regulatory decisions about food safety.
D.H.Bark, C.Abeyta, J.M.Hunt, PRL-NW, FDA, Bothell, WA
This work defines a simple method for the culture of Campylobacter that produces superior growth compared to the standard method described in the Bacteriological Analytical Manual. The study was initiated to look for alternate methods of culturing Campylobacter that do not require blood supplementation in either the broth enrichment or plate media. It was important that any new technique be as sensitive as the standard method without becoming more complicated. The methodology employs 35o C. incubations in the standard “campylobacter gas mixture”, stationary broth enrichments in 50 ml Erlenmeyer flasks, and bloodless broth and plate media. The data generated, with four different strains of Campylobacter jejuni, demonstrated that removal of blood from enrichment broth media significantly improved (p<0.01) growth, if replaced by a supplement such as FBP. Supplementation of plate media, on the other hand, had little effect on the cultural outcome. Additionally, enrichment in 50 ml Erlenmeyer flasks produced a dramatic increase in growth compared to the standard enrichment method. These alterations in methodology represent enhancements in efficacy of culturability, and ease of use, as compared to conventional methodology.
C.Cheng, J.Lin, K.Van, W.Lin, PRL-SW, FDA, Los Angeles, CA
Salmonella is one of the most important food and water-borne pathogen in the United States. The illness associated with this pathogen is acute gastrointestinal infection. The infective dose can be as low as 15-20 cells. A PCR method has been developed with custom designed primers to amplify a 420-bp fragment of Salmonella-specific invA gene. The system has been tested with 171samples of various fresh produces including cantaloupe, lettuce, sprouts, veggie packs, mixed fruits and salad bar entrees. The results showed that the method had high sensitivity ranging from 0.1 to 0.6 CFU/g. This newly developed PCR method is much more rapid, sensitive, and specific for detection of Salmonella spp. than the traditional BAM method and the AOAC-approved VIDAS method.
N.Sergeev1, V.Loparev2, A.Gonzalez2, K.M.Chumakov3, V.Chizhikov3, 1CFSAN, FDA, College Park, MD, 20740, 2CDC, Atlanta, GA 30333, 3CBER, FDA, Rockville, MD 20895
Rapid and sensitive microarray-based method for identification of three main genotypes of human herpesvirus 3 (Varicella-Zoster virus, VZV) has been developed. Sequence analysis of the open reading frame 22 (ORF 22) revealed five variable positions containing point mutations specific to each of the VSV genotypes: European (E), Japanese (J), and Mosaic (M). To discriminate these genotypes, we have developed a microchip that contained pairs of short oligonucleotide probes (oligoprobes) with sequences corresponding to all observed mutations. Evaluation of the CPM assay was conducted by using multiple VZV isolates obtained from different areas of the world. Fluorescently labeled ssDNA samples for microchip hybridization were prepared by PCR amplification of the target region of the ORF22 (400 bp), followed by primer extension of the PCR product in the presence of one primer and chemical labeling of ssDNA with cyanine Cy5. Ratios between hybridization signals from each pair of oligoprobes were used to identify sequences in variable positions. The results of this study showed that the method unambiguously identified each of the three VZV genotypes and can be a valuable tool for molecular epidemiology of VZV infection, post-marketing surveillance of the VZV vaccine and detection of adverse events associated with vaccination.
D.Volokov1, A.Rasooly1, K.M.Chumakov2, V.Chzhikov2, 1Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland 20740-3835, 2Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20895
Detection and identification of food-borne pathogens is an important food safety issue. In this communication we present data demonstrating utility of DNA microarrays, which were developed in our laboratory for other human pathogens, for reliable detection and discrimination of six Listeria species: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi, and four Campylobacter species: C. jejuni, C. coli, C. lari, and C. upsaliensis. Some of these bacteria are known as major food-borne pathogens of humans and animals. The approach used in this study involves PCR amplification of several target bacterial genes, synthesis of fluorescently labeled single-stranded DNA probes followed by their hybridization with multiple individual oligonucleotide probes immobilized on a glass surface and specific for each Listeria or Campylobacter species. Results of the microarray analysis of reference and clinical isolates of Listeria spp. and Campylobacter spp. demonstrated that this method resulted in unambiguous identification of all species based on sequence differences in the target genes. Our data illustrate the great potential of microarray approach for identification and characterization of bacterial pathogens in general.
N.Sergeev1, D.Volokhov2, K.M.Chumakov3, V.Chizhikov3, A.Rasooly2, 1Oak Ridge Institute for Science and Education, Oak Ridge, TN, 2CFSAN, FDA , College Park, MD, 3CBER, FDA, Rockville, MD
Staphylococcal enterotoxins (SEs) are a family of sixteen major serological types of heat stable enterotoxins that are a leading cause of gastroenteritis resulting from consumption of contaminated food. Serological typing of enterotoxins is a complex and time-consuming. We have developed a rapid and reliable one-tube assay for simultaneous detection and identification (genetic typing) of multiple SE genes in the bacterial genome. The SE genetic typing is based on PCR amplification of target region of the enterotoxin genes using only one pair of degenerate primers for all SEs followed by characterization of the PCR products by hybridization with a microchip containing specific oligonucleotide probes for each SE. Fluorescently labeled ssDNA probes for microarray assays were synthesized by the primer extension followed by chemical labeling with cyanine dyes. The use of degenerate primers allows the simultaneous amplification of as many as eleven different SE genes in one strain. The microarray assay was evaluated by analysis of bacterial DNA samples containing 15 previously characterized SE genes. The analysis of S. aureus clinical strains revealed several different combinations of SE genes, including one strain with genes for eleven enterotoxins. The results of this study demonstrated that the multi-toxigenic strains may be common for S. aureus, and that food poisoning may result from several enterotoxins expressed simultaneously in a single pathogenic S. aureus strain.
A.Debrabant1, N.Lee1, S.Bertholet2, R.Duncan1, H.L.Nakhasi1, 1DETTD, OBRR, CBER, FDA, Bethesda MD, 2LPD, NIAID, NIH, Bethesda MD
The protozoan parasite Leishmania donovani is the causative agent of human visceral leishmaniasis that is responsible for hundred of thousands of death per year worldwide. In addition, U.S. army and travelers to endemic areas are at risk for leishmaniasis and deferred for blood donations. However, there is no effective vaccine against Leishmania to date and parasites show resistance to current anti-leishmanial drugs. In multicellular organisms, cellular growth and development can be controlled by programmed cell death (PCD), which is defined by a sequence of regulated events. We report that a PCD pathway is initiated in stationary phase cultures of L. donovani parasites as well as upon treatment with anti-Leishmanial drugs (Pentostam and amphotericin B). The features of PCD in L. donovani are nuclear condensation, nicked DNA in the nucleus, DNA ladder formation, increase in plasma membrane permeability, decrease in the mitochondrial membrane potential, and induction of a PhiPhiLux (PPL)-cleavage activity (a caspase-like activity). Taken together, the existence of a PCD pathway in Leishmania suggests that the control of parasite growth plays an important role in L. donovani pathogenesis. Further, the molecules involved in this pathway could represent new targets for the development of novel drugs against human Leishmaniasis.
V.Dheenadhayalan, G.Delogu, T.Pickett, M.Brennan, CBER, FDA, Bethesda, MD 20892
Investigation of the highly homologous polymorphic PE multigene family, which account for ~5-10% of the genome of Mycobacterium tuberculosis (Mtb) remains an area of great interest. The PE_PGRS subfamily of genes (~65), which contain numerous Gly-Ala rich repeats have been implicated in antigenic variation. One of our research aim is to determine if any variation occurs in PE_PGRS gene expression in Mtb persisting within host tissues. Mice were infected with a low-dose aerosol of Mtb Erdman strain and sacrificed at different time points. Using polyclonal antisera produced by immunizing mice with a PE_PGRS DNA vaccine, we observed a specific and similar PE_PGRS banding pattern in immunoblot containing cell lysates from Mtb cultured from mice tissues 24 h after infection or Mtb grown in vitro. A major ~42kDa PE_PGRS band observed in the Mtb challenge strain was absent in Mtb isolated from the lungs and spleens of 3 mice 1-year after aerosol challenge. Molecular studies employing PCR and RFLP suggest that genetic alterations might be responsible for this apparent loss of PE_PGRS proteins in persistent Mtb. Further analysis are ongoing to identify if this loss of reactivity is due to specific gene alterations in PE_PGRS genes or to down-regulation of gene expression.
M.Peredelchuk1, R.Arena1, D.Volokhov2, V.Chizhikov2, G.Kaplan1, H.L.Nakhasi1, R.Duncan1, 1Division of Emerging and Transfusion Transmitted Diseases, CBER, 2Division of Viral Products, CBER
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 solution to this challenge.
PCR primer pairs were designed to amplify specific target sequences (amplicons) in parasites Trypanosoma cruzi, T. brucei, and Leishmania, as well as viruses HIV-1, HBV, HCV, and Vaccinia. Oligonucleotide probes designed to anneal specifically with target amplicons were printed on glass slides in a microarray. Amplification was tested with cultured pathogens spiked into human blood. Primer extension thermocycling in the presence of fluorescent nucleotide analogues directly labeled one strand of the amplicon, that was complementary to the probe on the microarray. Some pathogens have been detected by hybridization to prototype microarrays after multiplex PCR reactions to a level of sensitivity as low as 500 cells(or particles)/ml, though greater sensitivity can be achieved with this assay targeting one pathogen at a time.
S.S.Bhattacharya, M.Kulka, K.A.Lampel, T.A.Cebula, B.B.Goswami, OARSA, FDA, Laurel, MD
Hepatitis A virus (HAV) is a major cause of infectious hepatitis world-wide. Detection of infectious HAV in contaminated food or water has been a priority research area at CFSAN. Our laboratory has previously reported the development of RT-PCR based detection and typing methods for HAV in contaminated shellfish and produce. It is commonly held that RT-PCR can detect viral genome, however, this method cannot distinguish between infectious and inactivated virus. Therefore, signals obtained after PCR would be considered as false positive unless data on infectivity are available. We present data that show that this general assumption is not valid. Evidence is provided that demonstrate that signals generated after RT-PCR amplification of viral genome correlated well with the presence of infectious virus in the sample. Viral samples inactivated by heat or UV treatment produced significantly lower signal strength that paralleled infectivity of the sample in cultured cells. The loss of signal strength is most likely the result of damage to the viral RNA that renders it unsuitable for RT-PCR. The correlation between PCR signal and infectivity was better following UV inactivation than heat treatment. The procedure may be adapted to other viruses and inactivating agents.
Board G-09 Improved Enrichment for Enterohemorrhagic Escherichia coli (EHEC) by Exposure to Extremely Acidic Conditions
M.A.Grant, ORA, Pacific Regional Laboratory Northwest, FDA, Bothell, WA 98021
The current FDA procedure for isolation of E. coli O157:H7 utilizes E. coli Enrichment Broth (EEB) containing bile salts, cefsulodin, vancomycin and cefixime as selective agents. Food samples enriched in this medium are then streaked onto TCSMAC agar. Detection of possible EHEC colonies at the end of this process is often complicated by heavy growth of non-target microorganisms. A new procedure was developed for initial enrichment of EHEC by exposure to pH 2.00 for 2 hours at room temperature (20 - 22º C). When this method was compared to the current FDA method, 10 cultures of EHEC yielded larger populations via the acidification method by factors ranging from 1.6 to 4.4. Similarly, when the same cultures were age-stressed, larger populations were produced via the acidification method by factors ranging from 2.6 to 8.7. This improvement was seen both for O157:H7 strains and 4 other serotypes including O26 and O111. Additionally, experiments with 9 other species of enterics indicated they were more effectively inhibited by the acid enrichment procedure than the FDA method. Both pathogenic and non-pathogenic strains of E. coli were able to withstand exposure to pH 2.00 for as much as 5 hours. This ability to survive extreme acid shock was the basis for the improved enrichment procedure.
S.Gummalla1, P.R.Krause2, K.Merritt3, V.M.Hitchins3, D.J.Kopecko2, A.R.Mtungwa3, M.M.Wekell4, R.Bhiladvala5, H.G.Craighead5, R.H.Hall6, 1CFSAN, FDA, Laurel MD, 2CBER, FDA, Bethesda MD, 3CDRH, FDA, Rockville MD, 4ORA, FDA, Jamaica NY, 5Cornell University, Ithaca NY, 6NIAID, NIH, Bethesda NY
This study investigated the development of nanoelectromechanical cantilever (NEMC)-based attogram bio-detectors. Nanometer scale cantilevers are like mini diving boards and can be fabricated so that they naturally resonate in air (0.5-5 MHz). This frequency decreases in direct proportion to the mass attached to the cantilever, thereby forming a hypersensitive mass-sensing platform. Preliminary work included microbiology feasibility tests, confirmatory detection, and bio-specificity of cantilever surfaces. Results showed that silicon nitride (SiN) surface of cantilevers can be derivatized with anti-bacterial and anti-virus antibodies so as to serve as platforms for immunospecific target binding. PCR methods developed for detection of Escherichia coli O157 (EHEC) showed artificially inoculated EHEC in Apple juice and Beef extract, could be isolated by silicon nitride chips coated with antibody for these organisms. Bacillus thuringiensis spores targeted via anti-B. anthracis antibody were able to be microbiologically re-cultured confirming spores were immunospecifically bound to SiN surface. PCR and scanning electron microscopy showed Adenovirus (Adv) particles were bound to the SiN surface of chips via anti-AdV antibody. Poly vinyl pyrollidone (10%) was found to serve as a good blocking agent and enhanced the biospecificity of target attachment. Finally, we have developed a model for biofilm formation by showing that SiN is a suitable substrate for colonization of Staphylococcus epidermis. Further experiments are planned using already developed prototype pathogen detection chips with cantilevers for bacterial and spore detection.
Board G-12 Transfusion Related Fatalities from Bacterial Contamination of Blood Components
L.E.Simmons, MT(ASCP)1, M.A.Knippen2, L.G.Holness, M.D.3, 1OCBQ, CBER, FDA, Rockville, MD, 2OCBQ, CBER, FDA, Rockville, MD, 3OBRR, CBER, FDA, Rockville, MD
Introduction: Bacterial contamination of blood and blood components occurs infrequently but when contamination is present, the affected product has the potential to cause a septic reaction in the recipient when transfused. Transfusion associated sepsis may cause serious sequelae in the recipient which if not recognized and promptly treated can be fatal. In those fatalities where both the recipient and the implicated blood product are cultured, the same strain of the contaminant has often been isolated from both cultures.
Methods: CBER receives and monitors reports of transfusion-related fatalities and other serious outcomes reported by blood collection and transfusion facilities.
Findings: Transfusion related fatality reports submitted to us over the past 4 years [1999-2002] indicate an unexpectedly large number of deaths were attributed to bacterial contamination/sepsis. The fatality rate for transfusion related bacterial sepsis during this interval averaged more than 15%.
Conclusion: Because of the compromised state of health among blood product recipients and the potentially fatal outcomes, reducing the incidence of bacterial contamination in blood products is a critical concern for blood centers and hospital blood banks. Strategies are needed to determine how to best reduce the likelihood that blood products may become contaminated and information/recommendations provided to the industry.
K.J.Ishii1, F.Takeshita1, I.Gursel1, M.Gursel1, J.Conover1, A.Nussenzweig2, D.M.Klinman1, 1CBER, FDA, Bethesda, MD, 2NCI, NIH, Bethesda, MD
Unmethylated CpG motifs present in bacterial DNA stimulate a strong innate immune response. It was recently reported that toll like receptor 9 (TLR9)is the receptor of the CpG motifs. Independently, there is evidence that DNA dependent protein kinase (DNA-PK) mediates CpG signaling. Specifically, wortmannin (an inhibitor of phosphatidylinositol 3 (PI3) kinases including DNA-PK) interferes with CpG dependent cell activation, and DNA-PK KO mice fail to respond to CpG stimulation. Current studies confirm that human TLR9 confers the recognition of human CpG motifs and consequent cellular activations, and establish that wortmannin actually inhibits the uptake and co-localization of CpG DNA with Toll-like receptor 9 (TLR9) in endocytic vesicles, thereby preventing CpG induced activation of the NF-kB signaling cascade. We find that DNA-PK is not involved in this process, since 3 strains of DNA-PK KO mice responded normally to CpG DNA. These results support a model in which CpG signaling is mediated through TLR9 but not DNA-PK, and suggest that wortmannin sensitive member(s) of the PI3 kinase family play a critical role in shuttling CpG DNA to TLR9.
K.V.Mohan1, S.Kulkarni1, R.I.Glass2, Z.S.Bai3, C.D.Atreya1, 1OVRR, CBER, FDA, Rockville MD, 2CDC, Atlanta GA, 3LIBP, Lanzhou, China
A lamb strain of rotavirus has recently been licensed for use in China as a live vaccine to prevent rotavirus diarrhea in children. As rotavirus NSP4, especially the cytotoxic domain alone is considered to be associated with diarrhea, we sequenced gene segment 10, which encodes NSP4, of lamb rotavirus. Our comparative nucleotide sequence analysis suggests its close identity (91.17% homology) with that of group-A equine rotavirus (strain HI23). Multiple alignment of the deduced amino acid sequence of lamb NSP4 with that of other group A rotaviruses demonstrated homology ranging from 63.42% with that of porcine YM strain to 93.71% with equine HI23 strain of rotavirus. Phylogenetic analysis of the lamb rotavirus gene with 60 other NSP4 gene sequences of human and animal rotavirus strains, demonstrated that the lamb rotavirus strain belongs to genotype A. Comparative analysis also revealed that although it is a vaccine strain, the NSP4 cytotoxic domain of lamb strain demonstrated an overall amino acid conservation similar to that of other strains, whose NSP4 alone causes diarrhea in animal models. These results taken together with our previous observations clearly reaffirm the idea that the attenuation phenotype of rotaviruses does not involve NSP4 cytotoxic domain, perhaps due to the suppression of NSP4 cytotoxic activity by other rotaviral proteins.
S.Rubin1, G.Amexis1, M.Pletnikov2, K.M.Chumakov1, K.Carbone1, 1CBER, FDA, Rockcille, MD, 2Johns Hopkins University, Baltimore, MD
Mumps virus is highly neurotropic, and prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Mumps virus-associated CNS complications in vaccinees continue to be reported; outside the U.S., some of these complications have been attributed to vaccination with insufficiently attenuated neurovirulent vaccine strains. To examine the genetic basis for mumps virus neurovirulence we recently developed an in vivo mumps virus neurovirulence safety test in which the neurovirulence potential of two mumps virus strains and their neuroadapted and neuroattenuated variants were assessed. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the two mumps virus strains and their related variants with variable neurovirulence phenotypes were fully sequenced. A comparison at the nucleotide level associated three predicted amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain (nucleoprotein Phe Pro 468, matrix protein Val Ala 85 and polymerase Glu Asp 1165) and associated three predicted amino acid changes with neuroattenuation of the attenuated wild type strain (fusion protein Pro Thr 91, hemagglutinin-neuraminidase protein Ser Asn 466 and polymerase Ile Val 736). Neuroattenuation was also associated with two nucleotide substitutions in the 3’ nontranslated region. The potential role of these nucleotide and amino acid changes in neurotropism, neurovirulence and neuroattenuation is presented.
J.L.Skapik1, M.Pletnikov1, S.Rubin2, T.H.Moran2, K.Carbone2, 1FDA/ Johns Hopkins
Influenza virus has long been implicated in neurological disorders. To examine neurobiological changes due to influenza, we created a rat animal model using two inbred strains with differing environmental susceptibilities. Newborn Fisher344 and Lewis rats were inoculated intracranially with influenza (A/WSN/33). Viral antigens were detected in meninges, the subventricular zone, and the external germinal layer of the cerebellum. Infectious virus could be detected through post-natal day (PND) 6. Behavioral testing was performed on PND 30 and 60. Lewis rats showed elevated locomotor activity at PND 30, which was absent by PND 60. In contrast, Fisher344 showed marked hyperactivity at PND 30 that persisted at PND 60 as well as impaired habituation in acoustic startle tests at PND 60. No gross histological differences were seen in brains of infected Lewis rats, but moderate hydrocephalus was present in brains of infected Fisher344 rats. These preliminary findings indicate that the rat animal model provides evidence that transient neonatal infections pose real risks for neurodevelopmental damage.
D.M.Williams-HillFDA PRL-SW
Salmonella is a leading cause of foodborne illness around the world. FDA field laboratories utilize techniques outlined in the on-line Bacteriological Analytical Manual (BAM) to conduct surveillance of Salmonella and other foodborne pathogens. For seafood and other high flora food matrices, two types of enrichment media re used:
Tetrathionate (TT) and Rappaport-Vassiliadis (RV) Broth. The data presented here is a retrospective study of the distribution of Salmonella O-serotypes isolated from TT and RV media. Over 200 high-flora samples, primarily seafoods, were analyzed and the following parameters were quantitated for each sample: 1) the number of times the same serotypes were detected by RV and TT; 2) the number of times different serotypes were isolated in RV and TT; and 3) the number of times a serotype was detected in only one or other of the media. While the sample number is relatively small, the data indicate that the two media detect the same serotypes in approximately 70% of the samples. Approximately 18% of the samples are isolated from RV only while in 14% of the samples RV and TT each detect a different serotype. These data indicate the importance of using at least two media to enrich for Salmonella isolates. Further analysis of samples may also indicate whether specific serotypes are preferentially isolated in these media.
S.Zhao1, S.Qaiyumi1, S.Friedman1, R.Singh1, S.L.Foley1, D.G.White1, P.F.McDermott1, T.Donkar2, C.Bolin3, S.Munro4, E.J.Baron4, R.D.Walker1, 1Division of Animal and Food Microbiology, Office of Research, CVM/ FDA, Laurel, MD 20708, 2Division of Microbiological Studies, CFSAN/FDA, College Park, MD 20740, 3Animal Health Diagnostic Laboratory, Michigan State University, Elansing, MI 48824, 4Stanford University Medical Center, Stanford, CA 94305
Salmonella enterica Serotype Newport resistant to at least nine antimicrobials, including extended-spectrum cephalosporins, known as Newport-MDRAmpC, has been rapidly emerging in both animals and humans throughout the United States. In this study, 87 S. Newport isolated from human and food animals were characterized using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing, and examined for the presence of class-1 integron and blaCMY gene. Thirty-five PFGE patterns with XbaI were observed, three of which were indistinguishable among isolates from humans and animals. Fourteen antibiogram profiles were generated when tested with16 antibiotics. Fifty-three (60%) S. Newport isolates were identified as Newport-MDRAmpC, including 27 (27/29, 93%) from cattle, 7 (7/10, 70%) from swine, 3 (3/10, 30%) from chicken and 16 (16/30, 53%) from humans. None of the turkey isolates were Newport-MDRAmpC. However, 27 (31%) S. Newport isolates were susceptible to all 16 antibiotics. A blaCMY gene was present in all Newport-MDRAmpC isolates. Antibiotic resistance class 1 integron was common in S. Newport isolates. Plasmid-mediated blaCMY and integron were transferable via conjugation to the strain of E. coli. In conclusion, S. Newport-MDRAmpC was commonly identified among S. Newport isolates recovered from food animals. As with other Salmonella serotypes, there exists the possibility that this organism can be transmitted to humans through the food chain.
D.Volokhov1, V.Chizhikov2, K.M.Chumakov2, A.Rasooly1, 1CFSAN, FDA, College Park, MD, 2CBER, FDA, Rockville, MD
Over the past few decades, multiple antibiotics resistant pathogenic bacteria have emerged worldwide. Effective use of antibiotics often depends on the ability to determine the pathogen’s antibiotic resistance profile. We developed an oligonucleotide microarray that allows simultaneous detection and identification of six genes involved in microbial MLS (macrolide-lincosamide-streptogramin B) antibiotic resistance. The microarray was composed of oligonucleotide probes representing the sequences conserved among MLS resistance genes: ermA, ermB, ermC, ereA, ereB, and msrA/B. Each gene was represented on the microchip by seven individual oligonucleotides. The designed microarray was evaluated using six reference and clinical strains of S. aureus, S. pyrogenes and E. coli. Target regions from the DNA samples were amplified and simultaneously fluorescently labeled using one-tube multiplex PCR. The results of this study showed that the presence of MLS resistance genes in bacterial genomes could be unambiguously detected using this microarray assay. The ermC gene was found in all eleven clinical isolates tested, whereas the ermA and msrA/B genes were found in seven (64%) and five (45%) of the clinical isolates, respectively. Noteworthy, eight (72%) of the 11 strains contained two or three MLS resistance genes, but only three types of the gene combinations (ermA with ermC, ermC with msrA/B, and ermA with ermC and msrA/B) were observed. Our results suggest that microarray-based detection might be a powerful tool for profiling of antibiotic resistance genetic determinants in a wide range of bacterial strains.
H.Golding1, L.R.King1, J.Manischewitz1, J.Andersen2, J.Aliberti2, A.Sher2, 1DVP, CBER, FDA, 2NIAID, NIH
The activation of murine dendritic cells by Toxoplasma gondii has recently been shown to depend on a parasite protein that signals through the chemokine receptor CCR5. Here we demonstrate that this molecule, cyclophilin-18 (C-18) is a potent antagonist of HIV-1 fusion and infectivity. T.gondii C-18 efficiently blocked syncytia formation between human T cells and effector cells expressing R5 but not X4 envelopes while human or Plasmodium falciparum cyclophilins lacked this inhibitory activity. Importantly, C-18 protected peripheral blood leukocytes from infection with multiple HIV-1 R5 primary isolates from several clades. Cyclosporin A (CsA), a major ligand bound by cyclophilins, significantly reduced the ability of C-18 to inhibit both R5 mediated fusion and infection with R5 viruses. C-18 bound directly to human CCR5 and this interaction was partially competed by the b-chemokine MIP-1b and by HIV R5 gp120. In contrast to several other antagonists of HIV coreceptor function, C-18 mediated inhibition did not induce b-chemokines or cause CCR5-downmodulation, suggesting direct blocking of envelope binding to the receptor. These data support the further development of C-18 derivatives as HIV-1 inhibitors for blocking HIV-1 transmission and for post-exposure prophylaxis.
C.P.Giri1, R.B.Raybourne2, R.Thomas2, J.McDaniel3, D.J.Kopecko3, 1CFSAN, FDA, College Park, MD, 2CFSAN, FDA, Beltsville, MD, 3CBER, FDA, Bethesda, MD
Multi-antibiotic resistant S. typhimurium DT104 strains have recently gained prominence in food borne infections, raising the possibility of novel DT104 virulence genes. Our aim was to identify DT104 genes specifically induced intracellularly following infection into INT407 cells. Sau3A1-restricted fragments representing the DT104 chromosome were ligated into a promoter-trap, bacterial expression vector at a BamH1 cloning site upstream from GFPmut3.1/CAT reporter genes. Recombinant plasmids were electroporated into a DT104 strain and the resultant bacteria were used to infect INT407 cells. Following a 1-3 hr invasion, extracellular bacteria were killed by gentamicin and those intracellular bacteria expressing the CAT gene were selected in chloramphenicol (Cm). CmR, GFP+bacterial clones that were induced intracellularly were additionally enriched by FACS. To eliminate constitutive promoters, these GFP+ bacteria were subsequently grown in broth and resulting GFP- bacteria were sorted to enrich again only for intracellularly-activated promoter clones. All of 150 clones picked from the enriched library were found to express the reporter genes intracellularly at high levels, but not during growth in broth. Subsequent genetic analyses have revealed at least 6 different intracellularly-induced promoters. This double-selection, promoter-trap system has proven to be a powerful tool for identifying intracellularly-induced genes, which should lead to the identification of novel virulence attributes.
S.C.Sahu1, D.Gaines1, M.Wiedman2, K.G.Jinneman3, R.B.Raybourne1, 1FDA, CFSAN, Laurel MD, 2Cornell University, Ithaca NY, 3FDA, ORA, Seattle WA
The ability to quantify differences and patterns of virulence among isolates of Listeria monocytogenes (LM) would be of great value for quantitative risk assessment. Two collections of LM strains were studied, both of which had been characterized with respect to evolutionary group. These three recognized groups are characterized by nucleotide sequence differences in several virulence genes. One collection was composed exclusively of food isolates with no known history of human or animal infection. The other collection was composed mainly of strains derived from human and animal infections. Strains from each of the 3 evolutionary groups were tested for their ability to invade and replicate within monolayers of mouse hepatocyte cells, TIB73. Values for invasion were determined after 1 hr of invasion and replication was measured at 24 hr. An internal standard LM strain, EGD, was included in every assay and was used to normalize invasion and replication values in order to compensate for day-to-day variation in the assay. In an initial screening of 40 LM isolates from both collections, isolates from evolutionary group 3 exhibited less replication in hepatocytes than isolates from groups 1, with group 2 intermediary. This suggests that the three evolutionary groups may have inherently different virulence potentials.
P.Cullen1, S.D.McDermott1, P.J.Carter1, J.C.Paige2, D.D.Wagner1, 1CVM, FDA, Laurel MD, 2CVM, FDA, Rockville MD
Identification of environmental reservoirs of antibiotic resistance is critical to assessing human and animal health risk associated with drug use and in designing potential intervention and mitigation strategies to limit its spread. This survey is part of a continuing effort by FDA to better understand the role animal feed may play in the environmental persistence of antibiotic resistance and its dissemination. In this survey, one hundred and twenty-two samples of rendered products [meat and bone meal (72), blood meal (16), bone meal (2), feather meal (10), poultry meal (17), or fish meal (5)] were cultured for Enterococcus. Eighty-four percent (103/122) of the samples were positive for Enterococcus spp. with E. faecium comprising the highest percentage (86.5%) followed by E. faecalis (7.5%), E. gallinarum (2.0%), E. hirae (1.5%), and E. avium (0.5%). Four isolates were unidentified (2.0%). A total of 200 enterococci were recovered and tested for susceptibility to 17 antimicrobials. Resistance to erythromycin (4.5%), penicillin (2%), tetracycline (18.5%), ciprofloxacin (8%), and streptomycin (2.5%) was present in the isolates. There was no resistance detected to the streptogramin, quinupristin/dalfopristin (Synercid™) in any of the non-faecalis species. All isolates tested were also sensitive to vancomycin and linezolid. The isolates of E.gallinarum exhibited intermediate susceptibility to vancomycin (MICs up to 16ug/ml). Despite the low frequency of resistance found in this study, the high prevalence of enterococci distributed over a variety of rendered products indicates that this resilient organism may survive the rendering process and possibly contribute to the dispersion of resistance determinants in the food animal production environment.
C.M.Lathers, HFV-1, CVM, FDA, Rockville, MD
Veterinary medicine issues of food safety, transgenic animals, and antimicrobial resistance offer opportunities to develop new animal drugs. The industry can help by focusing research and development on new drugs for both humans and animals, including food animals, exotics, and minor species of fish, sheep, goats, birds, and bees. New companion animal drugs, pioneer and generics, are needed to treat diabetes, cancer, high blood pressure, renal failure, and arrhythmias. New central nervous system drugs are needed to alleviate pain, e.g. arthritis, induce sedation or anesthesia, prevent motion sickness, reduce stress and prevent death, or control seizures. Biotechnology offers opportunities for new animal drugs. Transgenic animals, e.g. Atlantic salmon, will be regulated as “new drugs.” All animal applications must address the environmental impact. Antimicrobials with novel actions to target mechanisms of resistance development are needed. Nanoscience and terahertz technology offer new pathogens biosensors. Cross over of drugs developed for humans using animal studies to establish PK/PD, safety and efficacy offer opportunities to develop new animal drugs. Review of these studies will determine which drugs are safe and efficacious for use in animals, whether or not approved for human use. Opinions are the author’s and do not represent policy of the FDA.
C.M.Lathers1, R.M.Levin2, H.L.DuPont3, P.C.Okhuysen3, Z.D.Jiang3, 1HFV-1, FDA, Rockville, MD, 2Albany College of Pharmacy, Albany, NY, 3Uni Texas, Houston, TX
Ongoing discussions focus on regulatory science and policy requirements to address the human and animal public health problem of antibiotic resistance. This study will develop a rabbit bladder model to study bacterial infections and resistance relevant to food-borne diseases. The model will compare virulence of resistant bacteria vs. the original strain, identify cross-sensitivity of resistant bacteria to specific classes of antibiotics, and predict virulence and susceptibility of resistant strains to antibiotics. Effects of ciprofloxacin and gatifloxacin on Escherichia coli, involved in food borne diseases, will be studied. The model will provide strains of resistant bacteria for pharmacological and molecular studies to devise genetic markers to predict sensitivity to antibiotics. The model allows study of: kinetics of bacterial growth and emergence resistance; cross-reactivity in a physiologically relevant model; mucosal defense mechanisms in bladders infected with resistant and non-resistant strains of bacteria; and measurement of urine toxins. Model advantages include: invasive methods to establish chronic infections are not needed; bacteria can be accessed via non-invasive techniques for linear studies; there are no endogenous flora to interfere; and toxins produced by bacteria will be excreted in the urine and limits toxic systemic effects. Opinions are the authors and do not reflect FDA policy.
R.M.Levin1, C.M.Lathers2, H.L.DuPont3, P.C.Okhuysen3, Z.D.Jiang3, 1Albany College of Pharmacy, Albany, NY, 2HFV-1, FDA, Rockville, MD, 3Uni Texas, Houston, TX
Ovariectomy in rabbits results in a marked thinning of the mucosal lining of the urinary bladder, increased bacterial adherence to the mucosa, and significantly increased mucosal permeability. These same changes occur in women post menopause, resulting in a significant incidence of recurrent and chronic infection. It is proposed that ovariectomy followed by acute overdistension of the rabbit bladder will induce a chronic infection and treatment with sub-therapeutic concentrations of antibiotics will cause emergence of antibiotic resistant organisms for utilization as an investigation model. Aspects of chronic infections and antibiotic resistance that can be studied include: a. Alterations in virulence of organisms associated with both chronic infection and resistance. b. Changes in phenotype associated with development of resistance. c. Changes in phenotype associated with development of chronic infection compared to acute infection. d. Development of cross-resistance and cross-sensitivity of the strain to specific classes of antibiotics. e. Rate of loss of antibiotic resistance once therapy is terminated. This model will provide data about bacterial membrane interactions in the presence and absence of resistance and will add to the data base evaluated when developing new antimicrobial agents. Opinions are the authors and do not reflect of the FDA.
S.Simjee1, D.G.White1, P.F.McDermott1, D.D.Wagner1, M.J.Zervos2, S.M.Donabedian2, L.L.English1, R.D.Walker1, 1U.S. Food and Drug Administration, Center for Veterinary Medicine, 8401 Muirkirk Road, Laurel, MD 20708, USA, 2William Beaumont Hospital, Royal Oak, MI 48073
The incidence of Gram-positive pathogens resistant to antimicrobial agents continues to be problematic due to their ability to acquire and transfer antibiotic resistance genes. As observed in human medicine, reports of antimicrobial resistant bacteria emerging in animal populations are increasing in frequency. Several studies have suggested that animal enterococci may be a potential reservoir of resistance genes, but has yet to be definitively proven. However, the transfer of resistance genes from humans to animals has never been reported in the USA. Over a two-year period (1996-1998), Thirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital over a two-year period (1996-1998). Isolated species included E. faecium (n=13), E. faecalis (n=7), E. gallinarum (n=11) and E. casseliflavus (n=4). Antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibiting resistance to 3 or more antibiotics. One E. faecium isolate, CVM1869, displayed high-level resistance to vancomycin (MIC>32 mg/ml) and gentamicin (MIC>2048 mg/m). Molecular analysis of this isolate revealed the presence of Tn1546 (vanA), responsible for high-level vancomycin resistance and Tn5281 carrying aac6'-aph2", conferring high-level aminoglycoside resistance. PFGE analysis revealed that CVM1869 was a canine E. faecium clone that had acquired Tn1546, perhaps from a human VREF. Tn5281 and Tn1546 transposons were located on two different conjugative plasmids. Sequence analysis revealed that in Tn1546, ORF1 had a 889 bp deletion and an IS1216V insertion at the 5’ end, and an IS1251 insertion between vanS and vanH. To date, this particular form of Tn1546 has only been described in human clinical VRE isolates unique to the USA. Additionally, this is the first report of a VREF isolated from a companion animal in the USA.
S.Simjee1, D.G.White1, P.J.Carter1, M.J.Zervos2, S.M.Donabedian2, P.F.McDermott1, 1U.S. Food and Drug Administration, Center for Veterinary Medicine, 8401 Muirkirk Road, Laurel, MD 20708, USA, 2William Beaumont Hospital, Royal Oak, MI 48073
In the last two decades enterococcal species have emerged as a leading cause of hospital-acquired infections. The major factor to contribute to this surge is the ability of enterococci to acquire and disseminate antibiotic resistance genes. Of greater concern is the fact that enterococci present in food animals may either colonize humans, leading to infections, or transfer their antibiotic resistance genes to human intestinal enterococci. Until recently little attention has been devoted to the study of enterococcal virulence factors which clearly play an important role in pathogenesis. In the present study we report on the prevalence of 8 enterococcal virulence genes (gelE, cylM, cylB, cylM, cpd, efaAfm, cob and esp) in streptogramin resistant E. faecium strains isolated from retail poultry (n=27) and humans (n=8) collected throughout the USA. In addition the presence of vatD and vatE was also examined. One human and 12 poultry E. faecium isolates tested positive for vatE while no vatD was detected by PCR. Only esp was detected in 2 human E. faecium isolates no other virulence genes were found. In contrast more virulence genes were found in poultry isolates, gelE (n=14), cylA (n=1), cpd (n=10), efaAfm (n=13). In addition, a single gelE PCR positive E. faecium tested positive for gelatinase activity. This study is the first to identify an E. faecium expressing gelE. In addition, the data presented here suggest that streptogramin resistant E. faecium recovered from retail poultry samples do not share the same virulence genes as streptogramin resistant E. faecium recovered from humans implying different reservoirs of origination.
J.J.Alexander, P.M.Colangelo, C.K.Cooper, R.Roberts, W.J.Rodriguez, M.D.Murphy, CDER, FDA, Rockville MD
BACKGROUND: Amox is not approved by the FDA for use in PEIA, nor was it studied in primate PEIA models. Various recommendations exist regarding the use of amox in PEIA for pediatric (ped) patients (pts) where ciprofloxacin and doxycycline may be contraindicated. The FDA believes it prudent to provide information about the safety and plasma pharmacokinetic (PK) data for amox.
OBJECTIVE: To provide clinicians with useful PK and safety data on amox for making therapeutic decisions.
METHODS: Safety and PK data for amox were reviewed from the literature and from internal data for amoxicillin/clavulanate (A/C). A dosing regimen was derived based on: (1) highest penicillin (PCN) MIC values for strains of B. anthracis reported in the literature (0.5 g/mL) and 11 recent intentional exposure isolates (0.12g/mL), (2) effective peak (2-3.5 g/mL) and trough (0.4-1.1g/mL) concentrations for PCN in non-human primates with PEIA, and (3) Amox time above the PCN MIC for B. anthracis.
RESULTS: PK data from the literature showed rapid plasma elimination with T of 1 to 2hr in ped pts 4 months to 4yr after single oral amox doses of 15 or 25mg/kg. Similar mean concentrations (0.6-0.7g/mL) were noted for both doses at the last reported timepoint (6hr post-dose). These data suggested that a dose of at least 15mg/kg would be adequate for PEIA. The issue of dosing interval for PEIA was addressed via review of internal PK data. Five pts (4mo to 12yr) received 22.5/3.2mg/kg A/C Q12hr for 10 days, and 6 other pts (1mo to 9yr) received 13.3/3.3mg/kg A/C Q8hr for 10 days. Either regimen resulted in peak plasma concentrations above the B. anthracis MIC (Q12hr=12g/mL; Q8hr=7g/mL). With the Q12hr regimen, 4/5 pts had plasma amox concentrations below the lower limit of assay quantitation (<0.2g/mL) at 12 hr post-dose. With the Q8hr regimen, fewer ped pts had plasma amox concentrations <0.2g/mL at the end of the dosing interval (1/6 at 6hr; 3/6 at 8hr). In 11 studies, 500 ped pts were treated for 1-6mo at various doses. Adverse events (AE) were 7 GI, 7 rashes, 2 GU and 1 allergic reaction. FDA’s database had 11 ped pts with AE when given amox for >21 days. No unusual signals were noted.
CONCLUSIONS: At least 45 mg/kg/day of Amox given in 3 divided doses to ped pts <40 kg should yield an adequate time above the MIC for susceptible B. anthracis over most of the dosing interval (75-100%). Doses <45 mg/kg/day and dosing intervals >Q8h should not be used for PEIA. Few AE were noted with long-term use. The AE were similar to those seen with short-term use.
D.Volokhov1, A.Pomerantsev2, S.Leppla2, A.Rasooly1, K.M.Chumakov3, V.Chizhikov3, 1CFSAN, FDA, College Park, MD, 2NIAID, NIH, Bethesda, MD, 3CBER, FDA, Rockville, MD
We have developed a rapid microarray-based assay for reliable identification of Bacillus anthracis and its accurate discrimination from other closely related bacterial species belonging to the Bacillus cereus group. The approach used in this study included PCR amplification of seven B. anthracis-specific genes encoding plasmid-associated virulence factors (cyaA, pagA, lef, and capA, capB, capC) and one chromosomal marker BA-5449, followed by analysis of the amplified DNA by hybridization with the multiple target gene-specific oligonucleotide probes (oligoprobes) immobilized on a glass surface. Evaluation of microchip was conducted using several B. anthracis strains with and without pOX1 and pOX2 plasmids as well as over 40 other Bacillus species including genetically related B. cereus, B. thuringiensis, B. subtillis and B. mycoides. The results showed that this method allowed us to (i) unambiguously identify and discriminate B. anthracis from other Bacillus species and (ii) distinguish between plasmid-containing and plasmid-free Bacillus anthracis strains.
L.E.Rodriguez-Saona1, F.S.Fry2, F.M.Khambaty3, E.M.Calvey4, 1JIFSAN, UM [present address: ARS, USDA, Betlsville, MD], 2OSAS, CFSAN, 3OPDFB, CFSAN, 4OSci, CFSAN, FDA, College Park, MD 20740
Recent advances in Fourier transform near-infrared (FT-NIR) spectroscopic instrumentation and pattern recognition techniques have significant potential for monitoring the presence of microbial pathogens. The complex cellular composition of bacteria yields FT-NIR vibrational transitions that might be useful for identification and sub-typing. NIR absorption spectroscopy allows fast, accurate and non-destructive measurements of chemical components and can provide information about structural and physical properties of materials. Sample protocols were developed for the reliable and reproducible identification and classification of bacterial strains by combining FT-NIR spectroscopy with multivariate methods. Bacteria including strains of Escherichia coli spp., Pseudomonas aeruginosa, Bacillus spp. and Listeria innocua were evaluated. The bacterial cells were either suspended in saline solution or treated with ethanol to reduce the safety concerns when analyzing pathogenic strains, and concentrated on an aluminum oxide membrane to obtain a thin bacterial film. FT-NIR measurements were made by using diffuse reflectance and the spectra were analyzed by Principal Component Analysis (PCA) and Soft Independent Modeling Class Analogy (SIMCA). A simple membrane filtration procedure yielded a thin bacterial film resulting in increased sensitivity and allowed for the rapid discrimination among closely related bacterial strains. PCA and SIMCA of second derivative spectra in the 5100 - 4200 cm-1 region exhibited clusters that discriminated between bacteria species at levels ~1 mg wet cells weight (~ 106 CFU/mg). Factors such as film thickness and stage of growth substantially affected the FT-NIR spectra and diminished the ability of PCA to differentiate among strains; this underscores the importance of developing robust sampling protocols. FT-NIR in conjunction with multivariate techniques can be used for the rapid and accurate evaluation of potential bacterial contamination in liquids with minimal sample manipulation. By generating a library of major food-borne pathogens and the refinement of the models, this approach could become a powerful tool for monitoring the safety of our food supply.
Board I-05 Isolation and Identification of Bacillus anthracis from food
B.D.Tall1, S.Monday1, M.H.Kothary2, D.Hao1, D.E.Hanes2, T.S.Hammack1, D.H.Burr2, 1FDA, College Park, MD, 20740, 2FDA, Laurel, MD, 20708
Recent events have reinforced the importance of FDA’s long-standing interest in the development of improved methods for the identification of potential threat agents from foods. Food, artificially contaminated with B. anthracis spores, was placed in a stomacher bag containing enrichment broth, and homogenized. A 10-ml aliquot of the homogenate was removed and placed at 65oC for 30 min. Aliquots of the heated and unheated homogenates were tested for the presence of spores using PCR. The sensitivity of the PCR assay was <10 spores/gm of food. Aliquots from the heated and unheated samples were plated onto PLET and MYP agar, and the plates and homogenates (as enrichments) were incubated overnight at 37oC. The overnight enrichments were plated onto PLET and MYP agar using a 10-l calibrated loop and a urine streak, and incubated at 37 oC. Colonies characteristic of B. anthracis were visible on MYP after 24 h of incubation, and on PLET after 48 h of incubation. However, B. anthracis was easier to identify on PLET than on MYP. Heating the homogenate and plating by the urine streak method decreased background thus increasing the detection of the pathogen in the food. This is the first of several methods being developed for the identification of threat agents in food.
M.Laassri1, V.Chizhikov1, S.Shchelkunov2, K.M.Chumakov1, 1CBER, FDA, Rockville MD, 2"Vector", Koltsovo Russia
The genus Orthopoxvirus (OPV) of the family Poxviridae contains several viruses potentially pathogenic for humans including Monkeypox, Cowpox, Vaccinia and Variola viruses. The most dangerous of them is Variola virus that causes smallpox. Although the disease was eradicated in 1977, there is a possibility that this virus can be used as a bio-weapon or for terrorist attacks. Therefore development of rapid methods for detection and discrimination of Orthopoxviruses and their differentiation from unrelated chickenpox virus (Varicella-Zoster virus, or VZV) that may produce similar clinical symptoms is an important task. Here we report the use of oligonucleotide microarray hybridization for rapid detection and discrimination of a variety of Orthopoxviruses.
The method is based on PCR amplification of viral genome segment with broadly specific primers and subsequent hybridization of the ssDNA with microarrays of immobilized oligonucleotides specific to individual orthopoxvirus species. The genotype-specific oligoprobes were selected by special custom software from the sequences of the B29R gene available for 44 isolates of Smallpox, Monkeypox, Cowpox, and Vaccinia viruses. Oligonucleotides specific for the VZV ORF-62 were also included to differentiate between OPV and VZV infections. The conditions for hybridization and analysis were optimized and found to be robust and reproducible.
We tested 24 coded samples of Orthopoxvirus DNAs, representing different OPV species and two VZV strains. The oligonucleotide microarray reliably discriminated between OPV species, and clearly differentiated them from chickenpox virus. The results can be obtained within few hours.
A.D.Lucas, V.M.Hitchins, K.Merritt, OST, FDA, Rockville MD
Because anthrax spores were distributed through the mail, decontamination of letters and packages can be expected to be performed. Ethylene oxide (EO) is commonly used to sterilize medical devices and is one method by which the mail may be treated. A major concern is the amount of residual EO and ECH (ethylene chlorohydrin) remaining. Sample preparation for EO and ECH analysis was challenging. For most samples, a small portion of the item was cut, weighed, and placed in a 20 ml vial for extraction. Items composed of more than one material were either prepared with all materials or run separately. For all items, sample preparation was performed with the idea that whatever part of the item people would come into contact most frequently was the area chosen for sampling and analysis. Following EO sterilization, EO and ECH residues were determined for various medical diagnostic kits, paper products, bandages, and other consumer items that may be sent by mail. Of the 69 samples, EO residues were above 250 ppm for 16 samples, 5 samples had detectable ECH residues. The data would suggest that EO sterilization may not be appropriate for some items.
K.Merritt, V.M.Hitchins, A.D.Lucas, CDRH, FDA, Rockville
An act of terrorism distributing anthrax spores through the mail raised the question of the effect(s) of sterilization protocols on items that might be mailed. Autoclaving was clearly not an acceptable method and was not included in this study. Various office items and medical items regulated by CDRH that might be damaged during standard sterilization protocols were examined in this study. All items were over the counter or routine office items. A large number of items were selected and only a few will be described here. Film was damaged by all protocols. Computer discs were not damaged by any of the protocols. ETO sterilization caused damage to some adhesive seals on packages. Gamma and E-beam sterilization caused color changes in many packages and in some fabrics. The color changes made the packages unattractive; however the contents of the packages were not damaged. No damage was apparent to paper, envelopes, or transparent tape. ETO caused damage to some first aid adhesive tapes. Only one type of bandage had changes in its ability to adhere to the skin. The OTC sniff test for Alzheimer’s was not altered by any of the protocols. However, no sterilization protocol was ideal in preventing damage.
Board I-10 Comparative analysis of the detection of vaccinia virus antigens by human polyclonal immune globulin preparations
M.G.Mikolajczyk, N.L.Eller, A.Jones-Trower, M.Kennedy, D.E.Scott, CBER, FDA, Bethesda, MD
With the possible threat of re-emergence of smallpox due to bioterrorist activities, vaccination of selected responders is beginning. Live vaccinia virus is the current vaccine for smallpox, and persons with compromised immune systems, atopic dermatitis and eczema, may be at risk for serious vaccine-related complications. Vaccinia Immune Globulin is recommended by the Advisory Committee on Immunization Practices as first-line treatment for these complications. In the setting of more widespread vaccination, or more adverse events than anticipated, supplies of VIGIV may be diminished. Anti-vaccinia antibodies could exist in the general population that contributes to plasma pools, as a result of persistent responses in remotely vaccinated people. If this were the case, and titers were sufficient, commercial preparations of Immune Globulin Intravenous (IGIV) would be a possible alternate source for the treatment of vaccinia-related complications. We have compared VIGIV and IGIV efficacy in vivo, and have found that some IGIV preparations contain anti-vaccinia antibody activity. We have also comparatively analyzed the reactivity of these antibody preparations, to each other and to serum from vaccinees, by western blot analysis. Briefly, vaccinia virus proteins were electrophoretically separated on a polyacrylamide gel, blotted unto a nitrocellulose membrane, and incubated with VIGIV, IGIV, and sera from immunized individuals. We identified 18 bands ranging in molecular mass from approximately 10- to 120-kDa, and found differences in the recognition of vaccinia antigens by the different immune globulin preparations, demonstrating differences in antibody profile between products from recently vs. remotely vaccinated donors. The clinical significance, if any, and the antigens of interest are the subjects of continued study.
E.Mitrojorgji, T.C.Smith, V.Krauthamer, CDRH, FDA, Rockville, MD, 20852
As part of FDA’s counter-terrorism efforts, studies are being performed to evaluate cell-based biosensors for toxins. These studies yield insight into mechanisms of toxin action, possibly enabling early detection. We initiated studies of excitable heart cells as a basis for biosensor operation, whereby spontaneous or evoked changes reflect physiologic changes associated with toxin exposure. Cardiac myocytes are isolated from chick embryo heart cells and cultured in a shaker bath to form aggregate balls.
Acetylcholine (Ach) is the primary autonomic neurotransmitter that regulates cardiac function. Many chemical warfare and terrorist treats involve substances that alter Ach activity. This makes the heart cell cultures a potentially useful sensor for these agents. Baseline characterization of the cultured myocytes anatomy and electrophysiology was necessary prior to evaluation of toxic effects. Microelectrode and fluorescence measurements were done to obtain resting potential, action potentials and cytosolic calcium levels of cells in both whole heart and cultured aggregates. From these techniques, and intracellular dye injection, we conclude that in vivo cell morphology, electrophysiology and mechanical properties are retained in vitro in the cell cultures. Initial studies of the effects of low concentrations of cardiovascular drugs demonstrated high sensitivity to the drugs. We are currently evaluating cholinergic effects in culture and their detection by optical, mechanical and electrophysiological sensors
J.G.Wilkes1, S.A.McCarthy2, F.Rafii3, R.Nayak3, L.Rushing1, A.Shvartsburg1, 1Chemistry, NCTR, Jefferson, AR, 2CFSAN, Dauphin Island, AL, 3Microbiology, NCTR, Jefferson, AR
The NCTR is building a capability for rapid MS-based characterization of bioterror agents or hoax samples. The instrumentation, personnel, and infrastructure needed for rapid bioterror analysis must be in place before the events occur. Practically, this implies that the methods need to be applicable to more routine food contamination events, such as occasionally occur, for example, in summer seafood harvests or for poultry products. These two problems are typically associated, respectively, with Vibrio and Salmonella strains. We collected a large number of environmental and clinical outbreak-associated non-pathogenic and pathogenic isolates (vibrios, salmonellae, and related genera), and have begun to assemble into a coherent database their mass spectral patterns acquired under standard operating procedures. We used two different MS instrumental platforms: pyrolysis metastable atom bombardment and matrix-assisted laser desorption/ionization, each with time-of-flight mass analysis. Various pattern recognition techniques are being tested that facilitate comparison of unknowns to database spectra. A novel element of this research is use of an algorithmic method for compensation of between-session spectral drift whether due to differences in instrument performance, microbial cell environment, or sample handling.
Z.K.Binienda1, B.T.Thorn2, W.Slikker, Jr.3, A.C.Scallet3, 1FDA, Jefferson, AR, 2ROW, Jefferson, AR, 3FDA, Jefferson, AR
Domoate (DOM) is a potent excitotoxic analogue of glutamate and kainate, present in some seafood (shellfish) as an algae-derived contaminant. DOM intoxication produces neurological symptoms including seizures, hence it is of interest to the FDA. Here, the ECoG was recorded in 15 non-anesthetized, adult, male Sprague-Dawley rats via bipolar, epidural electrodes. Following the 30 min baseline recording period, rats were injected i.p. with either saline, 2.2 or 4.4 mg/kg DOM while behavioral stereotypies and the ECoG was recorded for 2 hours. Rats were then sacrificed for c-fos immunohistochemistry, to visualize brain regions that might have contributed to the ECoG and behavioral alterations. The power spectra obtained by Fast Fourier Transformation analysis, revealed significant increases in delta (1.25-4.50 Hz) and theta (4.75-6.75 Hz) frequency bands. The power increase preceded the onset of behavioral stereotypies by approximately 30 min. Two hours after dosing, c-fos was activated throughout the limbic system in a dose-dependent manner. Data indicate that hippocampal lesions observed after DOM exposure result from limbic seizure-induced glutamate release.
D.R.Cawthon, M.Oetinger, J.T.Skinner, L.C.Schmued, W.Slikker, Jr., S.Z.Imam, S.F.Ali, NCTR, FDA, Jefferson AR
Methamphetamine (METH) is a widely used drug of abuse. In mice, METH administration (4x10 mg/kg at 2 h intervals) resulted in increased striatal levels of p53 protein and decreased levels of bcl-2 protein, which are key elements in activating the mitochondrial cell death pathway. Western blot analysis also revealed increases in the apoptotic effector protein caspase-3 in mouse striatum. A general caspase inhibitor (Z-DEVD-FMK) failed to alter the levels of these proteins following METH dosing, whereas a specific caspase-3 inhibitor (Ac-DMQD-CHO) significantly protected against METH-induced protein changes in the mouse striatum. Our data demonstrate that METH-induced dopaminergic neurotoxicity alters protein levels, which can lead to the onset of programmed cell death; however, it can be protected by specifically targeting the downstream effector action of Caspase-3.
T.F.Collins1, R.L.Sprando1, T.N.Black1, N.Olejnik1, M.E.Shackelford2, P.C.Howard3, M.Bryant2, D.I.Ruggles2, 1CFSAN, FDA, Laurel, MD, 2CFSAN, FDA, Washington, DC, 3NCTR, FDA, Jefferson, AR
Epidemiological data have suggested a correlation between high corn consumption and neural tube defects (NTDs) in infants. The mycotoxin, fumonisin B1(FB1), is an ubiquitous contaminant of corn. Its hydrolysis product is aminopentol (AP1), which is the main fumonisin product in tortillas. Although the toxicity of FB1 has been documented, no direct relation to NTDs has been established. In vitro studies have suggested that AP1 can increase NTDs, but prior to the study reported here, no developmental study of AP1 had been done. AP1 was given once daily by gavage on gestation days 3-16 at doses of 0, 15, 30, 60, or 120 mg/kg. Tissue weights and analyses of tissues for sphinganine-sphingosine (Sa/So) ratios were determined in day-17 dams and fetuses. Reproductive and developmental parameters were measured in day-20 fetuses. Conclusions: AP1was not teratogenic, less toxic than FB1, produced no variations in Sa/So ratios, did not affect reproductive or developmental parameters in fetuses, and produced no dose-related histopathological effects in dams. AP1 produced dose-related decreases in dam body weight gain, and increases in the number of fetuses with enlarged kidneys at the ureter at 60 and 120 mg/kg.
C.T.Jung, R.L.Bronaugh, J.J.Yourick, OCAC, FDA, Laurel, MD
Percutaneous absorption and metabolism of retinol from cosmetic formulations were studied to predict systemic absorption and to understand the significance of amounts remaining in skin. In vitro absorption studies were conducted using viable skin from fuzzy rat or human subjects, assembled in flow-through diffusion cells. Retinol (0.3%) formulations containing H3- retinol were applied for 24 hours. Extended studies were continued for 72 hours. Amounts found in receptor fluid and skin were determined using scintillation counting. Metabolites were analyzed by HPLC. Rat skin studies showed 23% of the applied dose in skin and 6% in receptor fluid. 72-hour studies demonstrated 18% in skin and 13% in receptor fluid. Significant amounts remained in rat skin and decreased over 72 hours, with proportional increases in receptor fluid. Human skin studies showed 7% in skin and 0.3—1.3% in receptor fluid at 24 hours. In human skin, 1.3% or less of the applied dose was found in receptor fluid, which did not increase over 72 hours. Metabolism in rat and human skin resulted in 3% polar metabolites and less than 1% retinoic acid. Retinol formed a skin reservoir, as expected of a lipophilic compound. This research was supported by FDA, Office of Women’s Health.
A.D.Papaconstantinou1, P.L.Goering1, T.H.Umbreit1, K.M.Brown2, 1CDRH, FDA, Rockville MD, 2Department of Biological Sciences, George Washington University, Washington DC
Bisphenol A (BPA) is used in the manufacture of medical tubing and other medical device plastics and is an endocrine disruptor. There is increased concern for patient exposure due to leaching of BPA from these medical plastics. We have shown that the effects of BPA and b-estradiol (E2) on uterine heat shock protein (hsps) levels are mediated through the estrogen receptor (ER), but the nature of the ER involvement remains unknown. The objective of the present experiment was to examine the role of PKC and ER on E2- and BPA-regulated hsp expression. Ovariectomized mice were treated subcutaneously with corn oil, E2 or BPA alone or in combination with the antiestrogen ICI 182, 780 (ICI) or the PKC inhibitor GF 109203X (GF), and uteri were collected at 6 or 24 hours post-administration. The results demonstrate that the effects of E2 and BPA on uterine hsps may be mediated through the ER, but only those of E2 may be mediated through heat shock factor-1, which controls transcription of hsp genes. PKC may be involved only in the regulation of hsp72 by E2 and of hsp90a by BPA. We conclude that E2 and BPA may be regulating uterine hsp90a and hsp72 expression through differential mechanisms.
H.M.Rhee1, M.A.Bae2, B.J.Song2, 1CDER, FDA, Rockville MD 20852, 2Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alchohol Abuse and Alcholism, Rockville, MD 20852
In an effort to document differential toxicity of troglitazone (TRO) and rosiglitazone (RSG), we investigated the relative toxicities of TRO and RSG on three different hepatoma cell lines, and observed that TRO, but not RSG, was cytotoxic. Additionally, we studied the mechanism by which TRO induced damage to HepG2 hepatoma cells. Our results indicated that TRO increased the levels of p53, p27, and p21, while it reduced the levels of cyclin D1 and phospho-Rb in a time-dependent manner. Increased p27 and p21 levels coincided with reduced activities of cell cycle dependent kinases (cdk) such as cdk2- and cyclin A-protein kinases 24 h after TRO treatment. These results demonstrate that TRO, but not RSG, causes G1 arrest of hepatoma cells, most likely through changing the levels of cell cycle regulators. Furthermore, TRO-mediated growth inhibition appears independent of PPAR activation because RSG did not affect the levels of cell cycle regulators.
A.C.Scallet1, L.C.Schmued1, W.Slikker, Jr.1, N.Grunberg2, P.J.Faustino3, H.Davis4, D.S.Lester5, P.S.Pine5, F.D.Sistare5, J.P.Hanig5, 1Div. of Neurotoxicology, NCTR, 2USUHS, Bethesda, MD, 3CDER, Kensington, MD, 4NIDA, Bethesda, MD, 5CDER, Laurel, MD
Ketamine is a pediatric anesthetic recently reported to enhance neuronal death in neonatal rats. To replicate those findings, PND7 rats were treated with saline, 10 or 20 mg/kg ketamine SC (1 or 7 doses given over 9 hrs), producing blood levels comparable to those in children during anesthesia. As previously reported, repeated doses of 20 mg/kg increased the number of dorsolateral thalamic neurons positive for neurodegeneration (De Olmos silver method). The number of degenerating neurons in the medial amygdala was also increased. Fewer or lower doses were ineffective. To investigate the mode of death of these neurons, coronal sections were stained with both Fluoro-Jade B (green) and DAPI (a blue DNA stain), as well as for caspase-3 (red). The results confirmed the developmental neurotoxicity of ketamine, demonstrated that FJ-B, like silver methods, successfully stained degenerating neurons in neonatal rats, and indicated that the mode of neonatal ketamine's neurotoxic action was to increase the rate of neuronal apoptosis.
J.S.Smith1, J.Tian1, J.Muller2, A.P.Byrnes1, 1CBER, FDA, Bethesda MD, 2CBER, FDA, Rockville MD
Recombinant adenoviral vectors (AdVs) are candidates for gene therapy of liver disease because of their hepatotropism. However, in humans severe liver damage can result in a decreased uptake of non-viral particles by the liver and an increased accumulation in the lung. Because AdVs can induce inflammation and tissue damage, an altered pulmonary tropism of AdV might cause dangerous respiratory complications. To study this phenomenon, we induced cirrhosis in rats by bile duct ligation (BDL) for four weeks, and evaluated the biodistribution of fluorescent-labeled AdV one hour after injection into the tail vein. In control rats, fluorescent AdV was seen concentrated in liver macrophages while very little AdV was visible in the lungs. In BDL rats, however, little fluorescence AdV was seen in the liver while the lungs contained large numbers of intravascular macrophages with fluorescent AdV. Electron microscopy confirmed the localization of AdV in pulmonary intravascular macrophages and quantitative PCR confirmed a significant increase in AdV in the lungs of BDL rats. Interestingly, compared to control animals, expression of AdV-encoded luciferase was significantly reduced in the livers of cirrhotic animals but was unchanged in the lungs of cirrotic animals. In sum, we suggest that AdVs may behave unexpectedly in patients with liver disease.
W.G.Wamer1, P.Vath2, D.E.Falvey2, 1University of Maryland
Various retinoids, including retinol, retinyl acetate and retinyl palmitate, are added to cosmetics that are applied to sun-exposed areas of the skin. It is well established that these retinoids absorb ultraviolet light and undergo a variety of photochemical reactions. The phototoxicological consequences of these photochemical transformations are less well understood. We have determined that incubation of human skin fibroblasts with 20 uM retinol or retinyl acetate, followed by irradiation with UVA light (320 nm to 400 nm), results in significant photocytotoxicity, measured as inhibition of cellular growth. Additional photochemical studies, using laser flash photolysis methods, suggest that the observed photocytotoxicity involves the retinyl cation, formed following absorption of UV light by retinyl acetate or retinol. We are currently conducting studies to determine the cellular targets damaged by photo-activated retinol and retinyl acetate.
Z.A.Xu1, D.R.Cawthon1, H.Duhart1, G.Newport2, W.Slikker, Jr.2, S.F.Ali1, 1Neurochemistry laboratory, Division of Neurotoxicology, NCTR, FDA, Jefferson, AR, 2Division of Neurotoxicology, NCTR, FDA, Jefferson, AR
PC12 cultured cells, mimicking dopaminergic function, have been used as a model for MPP+toxicity. However, the lowest dose level that produces dopamine (DA) depletion has not been determined. In this study, we evaluated the dose response and time course of MPP+ induced DA depletion and how it correlates with the changes in cellular transcription factors. PC12 cell viability, the content of DA, its major metabolite DOPAC, and DNA/protein binding were examined at different MPP+ dose levels after 24 and 48 hours treatment. We found a dose-dependent decrease of cellular DA, DOPAC and cell numbers in response to MPP+ treatment. In addition, transcription factors were also altered by MPP+.
J.Zhang, R.Honchel, E.H.Herman, J.L.Weaver, A.D.Knapton, F.D.Sistare, CDER, FDA, Laurel MD
Clinical development for certain pharmaceuticals is being inhibited by findings of vascular injury during pre-clinical testing. Fenoldopam (FP) has been shown previously to induce mesenteric and splanchnic arterial injury in rats. Adult male SD rats were given a single s. c. injection of 60 or 120 mg/kg FP or saline and euthanized 24 hr later. Vascular alterations included medial hemorrhage with smooth muscle cell death (apoptosis and necrosis) in muscular arteries and inflammation in small vessels (capillaries, venules, arterioles, and small veins). Rats treated with 60 or 120 mg/kg FP had an average vascular injury score of 3.4 or 3.8 on a scale of 0 (unaffected) to 5 (most severe) with an average of 30% or 53% of mesenteric vessels showing hemorrhage, respectively. Endothelial cell activation and injury, degranulation of mast cells, infiltration of eosinophils, and fibrin exudate were noted. A dose dependent increase in serum alpha-2-macroglobulin and granulocytosis (but not generalized activation) was observed. Findings of mast cell degranulation, accumulation of numerous eosinophils and widespread edema suggest that a localized pseudoallergic reaction can contribute to drug-induced rat mesenteric vascular injury. Better understanding of mechanism and identification of monitorable biomarkers will improve determination of human risk for drug-induced vascular damage.
Y.Hu1, I.Hirshfield2, 1U.S. Food and Drug Administration, Northeast Regional Laboratory, Jamaica, NY 11433, 2St. John's University, Jamaica, NY 11439
MO-B is an Epstein-Barr virus (EBV) transformed lymphoblast B-cell line from a 33 year old male Caucasian leukemia patient. It is positive for EBV viral capsid antigen (VCA) and nuclear antigen (EBNA). In the field of virology, there is a hypothesis that EBNA can be expressed in all known virus carrying cells and promotes hepatitis C virus (HCV) replication in vitro. In order to develop an efficient in vitro cell culture model for HCV replication, we established a cell culture system by using this EBV transformed human lymphoblast B-cell line (MO-B). This cell line was infected in vitro by using HCV-positive pooled patient serum samples. HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5’ untranslated region was analyzed. HCV minus-strand RNA was detected in the infected cell lines by highly strand-specific rTth (recombinant Thermus thermophilus DNA polymerase)-based reverse transcription followed by a novel, highly sensitive, single-tube nested polymerase chain reaction (PCR) method. PCR products were analyzed by direct DNA sequencing. Our results indicate that HCV can replicate in B lymphocytes transformed by EBV, and it may be evidence to support the hypothesis that EBV promotes HCV replication in vitro. We are the first to use MO-B EBV transformed lymphoblast human B cell line to propagate the HCV in vitro. Our procedure is available for routine use and this model should represent a valuable tool for the detailed study of HCV.
M.Kawakami, K.Kawakami, R.K.Puri, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, CBER, FDA, Bethesda MD
Recombinant IL-13 cytotoxin, composed of IL-13 and a truncated form of Pseudomonas exotoxin (PE), has been developed to target IL-13R-overexpressing tumors in vitro and in vivo. IL-13 cytotoxin shows extremely high and specific cytotoxicity to IL-13R-positive tumor cells, however, the mechanism of tumor cell death mediated by this agent is still not fully understood. We provided evidence of activation of two pathways of apoptosis in vitro and in vivo in immunodeficient animal models of human brain and head and neck tumors. These conclusions were drawn on the basis of observation of chromatin condensation, accumulation of sub-G1/G0 phase DNA population after treatment with IL-13 cytotoxin. Furthermore, intracytoplasmic molecules involved in apoptosis, e.g., caspase-3, -8, and -9, cytochrome c, and poly(ADP-ribose) polymerase (PARP) were activated or cleaved in a time-dependent manner after intratumor administration of IL-13 cytotoxin in xenografted animals. Our results indicate that apoptotic cell death may play a role in tumor regression after IL-13 cytotoxin treatment. In addition, apoptotic molecules may serve as surrogate molecular markers of tumor response to IL-13R-directed cytotoxin therapy.
J.N.LozierCBER, FDA, Rockville MD
Inhibitor antibodies are serious complications of replacement therapy for hemophilia, and potential hazards of gene therapy. There is a tendency toward inhibitors in some hemophilia kindred, suggesting a genetic influence on the immune response. Various mouse strains were injected with an adenovirus vector expressing human factor IX and assessed for their antibody response. Certain strains (e.g., C57Bl/6J)do not make antibodies to human factor IX, while others (e.g., A/J) make high-titer antibodies. Recombinant inbred mice derived from crosses between C57Bl/6J and A/J strains (AXB and BXA) permit mapping of gene(s) controlling the immune response to human factor IX. Testing of the antibody response to factor IX in AXB mice has been initiated so as to map gene(s) that may control the inhibitor response. The incidental finding of extreme sensitivity of AXB-23/Pgn mice to adenovirus (that is not seen in either parental strain) suggests gene(s) may affect adenovirus toxicity as well. Mapping of genes that influence inhibitor antibody responses or toxicity of adenovirus vectors in mice may help explain and predict toxicity in humans who use therapeutic products regulated by CBER.
J.Gao, R.Levis, B.Falgout, L.Markoff, CBER, FDA, Rockville, MD
The dengue virus capsid is a hydrophilic, positively-charged protein 99 amino acids in length. The capsid forms a complex with viral RNA in virus particles, and it contains a flavivirus-conserved centrally located hydrophobic domain that is functional for membrane insertion. A single positively charged amino acid (e.g., Arg at position 55 [Arg-55], in the context of the DEN2 genome) interrupts the hydrophobic domain in the DEN2 capsid protein and that of all mosquito-borne flaviviruses, except yellow fever virus. To investigate the requirement for this residue in virus replication, we made a series of point mutations in the context of a DEN2 infectious cDNA, so as to substitute Met, His, Leu, Ser or Ala for Arg. Wildtype and mutant DEN2 cDNAs were transcribed in vitro, and RNAs were used to transfect monkey kidney cells. Substitution of Arg-55 by hydrophobic nonpolar amino acids was lethal for DEN2 virus replication. Revertant viruses were recovered and contained spontaneous mutations resulting in the insertion of polar or hydrophilic amino acids other than Arg at position 55. The genomes of these viruses were completely sequenced in order to detect second-site mutations that might be indicative of virus protein-protein interactions involving capsid.
C.Frazar, P.A.Orlandi, DVA, OARSA, FDA, Laurel, MD
Cyclospora cayetanensis is a human parasitic pathogen that has been associated with outbreaks of food-borne illness attributed to contaminated fresh produce (raspberries, basil, mesculin lettuce). As with other pathogenic microorganisms associated with food- and waterborne contamination, this parasite has a direct impact on regulatory, economic and public health issues. Unlike most food-borne bacterial pathogens however, there is a considerable lack of information concerning its environmental biology, its host reservoirs, and the basic concepts of its pathophysiology. On a molecular level, only the small subunit rRNA gene and the first internal transcribed spacer region have been described. Beyond their usefulness in rapid molecular detection methods and phylogenetic analysis, however, such information has been of limited value. In contrast, the use of RT-PCR to detect differentially expressed molecular targets rather than genomic housekeeping genes may provide a means to assess parasite viability. From a regulatory perspective discrimination between an infectious oocyst and a non-infectious or a dead oocyst in a food sample is essential, as the latter two do not necessarily present a serious human health risk. As heat shock proteins (HSPs) are thought to play an important role in oocyst sporulation, mRNA expression may correlate oocyst detection with viability and infectivity. In the present study we identified a putative HSP70 homologue from C. cayetanensis. By aligning HSP70 gene sequences from closely related organisms such as Eimeria spp. and Cryptosporidium spp. and comparing highly conserved regions, we designed a series of DNA primers. PCR amplification using these primers yielded a 970 bp amplicon. BLAST analysis of this sequence revealed considerable homology to the HSP70 of C. parvum. Based on the information obtained thus far, we are currently developing a refined strategy to obtain and sequence the remainder of the C. cayetanensis HSP homologue. RT-PCR analysis will then be used to examine any correlation between the expression of this protein and oocyst sporulation and infectivity.
K.Herold1, A.Rasooly2, 1Department of Mechanical Engineering University of Maryland, 2CFSAN, FDA, College Park, MD
A tiled design of overlapping oligonucleotide probes on a microarray is important for gene analysis, especially when analyzing Single Nucleotide Polymorphisms (SNPs) by microarray. SNP detection depends on differences in the hybrid stability of short oligonucleotides where the matched/mismatched targets differ by only a single base. However, while many computer programs exist that can select an individual optimal oligonucleotide probe from a DNA sequence, there are few available programs for designing tiled probes. We developed a program, TILING, for designing overlapping oligonucleotide probes on the basis of their size and melting temperatures (Tm) for use in oligonucleotide microarrays. The three methods used for calculating Tm in this program are nearest neighbor analysis, salt-adjusted melting temperature calculation, and the Wallace equation. The user specifies the position of the fragment, the desired length of the oligonucleotide, the desired Tm, the position of the SNP, and the desired overlap among the probes. The program then scans the sequence and generates a list of suitable probes which are then further analyzed based on a series of design rules to arrive at the final recommended probes set.
S.Seetharam, R.K.Puri, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, CBER, Bethesda, MD 20892
Interleukin-13 (IL-13), a predominantly T cell derived cytokine elicits both pro and anti-inflammatory responses. IL-13 binds with high affinity to a cell membrane protein IL-13Ra2 chain. We have characterized this receptor chain with regard to its ligand binding, internalization and as a target for cancer therapy. To further characterize this receptor and to develop antibody reagents, highly purified protein is needed in milligram quantities. In this study, cDNA encoding the extra cellular domain (ECD) of hIL-13Ra2 chain was inserted into the pET expression vector with a hexa-histidine tag at the carboxyl end of the ECD. Upon induction with isopropyl b-D-thiogalactoside, the protein was localized in the inclusion bodies. By exploiting the metal binding property of histidine, the refolded protein was purified in a single capture step using immobilized metal ion chromatography The purified IL-13Ra2 ECD protein neutralized the cytotoxic activity of a chimeric fusion cytotoxin (IL-13PE) in an indicator cell line, renal cell carcinoma, in a concentration dependant manner. Furthermore, the proliferative activity of IL-13 in TF-1 erythroleukemic cells was inhibited. Our data suggest that the purified ECD of IL-13Ra2 chain can neutralize IL-13 activity and may be useful in various conditions including asthma where IL-13 plays a central role.
A.Selvapandiyan1, A.Debrabant1, R.Duncan1, S.Bertholet2, G.Sreenivas3, P.Salotra3, H.L.Nakhasi1, 1OBRR, FDA, Bethesda, MD, 2NIAID, NIH, Bethesda, MD, 3Institute of Pathology (ICMR) New Delhi, India
To address the pathogenesis of Leishmania parasite, we isolated a gene encoding centrin in Leishmania donovani (Ldcen). Centrin is a calcium binding protein involved in centrosome duplication in higher eukaryotes. The N-terminal region of the centrin in higher eukaryotes has a globular domain and is thought to be involved in protein-protein interaction. C-terminal region of the Ldcen protein binds 16 fold more Ca2+ than the N-terminal region. Immunofluorescence analysis localizes the protein in the area of basal body. Expression of centrin mRNA and protein correlated with the parasite growth. This correlation is further supported by the dominant negative effect on the growth of the parasite by over-expression of N-terminal deleted LdCen protein in the parasite. The intracellular survival of such a slow growing parasite in the human macrophages is affected. These studies suggest that Ldcen may have a role in the parasite growth and alteration of growth resulting from over expression of mutant centrin may have resulted in alteration of parasite virulence.
J.Simak, K.Holada, J.Zivny, J.G.Vostal, CBER, FDA, Bethesda MD
Membrane microparticles are submicron elements 0.1 —1 mm in size shed from the plasma membrane of eukaryotic cells that contain membrane phospholipids and express antigens characteristic of their cell of origin. Membrane microparticles derived from endothelial cells (ECMP) are present in circulating blood. We have designed a three color flow cytometry assay to analyze ECMP in human plasma and also in human umbilical vein endothelial cell (HUVEC) culture supernatant. At early stage of proapoptotic stimulation HUVEC release different populations of ECMP with respect to annexinV-binding and membrane antigen expression. We have shown that a subpopulation of ECMP from HUVEC as well as ECMP in plasma of blood donors express cellular prion protein (PrPc) which plays a key role in transmissible spongiform encephalopathy (TSE) diseases. The role of ECMP in disseminating of TSE infectivity in blood remains to be elucidated. We have also shown elevated counts of circulating ECMP in different groups of hematologic patients with hemolysis and a vascular injury component. ECMP which express VE-cadherin (CD144) may reflect acute vascular injury, while ECMP positive for ICAM-1 (CD54) may indicate inflammatory stimulation of vascular endothelium. Analysis of ECMP may serve as a diagnostic tool of vascular stimulation and injury.
D.M.Williams-Hill1, K.Felton2, V.Nguyen2, C.K.Hill2, 1FDA PRL-SW, 2USC Keck School of Medicine
Our earlier studies showed that BRCA1expression in the mammary gland of the cancer susceptible Wistar Furth (WF) rat strain is lower than in non-susceptible and resistant rat strains. In this study we examined the role of cycling hormone levels during the first six estrous cycles on BRCA1 and BRCA2 mRNA and protein expression in WF rat mammary epithelial cells (RMEC). RMECs were harvested from the mammary glands of rats of appropriate estrous stage. Total RNA was extracted and cDNA was synthesized using standard techniques. Quantitative analysis of cDNAs representing expression levels of BRCA1, BRCA2, TNF-a, b-actin and a ribosomal protein gene was accomplished using ABI-Prism real-time PCR. Protein was analyzed by western blot. In all sets of experiments concluded to date, there appears to be three maximum peaks of BRCA1 expression that coincides either with peak estrogen or prolactin levels during the estrous cycle. BRCA1/2 expression increases up to 150 fold between high and low expression periods. These peaks of expression may coincide with active periods of cell proliferation. We are currently looking at how susceptibility to induced mammary cancer is impacted by these waves of BRCA1 and BRCA2 expression.
J.A.Wilson1, A.B.Margolin2, P.M.Regan1, 1University of New Hampshire
There is a need to improve the AOAC Tuberculocidal test. A 60 min, single tube, one step RT-PCR hybridization assay incorporating a molecular beacon in the LightCylcer instrument was developed to detect the putative sigma factor gene A (sigA) of Mycobacterium bovis BCG. This assay is the first step in developing a quantitative RT-PCR assay to detect viable M. bovis BCG cells post-disinfection. SigA encodes two 70-like sigma factors of the RNA polymerase which are responsible for the transcription of most genes within the cell. The molecular beacon targets an internal fragment of the 160-bp amplicon. Cells were grown for 10 days in 5% CO2 95% air, at 37oC. Cells were harvested and prepared to 20% T at 650 nm. Purified RNA extracts were collected from serial 10-fold dilutions. Reverse transcription was performed on dilutions of RNA extracts at 55oC for 30 min followed by 45 cycles of PCR. Mean crossover point for 20% T Mycobacterium bovis BCG was 32.01 with a standard deviation of 1.88 (n = 10). The crossover point is defined as that point at which fluorescence is ten times greater than the background. This assay has the potential to become an alternative tool to the AOAC Tuberculocidal test method for the evaluation of liquid disinfectants.
M.J.Zhang1, J.Drenkow2, T.Gingeras2, K.Clouse1, A.I.Dayton1, 1CBER, FDA, Rockville, MD 20852, 2Affymetrix, Santa Clara, CA 95051
We have studied the effect of HIV-1 on host gene regulation in primary, human, monocyte derived macrophages (MDM), differentiated in macrophage colony stimulating factor (M-CSF) and infected in vitro. Here we report a novel, positive feedback loop, involving the IL-7 cytokine pathway, by which HIV upregulates its own replication in MDM. We demonstrate that HIV-1 infection, or treatment of MDM cultures with HIV-1 Tat(86) protein upregulates the IL-7 receptor (IL-7R) alpha-chain at the levels of steady state RNA and protein. This upregulation increases the activation of STAT-3 by IL-7 and increases the amount of HIV-1 released by infected MDM cultures. Furthermore, we present preliminary evidence that MDM cultures constitutively release physiologic levels of an IL-7-like activity, as documented by IL-7-specific EIA measurements of MDM culture supernatants and by the ability of anti-IL-7 antibody to inhibit baseline HIV release by infected MDM cultures. These results are consistent with a model in which IL-7 supports early HIV replication, which in turn produces Tat protein, which upregulates IL-7R. This results in increased intracellular signaling from IL-7R, which, in turn, enhances HIV replication. The overall effect of this mechanism on HIV replication can be as high as 50-fold.
J.Zivny1, R.Stopkova2, K.Holada1, J.Simak1, J.G.Vostal1, 1Laboratory of Cellular Hematology, CBER, FDA, Bethesda, MD, 2Dept. of Zoology, Charles University, Faculty of Natural Sciences, Prague, Czech Rep.
Cellular prion protein (PrPc) is involved in the pathogenesis of prion diseases. However, the mechanisms of PrPc regulation, its physiologic function, and the events leading to its conversion to the infectious form are not understood. We have characterized the expression of PrPc in erythroid differentiation on cells of mouse and human origin using real time RT-PCR and flow cytometry. Induction of erythroid differentiation in mouse erythroleukemia cell line led to the upregulation of PrP mRNA expression as well as the cell surface PrPc from 11, 700±1, 400 molecules/cell to 20, 400±2, 700 molecules/cell (p<0.01; n=3). The accelerated erythropoiesis in mice was associated with increased PrP mRNA expression in bone marrow and spleen. In humans the mean expression of PrPc on the surface of peripheral blood CD34+ cells increased from 5, 300±400 to 11, 700±3900 molecules/cell after 24 h of in vitro stimulation with erythropoietin (p<0.05; n=3) and remained upregulated up to six days of culture. While human mature erythrocytes express low levels of PrPc molecules, erythroid progenitors/precursors showed high expression of PrPc. These results suggest a potential role of PrPc in erythroid differentiation. The better understanding of PrPc physiology may lead to the development of new strategies for treatment and prevention of prion diseases.
S.F.Ali1, Y.Itzhak2, 1Div. of Neurotoxicology, NCTR, 2Dept. of Psychiatry and Behavioral Sciences, University of Miami School of Medicine, Miami, FL 33126
Gamma-hydroxybutyric acid (GHB) is a metabolite of gamma-aminobutyric acid (GABA) that fulfills the criteria of a neurotransmitter. The recreational use of GHB had markedly increased in many major cities in the US, particularly in the ‘rave’ parties. Recently, FDA and DEA has been approved GHB as schedule I and III. In the present study we investigated the behavioral and neurochemical outcome of chronic administration of GHB to Swiss Webster mice. Acute administration of 100, 200 and 300 mg/kg GHB produced dose-dependent hypolocomotion as determined by infrared beam interrupt. Animals treated with GHB (200 mg/kg/day) for 14 days developed a significant tolerance to the sedative effect of GHB as determined on day 6 and 14. These findings indicate that repeated administration of GHB produced tolerance to both the sedative and the cataleptic effect of GHB. The level of DA and HVA in tissue from GHB treated mice was significantly higher than control (+40 and +36%, respectively). The rewarding effect of GHB was investigated in the conditioned place preference (CPP) paradigm. The pairing of GHB (250 mg/kg) with one compartment of the CPP cage, in alternate days for 7 days, and with saline injections in the other compartment for 7 days induced significant place preference. Taken together, the present study indicates that repeated administration of GHB produces tolerance to the behavioral effects of the drug. This finding coupled with the rewarding effect of the drug support the abuse potential of GHB. The elevation of striatal DA concentration observed may be associated with GHB-induced decrease in DA release during the course of repeated exposure to the drug.O.A.Chiesa, K.E.Moulton, P.Chamberlain, J.O.Peggins, R.Idowu, D.N.Heller, J.D.von Bredow, CVM, Laurel, MD
Ciprofloxacin is a fluoroquinolone antibiotic commonly used for the treatment of urinary tract and gynecological infections associated with childbirth. Because fluoroquinolones have induced joint cartilage lesions in juvenile animals, diffusion of ciprofloxacin into breast milk and transfer to the nursing newborn raises concerns about potential damage to the developing cartilage of the nursing newborn. An assessment of the absorption and potential accumulation of ciprofloxacin in the nursing newborn can be accomplished in a dairy cow and her own newborn calf animal model to determine whether consumption of mother’s milk is safe during ciprofloxacin therapy. This unique model was developed to demonstrate the diffusion into and concentration of ciprofloxacin in milk and the oral absorption of ciprofloxacin by the nursing newborn calf.
O.A.Chiesa, J.D.von Bredow, P.Chamberlain, J.O.Peggins, R.Idowu, K.E.Moulton, CVM, FDA, Laurel, MD
Gentamicin, an aminoglycoside antibiotic, has often been used in human medicine to treat uterine infections following child-birth. The nursing newborn may be able to absorb gentamicin from the milk of the antibiotic treated mother. The sparse exposure data reported for the human newborn may be expanded significantly studying gentamicin transfer from mother to offspring using the dairy cow and her newborn calf as a large animal model for the mother and her new born infant. In these studies Gentamicin was administered daily to a Holstein cow by i.v. infusion for seven consecutive days following birth of her calf. Analysis of gentamicin in plasma and milk samples collected from the cow indicated diffusion of gentamicin into the milk. Plasma samples collected from the newborn calf also demonstrated significant oral absorption of the drug after each nursing with milk from the gentamicin treated cow.
C.D.Ellison, A.S.Carlin, L.X.Yu, CDER, FDA, Rockville, MD
Bioavailability of some oral drug products depends on whether the drug is administered with or without food. The current method of determining such food effects compares peak and total exposure (Cmax and AUC) of a drug product administered after fasting, and after a substantial meal. This study examines the effect of food on the variability of drug exposure. Exposure data from fasted/fed studies representing 339 immediate release drug products (42 active substances) were examined. All data were drawn from ANDA submissions to the agency, and are presented in blind fashion to protect confidentiality. Normalized variances (squared CV) were compared using a two-tailed F-test with 90% confidence. Compared to levels in fasted subjects, 59 products showed significant decrease in CV for peak and/or total exposure when administered after a substantial meal. 32 products showed a significant increase in CV for peak and/or total exposure. In conclusion, food potentially affects intersubject variability in drug exposure. Furthermore, such determination of food effect on drug exposure CV can be calculated using the data currently collected in clinical studies.
S.Hirschfeld, J.Alexander, D.Birenbaum, R.Johann-Liang, D.Shetty, D.Murphy, T.Crescenzi, R.Roberts, W.J.Rodriguez, CDER, FDA, Rockville , MD
The study of pediatric therapeutics can be assisted by using data from adult studies when supported by scientific evidence. In order to define the nature of the evidence used to extrapolate adult clinical data to a pediatric population, applications from 34 drugs in 23 indications that were granted Pediatric Exclusivity under the 1997 Food and Drug Administration Modernization Act were reviewed for the approaches used to relate diseases or conditions found in both adult and pediatric populations. Multiple sources of data were used. Clinical indications approved for adults for each drug were analyzed with respect to pathophysiology, non-clinical data, pharmacokinetics, exposure-response relationships, clinical signs and symptoms, laboratory and surrogate measures, natural history and response to therapy and then compared to the pediatric condition. Components important in determining relationships between adult and pediatric conditions were identified and classified into four categories:1)nonclinical data 2)pathophysiology 3)natural history or 4) response to therapy. Analysis showed that similarity of pathophysiology was the most common basis for supporting extrapolation, followed by similar response to therapy. This analysis defined the evidence used to support extrapolation of clinical efficacy data from an adult to a pediatric population with the same disease or condition.
J.C.Gorski1, S.M.Huang2, A.Pinto1, M.Miller3, M.Desai1, S.D.Hall1, 1Indiana University, Indianapolis, 2Office of Clinical Pharmacology and Biopharmaceutics, CDER, FDA, , 3Office of Women's Health, FDA, Rockville
We determined if Echinacea (E), an OTC herbal remedy, alters CYP activity in vivo using the disposition of oral caffeine (CF), tolbutamide (TB), dextromethorphan (DM), and I.V. and oral midazolam (M). 12 subjects received CF, TB, DM, and I.V. M on days 1 & 6 and oral M on days 2 & 7 before and after E dosing (400 mg QID x 8 days). E significantly (p 0.05) reduced CF oral clearance (CLPO) from 6.63.8 L/hr to 4.9 2.3 L/hr. TB CLPO was significantly reduced from 0.820.16 to 0.71 0.18 L/hr following E dosing. The CLPO of DM was not altered by E (1289 414 vs 1281 483 L/hr). M systemic clearance was significantly increased from 32 7 L/hr to 44 15 L/hr but M CLPO was not altered 137 19 L/hr vs.146 71 L/hr by E dosing. These data indicate that E can alter the in vivo catalytic activity of CYP3A, CYP1A2 and CYP2C9 but not CYP2D6.
J.C.Gorski1, M.A.Hamman1, Z.Wang1, N.Vasavada1, S.M.Huang2, S.D.Hall1, 1Indiana University, Indianapolis, 2Office of Clinical Pharmacology and Biopharmaceutics, CDER, FDA, Rockville
To determine if SJW alters the disposition and efficacy of an OC, 12 females received an OC (ethinylestradiol (EE2)/ norethindrone (N) for 3 months. SJW (300 mg TID) was given during months 2 and 3. EE2, N concentrations, and CYP3A activity were assessed in months 1 and 3. SJW increased the oral clearance of N ( p 0.05; 8.2 ± 2.7 to 9.5 ± 3.4 L/hr) and reduced the half-life of EE2 (p 0.05; 23.4 ± 19.5 to 12.2 ± 7.1 hr). SJW increased the oral clearance of midazolam (M; p 0.05, 109.2 ± 47.9 to166.7 ± 81.3 L/hr) but not it’s systemic clearance (37.7 ± 11.3 to 39.0 ±10.3 L/hr). 2/12 women had breakthrough bleeding in month 1 compared to 7/12 women in month 3. M oral clearance was greater in women experiencing breakthrough bleeding (215.9 ± 66.5 L/hr) compared to those who did not (97.5 ± 37.2 L/hr). In conclusion, SJW alters the disposition and efficacy of OCs and should be used cautiously together.
C.M.Lathers1, G.L.Drusano2, 1HFV-1, CVM, FDA, Rockville, MD, 2Albany Medical College, Albany, NY
Models like Markov-Chain Monte Carlo evaluate drug pharmacokinetic and pharmacodynamic effects on Earth and in different gravity loads. Population studies using average bioequivalence methodology may not fully reflect true pharmacokinetic and pharmacodynamic “intra and inter — individual” differences since bioavailability differs not only among persons but also within the same person. There is need to determine individual bioequivalence when evaluating different formulations or effects of same formulation in different environments, i.e., Earth 1-g vs. outer space 0-g, or normals vs. disease. This study uses Earth individual bioequivalence measurements to predict drug levels in a given astronaut in space using population pharmacokinetic modeling with Maximal A-posteriori Probability (MAP) Bayesian estimation to evaluate bioavailability. Point estimates for individual patients result and allow 1-g and 0-g measurements to be correlated with outcome. Principle is demonstrated using a bioavailability study of stable-labeled and cold isotope. 168 hours of plasma and urine values were modeled with a 9-differential equation, four output pharmacokinetic model. Our new model uses individual bioequivalence data and correlates in vivo and in vitro ground-based data to predict drug levels in a given astronaut in microgravity. This model may be used to compare normals vs. disease patients. Opinions are authors’ and not FDA’s.
A.R.Morrison1, C.M.Lathers2, 1Sch Vet Med, Univ Penn, Philadelphia, PA, 2HFV-1, CVM, FDA, Rockville, MD
Homeostatic and circadian processes guide internal drive for sleep and regulate light effects on normal sleep/wake distribution. External stimuli and conditioning impact sleep quality. The amygdala interacts with sleep brain stem nuclei and brain autonomic neural control of blood pressure, heart rate and rhythm. Chronic rat recordings found central nucleus amygdala (CNA) serotonin induced rapid changes of state when injected in rapid-eye-movement sleep (REM) vs. non-REM. A serotonin antagonist released ponto-geniculo-occipital (PGO) waves, which occur spontaneously during REM into non-REM. Stimuli conditioned by pairing with aversive stimuli in a fear-conditioning paradigm increased sound-elicited PGO waves, signals of alerting, and reduced REM. Abrupt electrographic changes and large brief cardio-respiratory activations at awakening suggest a distinct, transiently aroused awake state may exist, compared to later wakefulness. Transition was associated with reduced gating of motor responses to sensory inputs. CNA electrical stimulation enhanced acoustic startle response and spontaneous PGO waves, suggesting CNA modulates alerting mechanisms. Understanding sleep neural mechanisms will allow therapeutic development to correct flight sleep disorders. Good sleep and awakening patterns while living in space will ensure task completion, protect cardiovascular function and maintain astronauts in a ready state to respond to threatening stimuli. MH42903Support. Opinions are authors’ and not FDA’s.
C.M.LathersCVM, FDA, Rockville, MD
Head-out water immersion (HOWI) simulates weightlessness and is used to study renal patients, inducing primary central hypervolemia due to cephalad fluid redistribution, diuresis and natriuresis, and decreased plasma volume (PV). Decreased colloidal osmotic pressure due to leg capillary bed fluid shifts accounts for 25% increased sodium excretion. Healthy subjects but not acute renal failure exhibit a correlation between HOWI-induced changes in volume regulatory hormones. Orthostatic tolerance post space flight exhibits decreased PV and total body water. Frey et al (J Clin Pharmacol 1991, 1994) compared oral rehydration solutions of varying osmolality and sugar content. Urine flow was greater after water and glucose containing 1% solutions and lowest after saline with no glucose. PV increased more at 2, 3, and 4-hrs after 1.07% saline than after other solutions, including the 0.9% saline countermeasure (3740 vs. 3520 mL). Systolic blood pressure increased upon standing after saline but decreased when hypohydrated (20 mg iv Lasix) or euhydrated. Skylab 24-hr urine revealed elevated sodium in-flight and post flight. Acute renal disease decreases sodium excretion; hypotonic 0.45% solutions correct it. 1.07% saline may be better than 0.9% saline as a post space flight countermeasure for orthostatic intolerance. Opinions are the author’s and not FDA’s.
J.A.Smith1, C.M.Lathers2, 1UT MD Anderson Cancer Center, Houston, TX 77030, 2Office of the Director, Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD 20855
Physiological changes in microgravity may make astronauts more susceptible to unnecessary toxicity due to decreased clearance of drugs. Microgravity-induced alterations in renal, circulatory, liver, and body function and composition may impact drug disposition. Numerous drugs alter metabolic activity of CYP450 isoenzymes, exerting significant effects on efficacy and toxicity. We will assay cytochrome P450 isoenzyme activity in vivo to determine variability in hepatic function in microgravity. Serum creatinine measurements alone will not accurately represent GFR decline due to muscle mass loss during long-term space travel. Renal function decline is measurable and has predictable effects on drug elimination. We will monitor estimated creatinine clearance and duration of time in microgravity. Albumin (HSA) is the major plasma protein responsible for drug binding but alpha-acidic glycoprotein (AAG) is also involved. Stress-induced increases and decreases of AAG occur. Differences in plasma protein drug binding are important because fluctuations in drug free fraction correspond with the therapeutic effect. We propose to screen conventional plasma proteins under different microgravity environments associated with space travel and associated stress. Knowledge gained will minimize unnecessary drug toxicity, optimize duration and quality of life in long-term space travel, and improve patient management on Earth. Opinions are author’s and not the FDA’s.
P.E.Nwakama, Y.Yang, B.M.Davit, D.P.Conner, L.X.Yu, CDER, FDA, Rockville MD
To demonstrate that a generic drug product is bioequivalent (BE) to the corresponding innovator product, a sponsor must show comparable rate and extent of absorption of the two products. Peak drug plasma concentrations (Cmax) and the area under the plasma concentration-time curve (AUC) are used as indices of the rate and extent of absorption, respectively. The purpose of this study was to determine how closely plasma concentrations from generic versions of drugs regarded by many experts as having a narrow therapeutic index (or critical dose range) compared to values from the corresponding innovator products. We performed a retrospective analysis of BE data from all such generic drugs approved from 1996 to 2002. This included data from 35 BE studies, representing 7 different drugs and 20 drug products. BE criteria are that the AUC and Cmax ratios (generic/innovator) should fall within a 90% confidence interval (CI) of 80% to 125%. For AUC, generic/innovator ratios averaged 0.99 (3.3% CV). For Cmax, the generic/innovator ratios averaged 0.99 (7.0% CV). All were well within the 90% CI criteria of 80% to 125%. Thus, the average differences between the mean AUCs and Cmax of the generic and innovator critical dose drug products are very small.
P.M.Sathe1, L.X.Yu1, A.S.Hussain2, W.H.Barr3, B.Agoram4, W.Waltosz4, 1OGD, CDER, FDA, 2OPS, CDER, FDA, Rockville, MD 20852, 3Virginia Commonwealth University, 4Simulations-Plus Inc.
The influence of ‘food’ on the zero order (sustained release) input oral propranolol was evaluated in six healthy subjects using a two way pilot study. Two dose loads, 60mg and 120mg corresponding to dosing regimens of 5mg/hr for 12 consecutive hours and 10mg/hr for 12 consecutive hours were studied, with food given at 4 hours after the first dose. A sharp spike in plasma levels occurred at the time of food ingestion in every subject for both inputs. This amounted to an increase in the absorption fraction in the average profiles of 157% and 164% respectively. The normalized mean area under the curve up to last detectable sample point and infinite time for the ‘fed’ 10mg/hr treatment were 36.29% and 24.88% greater than the ‘fed’ 5mg/hr treatment, reflecting an average decrease in oral clearance of 26.6% and 19.9% respectively. A simulation of the data using Gastro-Plus© program which used a Compartment Absorption and Transit model by Yu and Amidon1, indicated that the observed spikes in plasma levels were primarily due to changes in Gut and Liver pooled Vmax scale factors. This suggests that the intrinsic clearance of drug is the main contributor of the altered oral availability.
1. Yu L.X. and Amidon G.L. A compartmental absorption and transit model for estimating oral drug absorption. Int.J.Pharm. 1999, 186(2):119-125
A.Selen, S.M.Huang, J.A.Lazor, L.J.Lesko, D.Murphy, R.Roberts, W.J.Rodriguez, V.Tandon
A pediatric pharmacokinetic and pharmacodynamic knowledge base is being built. Currently, the knowledge base consists of data from 12 pediatric studies. Various statistical methods including correlation and regression analyses are being applied to determine the effects of age, gender and race (as appropriate) on drug clearance and elimination half-lives. Descriptor equations correlating drug PK parameters with patient characteristics (age, gender, body-weight and body-surface area) will be presented for five model drugs that display a spectrum of drug elimination characteristics. Overall, the study size is a key factor in determining age-related effects in drug elimination. An approach to "enrich" age groups further support the need for optimized pediatric studies or unique pediatric study designs for better characterization of drug pharmacokinetics which may lead to the "right dose" for the pediatric patients and to significant public health benefit.
S.J.Tannenbaum1, P.I.Lee2, H.Sun2, G.Williams2, R.Temple2, L.J.Lesko2, 1Center for Drug Development Science, Georgetown University, Washington DC 20057, 2CDER, FDA, Rockville, MD, 20857
Clinical trial simulation was used to compare a FD design (all patients receive the same dose) to a PKMD design (individual doses based upon each subject’s pharmacokinetic parameters); the percentage of patients falling within a specified target exposure range (%P) was compared. Doses were determined by D=CL·AUCtarget, where AUCtarget (134 mg·h/L) is the geometric mean of the exposure range (80-230 mg·h/L), and CL is the population average clearance (CLpop = 0.385 L/h) for FD or the individual predicted clearance, a function of CLpop, age, and weight, for PKMD. %P was 95.9% in the PKMD group (25-200 mg) versus 79.9% in the FD group (50 mg). To generalize this finding, an additional simulation was conducted using 81 combinations of target ranges and %CVs for population AUC values (AUCpop). The results showed that %P increased as the variability of AUCpop decreased and as the target exposure range widened. In conclusion, by assigning individual doses to account for the variability in clearance, the variability in AUCpop can be reduced. The extent of this reduction, in combination with a known target exposure range, can be used to determine how much a PKMD design would increase %P compared to a FD design.
L.J.Lesko1, R.N.Patnaik1, S.J.Wang2, 1OPS/CDER, FDA, Rockville MD 20852, 2OB/OPaSS/CDER, Rockville, MD 20852
Effectiveness and safety of drug therapy is highly dependent on dose and resulting plasma drug concentrations. Variability in the dose-plasma concentration relationship between- and within-individuals leads to lack of a response or adverse events. Inter- and intraindividual variability is due to environmental and/or genetic factors. We evaluated 41 bioequivalence studies that reported repeated drug administration to estimate the magnitude of genetic contribution to variability in drug disposition. We used an analytic method that compares variances for between- and within-person variation in the area-under-plasma concentration-time curve that is an index of systemic exposure. The drugs included substrates for CYP 2C9, 2C19, 2D6, and 3A4, and for UGT2B7. Drugs excreted by renal excretion with little hepatic metabolism were also included. We found the highest degree of genetic contribution to variability in the disposition of omeprazole, phenytoin, loratidine, morphine, dextroamphetamine and propafenone; each is a substrate for hepatic enzymes known to have gene variants. The lowest degree of genetic contribution to variability was found for those drugs where disposition is dependent on active transport or renal excretion. These data suggest that individual replicate design pharmacokinetic studies can be recommended in drug development to assess genetic impact on exposure-response relationships.
J.A.Arcidiacono, S.Tsuyuki, E.T.Bloom, OCTGT, FDA, Bethesda, MD
Xenotransplantation using porcine sources has been proposed as a means to alleviate the shortage of human organs for transplantation. NK cells appear to be important mediators of the human anti-pig immune response. The results of this study show that the interaction of human NK cells with porcine target cells is regulated by redox. Thiol-deprivation strongly diminished the capacity of IL-2-activated human NK cells to kill porcine endothelial cells independent of the duration of IL-2 activation. This finding correlated with reduced proliferation and interferon (IFN)- production. For fresh NK cells, thiol depletion also reduced lysis of porcine and human targets. Because many adhesion molecules exhibit interspecies recognition and can be modulated by redox, we investigated whether changes in expression of adhesion molecules might explain our observations. Thiol depletion reduced CD11b and CD29 expression on fresh NK cells. Blocking studies showed that antibodies to CD11b or CD2 decreased NK lytic activity against PAEC while anti-CD18 more strongly reduced killing of K562. The combination of mAb to CD11b and CD18 reduced lytic activity against both PAEC and K562. These findings suggest that manipulation of redox status may enhance the survival of nonhuman animal tissues in humans through modulation of adhesion molecule expression/interactions.
R.Raghunandan, P.Riggins, E.Rudikoff, S.R.Bauer, DCGT, OCTGT, CBER, FDA
Cellular therapies hold tremendous potential for treatment of disease. Many cell therapies rely on ex vivo expansion and differentiation to achieve the desired number and types of cells. Notch and Delta family molecules mediate cell-cell interactions that influence proliferation and differentiation of many of these cell lineages. Dlk is a member of the Notch-Delta family and can influence proliferation and differentiation of hematopoietic stem cells, T cells and B cells. We study the role of dlk both in vitro and in vivo. Our in vitro studies show that decreased dlk protein on stromal cells removes the precursor-B cell requirement for interleukin-7 while causing upregulated pre-B notch-3 mRNA. Dlk knockout mice were generated to understand the in vivo role of dlk in B cell development. Immature B cells population in bone marrow and spleen increased while marginal zone B cells were altered. Amounts of serum IgG subtypes were altered indicate changes in the cell intrinsic /extrinsic B cell function. Consistent with our in vitro results, dlk -/- mice have perturbed B cell development and function. We expect that knowledge gained will ultimately lead to the ability to expand precursor cell populations selectively for use in treatment of a variety of human diseases.
S.Tsuyuki, J.A.Arcidiacono, E.T.Bloom, OCTGT, FDA, Bethesda, MD
Endothelial cells (EC) are primary targets of the recipient's immune response to transplanted organs and constitutively express Fas (CD95) ligand (FasL) on their surface. We investigated the role of porcine FasL in the generation of the human anti-pig response in vitro. Porcine aortic endothelial cells (PAEC) lysed a Fas+ human T cell line, Jurkat. Anti-human Fas monoclonal antibody specifically inhibited this killing suggesting that porcine FasL recognizes and binds human Fas to induce apoptosis of human Fas+ cells. We cloned porcine FasL, and found that PAEC expressed both FasL mRNA and protein. Transfection of PAEC with a plasmid encoding porcine FasL increased their ability to induce apoptosis in Jurkat cells, fresh human T cells activated with IL-2 and anti-CD3, and fresh IL-2-activated human NK cells. Moreover, porcine Fas L-transfected COS-7 cells induced significant apoptosis in Jurkat cells compared to that induced by mock-transfected COS-7 cells. The over-expression of porcine FasL in PAEC reduced their susceptibility to lysis by activated human NK or T cells, probably through killing of the immune effector cells, a phenomenon sometimes referred to as counterattack. These findings suggest that porcine FasL over-expression in EC of vascularized xenografts may provide protection from cellular xenograft rejection.
I.G.Berthold1, M.L.Pombo2, L.D.Wagner1, M.T.Jaramillo3, J.L.Arciniega1, 1FDA, Bethesda MD, 2FDA, Bethesda MD. National Institute of Hygiene "Rafael Rangel", Venezuela, 3FDA, Bethesda MD. National Public Health Laboratory, Mexico
Biochemical and immunologic characterization of antigens in final vaccine formulations would be significantly enhanced by desorption from an adjuvant. In the only anthrax vaccine currently licensed for human use in the US the antigen is adsorbed on aluminum hydroxide. Aluminum phosphate was studied as an alternative adjuvant for a potential anthrax vaccine based on rPA (recombinant protective antigen) because, contrary to aluminum hydroxide, the antigen can be desorbed in a mild and quantitative way. In addition to the effect of the two different salts on adjuvanticity, the effect of different adjuvant amounts and antigen-adjuvant binding on the immunogenicity were studied. Results indicate that comparable antibody responses to rPA were obtained with both aluminum salts. Additionally, results with aluminum phosphate as adjuvant suggest that, in this mouse model, binding of the protein to the adjuvant is not essential for adjuvanticity, whereas the amount of adjuvant has an influence on the antibody response.
S.L.Epstein1, T.M.Tumpey2, J.M.Misplon1, C.Y.Lo1, L.A.Cooper3, K.Subbarao3, M.Renshaw3, S.Sambhara3, J.M.Katz3, 1CBER, FDA, Rockville, MD, 2USDA, Athens, GA 30605, 3CDC, Atlanta, GA 30333
Current influenza vaccines are based on viral strains predicted to circulate during the coming season. If an unexpected strain or even a pandemic emerged, those vaccines could not be prepared quickly enough, so strategies based on conserved antigens should be explored. Viruses from the 1997 H5N1 Hong Kong outbreak provide a model of unexpected emerging strains. We immunized mice with DNA vaccines expressing conserved nucleoprotein (NP) and matrix (M) from A/H1N1, and challenged with A/H5N1 viruses. NP+M DNA vaccination reduced replication of A/Hong Kong/486/97, a nonlethal H5N1 strain, and protected against lethal challenge with A/Hong Kong/156/97. DNA vaccination protected only partially against extremely virulent A/Hong Kong/483/97. Mice given NP+M DNA and then exposed to HK/156 survived subsequent rechallenge with A/Hong Kong/483/97. In the absence of strain-matched vaccines, DNA vaccination to conserved influenza components may provide a useful first line of defense against a rapidly spreading pandemic virus. Immunity could be supplemented when strain-matched vaccines became available.
J.C.Byrne, R.Levis, Division of Viral Products, CBER, FDA, Bethesda, MD 20892
We are currently working in a collaborative study to develop an alternative potency test for rabies vaccine products. A critical assay for rabies vaccine product release is the vaccine potency assay. The current test, the NIH potency test, is an animal immunogenicity assay. There are several difficulties with this test. First, due to the inherent variability of mice, there is a high degree of variation between test results. There is no way to control for this, and therefore large numbers of animals must be used. Second, the current test takes up to ten weeks to complete. This has an impact on the time required for release of product. Third, the current test requires the use of virulent rabies virus to challenge immunized mice. This causes extreme discomfort for the animals, requires highly trained personnel, and specialized containment facilities. The goals of our collaborative studies, results of which will be presented, are: identification of antibody reagents that recognize all vaccine strains and the development of an antibody binding assay which is reliable and consistent with vaccine potency. Ultimately, in collaboration with other regulatory agencies and manufacturers, we hope to develop a new in vitro assay that will replace the current test.
L.Yu, L.Markoff, CBER, FDA, Rockville, MD
All flavivirus genomes contain a 3’terminal “stem and loop” secondary structure (3’SL), formed by the last ~100 nucleotides of the 3’noncoding region in the 10, 500-nucleotide viral RNA. The 3’SL secondary structure is required for virus replication. We employed an infectious cDNA derived from the genome of West Nile (WN) virus to study the effect of altering the secondary structure of the 3’SL on WN virus replication. RNAs derived from mutated WN cDNAs were used to produce mutant WN viruses by transfection. Mutants were characterized for replication in both hamster kidney (BHK) and cultured mosquito (C6/36) cells. Results showed that a “bulge” formed by apposed noncomplementary nucleotides near the mid-point of the long double-stranded region of the wt WN 3’SL was essential for production of viable mutant WN viruses. Introduction of additional bulges at two different locations into the wt WN 3’SL in one case resulted in a mutant WN virus that outgrew wt WN virus in C6/36 cells, and in a second case resulted in a mutant WN virus that was severely impaired for replication in this same cell type. Thus bulges and their topological location were determinants of replication of WN viruses in both BHK and C6/36 cells.
B.J.McWatters1, F.Voulgaropoulou2, P.R.Krause1, 1CBER/OVRR/DVP, FDA, Bethesda, MD, 2NIH, NIAID, Rockville, MD
The potential presence of unknown or modified viral pathogens represents a significant issue in the development of vaccines and other biological products, and also has relevance to food safety. The ability to detect unknown pathogens also has value in biothreat response, and in understanding the pathogenesis of human and animal diseases. To detect such agents in biological samples, our laboratory has developed a method based on purifying potential viruses free of cellular nucleic acids, using a non-specific PCR method to amplify non-cellular nucleic acids, and identifying these sequences using available databases.
Preliminary studies used this approach to identify a range of known RNA and DNA-containing viruses constitutively expressed from or spiked into cell culture. Real-time PCR is being used to optimize assay conditions and increase assay sensitivity. Clinical samples have been screened to attempt to identify unknown viral pathogens that cause human disease. Work continues on each of these fronts to further increase the sensitivity of this assay, determine the range of viruses that can be detected, determine what percentage of a virus is identified, and to continue to attempt to identify viruses in clinical samples.
R.Vassell, Y.He, L.R.King, A.Saez-Cirion, H.Golding, C.D.Weiss, CBER, FDA, Bethesda, MD
Gp120 binding to cellular receptors is thought to cause gp41 to fold into a six-helix bundle (hairpin) structure composed of two heptad-repeat domains that assemble into a trimer-of-hairpins. Bundle formation pulls viral and cellular membranes close together for membrane fusion. We designed a novel gp41 construct that is thought to mimic bundle and prehairpin conformations of gp41. This construct binds receptor-activated Env and potently inhibits HIV entry (~35 nM IC50). Antibodies from rabbits immunized with this construct bind receptor activated Env. Preliminary data in some assays suggests these antibodies have broadly neutralizing ability.
L.Zhang1, S.Kozlowski2, 1DMA, OTRR, CBER, FDA, 2DMA, OTRR, FDA
Peptide-based vaccine for cancer or autoimmunity have many attractive features such as ease of manufacturing and characterization as well as excellent safety documentation in clinical trials. However, ambiguous results from initial clinical trials suggest that peptide-base vaccine are far from optimal. The insufficient immunity generated by peptide vaccines can be attributed to many problems inherent for any peptide-based immune manipulation including identification and selection of the most appropriate T cells epitopes, lack of immunogenicity, generating immune tolerance and apoptosis of antigen specific T cells.
Recent years of studies on T cell migration and chemokine/cytokine expression have clear established that microenvironment surrounding antigen-specific T cells in second lymphoid organ or in inflamed site play critical role in the outcome of T cells (activation, anergy, apoptosis or type of cytokine production). Where the T cells located decide what signals or cytokine of APC can be received. By using TCR transgenic T cells (CD4 and CD8+) adoptive transfer model, we in situ examined the dynamic homing of transgenic T cells in second lymphoid organ, spleen and lymph nodes after immunization with appropriate peptides and protein and the location of cytokine and chemokines production and apoptosis. The major findings of the study are 1) there is a distinct difference in homing of DO11 TCR-transgenic T cells in spleen between immunization with ova peptide and with ova protein (both in CFA). DO11 T cells immunized with peptide were found exclusively in red pulp but not in T and B zone (white pulp), while 90% of DO11 T cells (in ova protein immunization) were found in T and B zone. 2) distribution of cytokine production were very different though both type of immunizations induce strong expression of IL-4, IL-2 and IFN-gamma. With peptide immunization, cytokine producing cells were found exclusively in red pulp but not in T and B zones. In contrast, with ova protein immunization, significant numbers of cells producing cytokines IL-4, IL-2 and IFN-gamma were found in T and B zone. The similar distribution patterns were also observed for cells expressing CD40 ligand. 3) Fas ligand expressing cells were found exclusively in red pulp with ova peptide or protein immunization. The difference between the two types of immunization is that lot more cells expressing fas ligand were observed in ova peptide immunization and lot more in 10 day compared with 5 days post (only a few Fas ligand expressing cells in red pulp can be found in 2C/peptide). 4) in model of 2C T cells, the similar results as with ova peptide immunization were obtained in QY5 or SYIR peptide immunization (not protein challenge), suggesting that the peptide immunization-induced migration or distribution of antigen specific T cells are not just limited to CD4+ T cells. 5) very different apoptotic dynamics of transgenic T cells were found between 2C and DO11 peptide immunization (DO11 protein immunization of apoptosis still ongoing experiment). While there are basically few apoptotic cells observed in 5 days postimmunization 2C/peptide, DO11/peptide and DO11/protein, a huge numbers of apoptotic cells were found 10 days post immunization in DO11/peptide in red pulp exclusively but not in 2C /peptide (it is interesting to see DO11/protein at 10 days). These results have tremendous significance in our understanding and design of peptide based immune manipulation. It has been established that it is important for T cells to be antigen presented by professional DC in T zone. Bypassing this may result in suboptimal activation and dysfunction of T cells. The unique homing of cytokine producing cells and antigen specific T cells in peptide immunization may partially explained the ineffectiveness of peptide based manipulation and strong suggest that any design of peptide vaccine should have a consideration of appropriate homing property of T cell.
N.E.Kay1, N.Bone1, D.F.Jelinek2, P.Leland3, D.A.Frank2, T.E.Battle2, R.K.Puri3, 1Mayo Clinic, Rochester, MN, 2Dana Farber Cancer Institute, Boston, MA, 3Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, CBER, FDA, Bethesda, MD
Current treatments for B-Chronic Lymphocytic Leukemia (B-CLL) can slow progression and alleviate symptoms, but none are curative. Utilizing RT-PCR, 9 highly purified B-CLL primary cultures were found to express type III Interleukin-4 receptors (IL-4R). These receptors were shown to be functional in STAT signaling assays and IL-4 treatment of B-CLL enhanced in vitro resistance to apoptosis. We produced a chimeric protein composed of circular-permuted IL-4 and a mutated form of Pseudomonas exotoxin (termed cpIL4-PE) to target these receptors. cpIL4-PE binds to IL-4 cell surface receptors, internalizes and mediates cytotoxicity. Analysis of viability by annexin/PI flow cytometry of B-CLL cells treated with cpIL4-PE demonstrated induction of apoptosis and/or cell death in 6/7 B-CLL tested. When 9 B-CLL primary cultures were incubated with low concentrations of cpIL4-PE in a protein synthesis inhibition assay, an agonist activity was seen in 7 B-CLL. However, at higher concentrations, there was a consistent drop in protein synthesis from the elevated levels. The IC50 (protein concentration of cpIL4-PE causing 50% protein synthesis inhibition) ranged between 10-1, 000 ng/ml indicating that B-CLL are differentially susceptible to the cytotoxic effects of cpIL4-PE. These results suggest that IL-4R on B-CLL cells may be targeted by cytotoxins as a potential therapy for this currently incurable disease.
S.R.Husain, R.K.Puri, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, CBER, Bethesda, MD 20892
Brain tumors, in particular, glioblastoma multiforme (GBM) have defied all current therapeutic modalities and pose an extremely difficult public health problem. New approaches of targeting the drug to tumor cells via receptor specific ligands are being explored. A recombinant fusion protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (termed IL-13 cytotoxin, IL13-CTx) was developed in our laboratory to target IL-13 receptors on GBM. IL13-CTx is selectively potent in killing GBM cells in vitro as they express high number of these receptors on their cell surface. Normal brain cells lacking IL-13 receptors were spared of killing. The direct injection of IL13-CTx in to subcutaneous human GBM tumors caused complete and durable regression of tumors in all mice. Intravenous and intraperitoneal administration of IL-13 cytotoxin also reduced tumor burden significantly. In preclinical safety and toxicity studies, systemic administration of IL13-CTx in mice appeared to be well tolerated in high doses. Intracerebral injections of IL13-CTx in doses up to100 µg/ml did not show evidence of gross or microscopic necrosis, while at 500 µg/ml caused regional necrosis in rat brain. These studies produced scientific basis for the development of Phase I/II clinical trials for malignant glioma therapy. Currently, three multicenter/multinational clinical trials have been initiated by our Cooperative Research and Development Agreeement (CRADA) partner.
B.H.Joshi, R.M.Goel, P.Leland, R.K.Puri, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, US FDA, CBER, Bethesda, MD
Effective therapy is lacking for human bladder carcinoma. We have discovered earlier that a variety of murine and human carcinoma cells express high levels of receptors for interleukin-4 in vitro and in vivo. Here, we demonstrate that human bladder carcinoma cell lines express IL-4 receptors as assessed by reverse transcriptase PCR. Five human bladder carcinoma cell lines expressed mRNA for IL-4Ra and IL-13 receptor a1 chains. mRNA for IL-2g c chain known to form a complex with IL-4Ra chain in immune cells was absent in these cell lines. Indirect immunofluorescence analysis for three different receptor chains confirmed RT-PCR results and all three cell lines were equally positive for IL-13R a1 and IL-4R a chains. A chimeric protein composed of circularly permutated IL-4 and a truncated form of Pseudomonas exotoxin [IL-4(38-37)-PE38KDEL] was highly cytotoxic (killing 50% of the cell population at <20 ng/ml of IL-4 cytotoxin) to three IL-4R positive cell lines. However, two other cell lines were moderately sensitive to IL-4 cytotoxin. In addition, paraffin sections prepared from human bladder cancer specimens showed in situ expression of the protein and transcripts of IL-4R chains. These results suggest that IL-4-cytotoxin may be a useful agent for the treatment of human bladder cancer and further studies should be performed to explore this possibility.
J.W.Karanian, D.Wray-Cahen, A.Ashby, S.L.Hilbert, P.Abii, W.F.Pritchard, OST, CDRH, FDA
Pre-clinical animal studies of medical devices are used to evaluate safety, failure modes, effectiveness, and surgical handling characteristics of medical devices prior to the initiation of clinical trials. The CDRH Laboratory of Large Animal Research (LLAR) provides (i) development and understanding of animal models of human disease and device intervention, (ii) applied research in support of medical and hybrid device regulatory issues, (iii) education through courses and workshops and (iv) regulatory support through review of pre-clinical animal studies. LLAR is establishing animal models of disease and investigates the safety and effectiveness of diagnostic and therapeutic devices and combination products. LLAR is located at the MODII Animal Research Facility with animal management and veterinary support provided by the CVM Office of Research. The LLAR facility includes angiography and surgery suites, laboratories, pre- and post-operative animal holding, darkroom, and steam and gas sterilization. Specific capabilities include diagnostic ultrasound, interventional radiology, animal surgery, in vivo monitoring and hemodynamics, in vitro physiology and pathology, and nutritional manipulation. LLAR represents a collaborative effort integrating the science and technology of multiple disciplines in device evaluation, model development, and applied research in support of current and evolving regulatory issues.
J.W.Karanian1, D.Wray-Cahen1, S.L.Hilbert1, A.Ashby1, P.Abii1, M.J.Hallisey2, W.F.Pritchard1, 1OST, CDRH, FDA, 2Hartford Hospital, CT
The predictive value of existing animal models for evaluating interventional cardiovascular device safety is limited. Preclinical trials are typically performed in normal healthy vessels of juvenile female and castrate male swine. Our current studies in swine models are designed to evaluate differences between animal gender and age, hormone status, short-term versus long-term healing, and normal versus diseased vessels on interventional device performance. In addition, we are comparing angioplasty and stent performance in experimentally-induced de novo lesions and diabetic lesions. These combined efforts have resulted in development of an in vivo animal model which adequately predicts the interactions between interventional cardiovascular devices and patients prior to initiation of clinical trial. In addition, an in vitro model of balloon angioplasty-induced vascular stenosis has been developed which allows for the study of combination therapeutics, drug delivery, drug pharmacodynamics and pharmacokinetics. This combination of a variety of models and techniques allows us to address the diversity of emerging issues associated with new technology to treat cardiovascular disease.
W.F.Pritchard1, B.J.Wood2, J.R.Cebral3, R.J.Lutz2, A.Sofer3, D.Wray-Cahen1, D.Perkowski2, A.Ashby1, J.W.Karanian1, 1OST, CDRH, FDA, 2Clinical Center, NIH, 3GMU
Thermal ablation of tumors is a rapidly developing therapeutic intervention. Treatment failure, or tumor recurrence, typically occurs at the margins of the ablation. Tissue perfusion and vascular flow in or near the target tissue can significantly alter the size and shape of the ablation by cooling the tissue, potentially leading to incomplete ablation and an increased risk of tumor recurrence. We have developed predictive in vivo models in swine for hepatic and renal ablation to quantify the relationship between blood flow, tissue thermal and dielectric properties and probe placement on the extent of ablation. Finite element computational models predict temperature distributions in tissue during ablation by using biothermal and electric field equations and incorporating the effects of blood flow. In vitro models of tissue, e.g., gels and egg white, corroborate the predictions of the numerical models and provide insight into the in vivo studies. Taken together, these models contribute to our understanding of factors affecting thermal ablation and can lead to more effective treatment. This combination of a variety of models and techniques allows us to address the diversity of emerging issues associated with new technology to treat cardiovascular disease.
Board O-07 Systemic and Local Drug Delivery Inhibit Vascular Stenosis Following Angioplasty and Grafting: Safety and Effectiveness and Routes of Administration
J.W.Karanian1, N.Kipshidze2, D.Wray-Cahen1, S.L.Hilbert1, A.Ashby1, W.F.Pritchard1, 1OST, CDRH, FDA, 2Lenox Hill Hospital , NY, NY
Swine studies were designed to assess the safety and effectiveness of systemic versus local administration of drugs to inhibit vascular occlusion due to stenosis. Stenosis resulting from neointimal hyperplasia is a typical failure mode associated with balloon angioplasty, stenting and vascular grafting. We have shown balloon angioplasty of swine coronary arteries is followed by the development of stenosis (neointimal hyperplasia) by 30 days. Systemic estrogen replacement therapy (ERT) was shown to moderately reduce the angioplasty-induced coronary stenosis in swine. Recent reports analyzing the risks/benefits of ERT have focused on chronic systemic administration, not localized single dose delivery. However, we have shown that local delivery of an immunosuppressent drug markedly inhibits stenosis at the venous anastomosis of a vascular graft. We are currently studying the pharmacokinetics and pharmacodynamics of local drug delivery (e.g., steroid, immunosupressent and cytostatic drug) as a method of reducing the neointimal hyperplasia that leads to vascular stenosis in our swine models. These results are consistent with the proposition that local drug delivery, via an intraluminal catheter or drug-eluting device, may provide a more safe and efficacious therapy for the treatment of occlusive vascular disease.
K.Kawakami, M.Kawakami, R.K.Puri, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, CBER, FDA, Bethesda MD
A predominant immune cell-derived cytokine interleukin-13 (IL-13) binds with high affinity to a cell surface protein termed IL-13Ra2 chain. This chain plays a key role in ligand binding and internalization in some human tumor cells. We have recently demonstrated that the introduction of this cytokine receptor chain highly sensitizes tumor cells that do not or express low levels of this chain to a recombinant fusion therapeutic cytotoxin comprised of IL-13 and Pseudomonas exotoxin (IL13-PE). We have exploited this observation and established a novel approach for cancer therapy. For this, a plasmid encoding the IL-13Ra2 chain gene was mixed with liposomes (DOTAP) and injected into subcutaneously or orthotopically xenografted human prostate, breast, pancreatic, and head and neck tumors in immunodeficient mice, followed by systemic or local therapy by IL13-PE. Only tumors enforced to express IL-13Ra2 chain in vivo acquired extreme susceptibility to the antitumor effect of IL13-PE. There was a dominant infiltration of inflammatory cells including macrophages and natural killer cells in the regressing tumors. Because macrophages were found to produce nitric oxide, IL-13Ra2 targeted cancer therapy involved not only a direct tumor cell killing by IL13-PE but also activation of innate immune response at the tumor site. Therefore, this approach may be a new powerful approach for cancer therapy.
D.L.Rowley1, D.D.Levy2, K.A.Lampe1, P.A.Orlandi1, 1DVA, OARSA, FDA, Laurel, MD, 2DMB, OARSA, FDA, Laurel, MD
The aim of this project is to develop a robust molecular (PCR) detection and quantitation system for the presence of bioengineered events found in food. Initial experiments were designed to evaluate and compare both standard and commercially available DNA isolation kits for the detection of bioengineered events in plant products and processed foods. DNA extraction kits were used on yellow corn and yellow corn products to assess extraction efficiency, relative yield, relative initial quality and stability of template from produce and processed foods for use in PCR analysis. Course ground unprocessed corn “grits”, the meal resulting from whole kernels processed in a coffee grinder, corn meal, corn flour, a muffin mix and taco shells were processed using four different standard or commercially available extraction methods. These methods included 1) a guanidine-HCl lysis/Wizard PCR Prep purification; 2) Wizard Magnetic DNA purification system for Food; 3) High Pure GMA Sample Preparation Kit; and 4) DNeasy Plant Mini Kit. Extracted DNA was evaluated by quantitative fluorometric analysis of total double stranded DNA, evaluation of fragmentation using agarose gel electrophoresis, and suitability as a PCR template by both standard and real-time PCR techniques. The efficacy of each extraction method varied with the type of sample being analyzed. These results provide a first step towards development of a standard operating procedure for developing methods to detect a novel or unapproved bioengineered event in a plant or processed food.
L.Rudenko, J.C.Matheson, A.Adams, M.Alewynse, G.Claycamp, E.Dubbin, B.Hooberman, L.Hungerford, W.Jones, T.Modric, G.Sherman, CVM, FDA, Rockville, MD
Animal clones (AC) derived from somatic cell nuclear transfer (SCNT) are likely to be used as elite breeding stock. CVM has asked developers not to introduce ACs or their progeny (P) into food until the Agency has completed its food consumption risk analysis. Part of this analysis includes the development of a transparent, science-based review of publicly available information, and is composed of two components. The Critical Biological Systems Analysis (CBSA) evaluates the health of these animals, and is based on the hypothesis that a healthy animal is likely to produce safe food products. The Compositional Analysis (CA), reviews AC/P meat and milk constituents. The CBSA suggests that if animal clones survive weaning and the juvenile period, they will likely reproduce and function normally. No specific data were available on the composition on meat or milk from AC/P. CVM is in substantial agreement with NAS's 2002 opinion that animal clones and progeny are likely to be acceptable for food consumption, although further data would decrease the uncertainty associated with such a judgment. Based on the scientific framework derived from this risk analysis, CVM has developed a series of recommendations for the additional data that could significantly decrease these uncertainties.
W.H.Chou, S.M.Huang, C.G.Sahajwalla, L.J.Lesko, CDER, FDA, Rockville MD, 20852
Pharmacogenetics/Pharmacogenomics (PGtx) is positioned to be an important scientific tool in drug development and regulatory decision-making to improve the efficacy of new drugs, to personalize drug dosing, and to minimize adverse drug reactions. In order to gain insight into how companies are utilizing PGtx in drug development, we examined a subset of INDs and NDAs submitted to the Agency over the past 10 years. The following information was collected: therapeutic class, genetic variations, methods of testing, types of study, metabolic fates of the drug, results, and the clinical utility. A total of 70 INDs and or NDAs have been reviewed. Overall, genes encoding CYP2D6 activity are the most frequently examined and accounted for 73% of all tests, followed by CYP2C19, CYP3A4, transferases, CYP1A2, certain receptors, CYP2C9, and Pgp. In many occasions, multiple genetic variations were tested and both phenotyping and genotyping methods were used in the same submission. This informal survey indicated increased integration of PGtx in the drug development process over the last two to three years. Recent, major advancement in PGtx technologies and wide availability of routine PGtx testing appear to contribute to this increased integration.
D.Ranamukhaarachchi1, R.A.Bright1, M.Chan1, S.Farr2, J.J.Langone1, R.Neft2, J.P.O'Neill3, J.Slater4, S.C.Wood1, R.K.Elespuru1, 1CDRH, FDA, Rockville MD, 2Phase 1 Toxicology, Santa Fe NM, 3University of Vermont, Burlington VT, 4CBER, FDA, Bethesda MD
Allergy to latex is a significant problem among millions of health care workers and patients repeatedly exposed to medical devices containing natural rubber latex, most commonly latex medical gloves. New genomic technologies have the potential to identify differentially responding individuals and to offer a pathway for prevention of the adverse event. We describe our overall strategy for the development of a differential gene expression profile characteristic of the more serious, life-threatening Type 1 allergy, obstacles encountered, and problems resolved. Peripheral blood lymphocytes from allergic and non-allergic health care workers are cultured with or without latex allergen. RNA is isolated and used for gene expression analysis by differential display PCR and real-time PCR. Results of gene mining studies and genes expected to play a role in allergy development will be used to create a model microarray diagnostic device. This device will be used to explore issues in microarray device validation, including RNA isolation, diagnostic sensitivity, array generation, and bioinformatics. The data on differentially expressed genes may also be informative relative to mechanism of allergy development, gender differences, and genetic pre-disposition to latex allergy. Supported by the FDA’s Office of Women’s Health.
D.Ranamukhaarachchi, J.J.Langone, R.K.Elespuru, CDRH, FDA, Rockville MD
Molecular diagnosis of disease using differential gene expression requires targeting and detection of RNA synthesized by affected cells against a background of other cells that usually predominate by orders of magnitude. Detection of antigen-specific lymphocytes within the general pool of lymphocytes by this approach is a difficult task in the diagnosis of immunological disorders. Using real-time PCR, we evaluated the detection limits of gene expression by spiking phytohemagglutinin (PHA)-stimulated lymphocytes into aliquots of unstimulated lymphocytes from the same sample in ratios of 1/1 to 1/1, 000, 000. Differentially spiked lymphocytes were normalized to 2X106 cells each. RNA was extracted, reverse transcribed into c-DNA, and analyzed for proliferation genes expressed exclusively or in large copy number in PHA-treated cells using real-time PCR. Results indicated the detection of a single stimulated lymphocyte in a background of 1000 unstimulated cells for low expressed genes, and one in 100, 000 for highly expressed genes. In other experiments using mRNA with known abundance, we were able to detect genes at a copy number as low as 10 after reverse transcription of mRNA. In addition to disease diagnosis, these findings have broad application in determining efficacy and quality control standards for diagnostic devices, and detection of pathogens.
R.K.Elespuru, CDRH, FDA, Rockville MD
New genetic and genomic technologies provide challenges for FDA, e.g. in the development of review standards for genomic diagnostic devices at CDRH. However, these technologies also provide opportunities for innovative approaches to evaluating safety and efficacy of many FDA-regulated products. Because genetic and genomic data are both generated using microarrays, these dual technologies are often considered as one. However, the technical issues, types of data, complexity, clinical application, ethical issues, need for bioinformatics, and previous track record for these technologies are often quite distinct. This presentation will provide a framework for comparing and contrasting these technologies. This framework is offered as a means of dissecting the technologies into important elements, as a general tool for education of scientists in different disciplines, and as a focus for dialogue among review and bench scientists. Discussions of the paradigm may help focus approaches to the development of review standards for acceptance of genomic and genetic data, and for validation of genomic and genetic diagnostic devices.
H.Fang, W.Tong, J.F.Bowyer, NCTR, FDA, 3900 NCTR Road, Jefferson, AR 72079
Studying the functional roles of the various brain regions has been a major focus of investigation in neurobiology. Relating to this endeavor, we have investigated the gene expression involved in the regional differences in brain using a cDNA microarray technology. The gene expression profiles for 4 brain regions (S. Nigra, Striatum, Parietal Cortex and PLCo) were determined from 7 to 9 unique pools of total RNA, with each pool derived from 2 to 3 individual rats, for each brain region using a membrane-based cDNA microarray that contains 1185 unique probes. Three hundred and thirty-nine genes were differentially expressed across the four brain regions based on an ANOVA analysis. The differences in the expression pattern of 4 brain regions were assessed using Principle Component Analysis (PCA) based on these genes. The resulting PCA model correctly predicts the regional classification of new samples. Furthermore, we identified 63 genes out of 339 differentially expressed genes using SIMCA, which demonstrated the highest discriminating power to differentiate 4 brain regions. Hierarchical Clustering and Pearson correlation analysis using these 63 genes revealed that S.Nigra and Striatum regions differed from each other as well as differing from the Parietal Cortex and PLCo regions with respect to the expression patterns. In contrast, the gene expression patterns for the Parietal Cortex and PLCo are more similar, indicating that their functional roles in brain are more related. Importantly, the hypergeometric distribution calculation on Clontech gene function term in each cluster demonstrated that the clustering is significant for certain biological functions of the genes, such as neurotransmitter receptors, membrane channels and transporters, and intracellular modulators or second messengers. Our studies confirm and extend the recent work showing that functionally distinct brain regions can be recognized by gene expression profiles and that the cDNA microarray technique is useful to study the relationship of the functional roles of brain regions with gene expression patterns.
J.Han1, R.K.Puri1, R.L.Farnsworth2, P.Ikonomi2, 1Microarray Program, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, CBER, FDA, Bethesda, MD 20892, 2ATCC, Manassas, VA 20110
The human embryonic kidney (HEK) 293 cells are the most commonly used cell line in the production of biologics. When the 293 cells are grown to over-confluence, they exhibit a transformed phenotype. In addition, changes in cell culture conditions can also influence metabolism, growth rate and the quality of biological product produced by these cells. No effective techniques are available that can monitor global quality of cells in culture. By using the DNA microarray technology, we hypothesized that monitoring of gene expression profiles of cells during various growth conditions may provide a novel approach for quality assessment. The 293 cells cultured in different confluence states (40%, 90%, and over confluence) were harvested, and total RNA extracted. Labeled probes were hybridized with high quality 10K cDNA microarray chips produced at the ATC/NCI. The results were analyzed by GenPix, and mAdb database software tools developed by CIT/NIH. The gene expression patterns of the 293 cells under different confluence conditions were compared. Significant differences in gene expression level were observed in over-confluence cells, comparing with 40% confluence cells. As increases in gene expression belonging to metabolism pathway, such as ALDOA, PFKP, LDHA, transcription such as ATF3, and stress related genes such as PLOD2, DSIPI, PLEC1 etc, were observed in over-confluence cells. These changes were validated by quantitative Real-Time PCR analysis. Additional studies are ongoing to evaluate the quality of adenoviral vectors produced by over-confluence cells.
H.Hong, H.Fang, Q.Xie, R.Perkins, W.Tong, NCTR, FDA, Jefferson AR
DNA microarray technology enables investigating the expression of thousands of genes in a single experiment. Accurate analysis and meaningful interpretation of massive information from this technology pose a unique challenge. Classification using the pattern recognition methods can be used to predict disease type and/or stage and chemical-induced toxicity based on the gene expression data from microarray experiment, which shows promising to correlate genotype with phenotype. The classification model development consists of two steps: model construction and validation. Most classification models based on microarray data in the literature are developed using only a small set of genes out of a large set of genes on a chip through a gene selection procedure first. The same set of genes is then used in both model development and validation steps, which has been proved inappropriate to validate the robustness of a model. In this poster, a novel classification method, named Decision Forest is presented, which combines the gene selection and model construction into a single step. The method offers a number of advantages over traditional classification approaches. More specifically, the cross-validation results of Decision Forest have better indication to the quality of a classification model.
C.D.Atreya, K.V.Mohan, OVRR, CBER, FDA, Rockville MD
The function of a protein is intimately associated with its subcellular localization, which is a signal-dependent event. Signals for targeting cellular proteins to the endoplasmic reticulum, mitochondria, nucleus or the cell membrane have already been identified and the most recent inclusion to this list of organelle-targeting signals is the peroxisomal targeting signal (PTS). Peroxisomes are vital cellular organelles controlling important metabolic functions like hydrogen peroxide degradation, fatty acid metabolism, gluconeogenesis, toxin degradation, etc. Certain human metabolic disorders such as Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and rhizomelic chondrodysplasia punctata are associated with the peroxisomal defect in protein import and/ or biogenesis. The peroxisomal targeting signals are of two types, PTS1 and PTS2. Type 1 signal sequence is a tripeptide motif present near or at the carboxy terminal of the protein whereas, PTS2 constitutes a combination of nine amino acid bipartite sequence in the N-terminal half of the protein. Proteins from mammals, plants, fungi, parasites have all been identified to contain PTS. However, so far these signals have not been reported among viral proteins. We report here the identification and conservation of both PTS1 and PTS2 among a variety of viral proteins.
P.S.Pine1, E.H.Herman1, J.Zhang1, A.D.Knapton1, G.Holt2, F.D.Sistare1, 1CDER, FDA, Laurel, MD, 2Oxford GlycoSciences, UK
The present study investigates whether two-dimensional gel electrophoresis can be used to discover biomarkers associated with doxorubicin (DOX)-induced cardiotoxicity. Serum samples were collected from spontaneously hypertensive rats (SHR) treated with DOX, with/without the cardioprotectant dexrazoxane (DZR). Features from the 2-D gels were then fluorescently stained and measured using quantitative image analysis to create protein expression maps (PEMs). Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to test the ability of the PEMs to discriminate between treatments. Animals treated with either saline or DZR clustered together, indicating that the cardioprotectant had little effect on serum proteins by itself. The animals treated with DZR-DOX clustered together but fell between the controls and the DOX cluster of animals, consistent with the partial attenuation seen in the myocardial lesion scores and cardiac troponin T levels observed with DZR pretreatment. Features that correlated with a pattern of complete or partial protection by DZR pretreatment could be identified using expression profile matching. Some of those features have been identified and annotated, including: alpha-1-antiproteinase, fibrinogen, anti-thrombin III, and several apolipoproteins. These data indicate that 2-D gel analysis of serum identified a consistent set of protein features correlating with both myocardial injury and cardioprotectant activity.
K.L.Thompson1, B.A.Rosenzweig1, P.S.Pine1, J.Zhang1, E.H.Herman1, A.D.Knapton1, R.Honchel1, B.Shimada2, S.Kassam2, D.F.Finkelstein2, J.Lescallet2, J.Retief2, F.D.Sistare1, 1CDER, FDA, Laurel, MD, 2Affymetrix, Santa Clara, CA
DOX is a cardiotoxic anti-neoplastic drug that induces diffuse cardiac pathology characterized by myofibrillar loss and cytoplasmic vacuolization. Administration of DZR 30 min prior to DOX significantly protects the heart from injury. We investigated the gene expression changes in heart tissue on Affymetrix RGU34A arrays in spontaneously hypertensive rats (SHR). Adult male SHR were given 2 or 3 weekly injections of 3 mg/kg DOX +/- 50 mg/kg DZR, or 1 mg/kg/wk DOX for 9 weeks. After 9 weeks of DOX treatment, significant cardiac pathology is observed, and genes involved in tissue injury and immune cell markers are upregulated. No significant effect on gene expression, pathology, or clinical chemistry is seen after 2 injections of DOX. Alterations in serum levels of cardiac troponin T, cholesterol, triglycerides, and total protein along with minimal myocyte damage were detected after 3 weekly injections of DOX but not DOX+DZR. DZR, which alone had little effect on gene expression, significantly reduced the magnitude of change in a subset of genes significantly altered by DOX alone. The subset of genes significantly changed by DOX treatment +/- DZR may provide mechanistic clues for the rational design of clinical strategies to further reduce DOX cardiotoxicity.
W.Tong, X.Cao, S.C.Harris, H.Hong, H.Fang, Q.Xie, R.Perkins, D.Casciano, NCTR, FDA, Little Rock AR
ArrayTrack is in-house software for management and analysis of microarray experiment data through an interactive user-friendly interface. The software is comprised of three integrated components: (1) the MicroarrayDB stores microarray experiment information that is MIAME (Minimal Information About Microarray Experiments) compliant (the de facto standard for microarray data); (2) the GeneTOOL provides analysis capabilities for data visualization, normalization, significance analysis, clustering, and classification; and, (3) the LIBs contain information (e.g, gene and protein annotation, classification, and metabolic pathway) from public repositories. Using ArrayTrack, we can select an analysis method from the GeneTOOL, apply the method to selected microarray data stored in the MicroarrayDB, and the analysis results can be directly linked to gene information in the LIBs. Moreover, ArrayTrack also allows data to be linked with other public databases.
The software will be demonstrated on-site and is available through the FDA Intranet. We welcome anyone to use the system for collaboration.
T.L.Williams1, P.E.Leopold2, S.M.Musser1, 1CFSAN, FDA, College Park, MD, 2Bioanalyte, Inc.
We describe an integrated approach for automating protein analysis of bacterial cell extracts. The method uses electrospray LC/MS to generate chromatographic profiles of proteins present in an extract, along with a software program which automates the data analysis. The software program, ProteinTrawler, automates the sequential summing, centroiding and deconvolution of multiply-charged proteins present in consecutive scans of the LC/MS analysis. This procedure generates a concise, single spectrum of proteins present in the extract, along with their retention time and relative abundance. A comparison of the method with “whole cell” MALDI analysis, demonstrates improved mass resolution and mass accuracy, along with the appearance of a greater number of proteins. Additionally, it is possible to compare protein expression among strains of bacteria by normalizing the relative abundance of similar proteins in each analysis.
B.A.Rosenzweig1, P.S. Pine1,O.E. Domon2, S.Morris2, F.D. Sistare1, 1CDER, FDA, Laurel, MD and 2NCTR, FDA, Jefferson, Arkansas
A significant limitation to the use of dual-labeled spotted cDNA microarrays is the introduction of signal variance by dye bias. Several approaches were used to assess the effects of dye bias on fluorescent signals hybridizations. Human TK6 cells were exposed to vehicle or benzo[a]pyrene diol epoxide (BPDE) for 4 or 24h, total RNA was isolated, labeled with Cy3 or Cy5, and hybridized to Human 350 Microarrays (Phase-1, Santa Fe,NM). Dye bias alone accounted for a significant component of measured differences and introduced unacceptably high numbers of both false positive and false negative statistically significant signals. However, we have found that within a given set of concurrently processed hybridizations, the bias is remarkably consistent and, therefore, measurable and correctable. We demonstrate a practical experimental design to measure and mathematically eliminate this dye bias for each set of concurrently processed microarrays by including split control sample hybridizations We show that approximately 25 - 30% of the genes identified as statistically significantly altered by BPDE treatment on a pair of microarrays can differ depending on the approach chosen to eliminate dye-bias effects. Incorporating split control microarrays within a set of concurrently processed hybridizations can eliminate the need for dye-swaps and improve experimental precision.
G.C.Jacobs1, M.A.Nicholl1, S.M.Bodeis2, J.N.Johannessen3, 1Glenelg HS, Glenelg, MD 21737, 2CVM, Laurel, MD 20708, 3CFSAN, Laurel, MD 20708
Pathogen-induced spoilage in eggs is an important economic and food safety concern. MRI technology may help clarify the process of bacterial growth and spoilage inside whole shell eggs. To evaluate MRI endpoints for following bacterial growth, eggs were inoculated with 104 CFU Aeromonas hydrophila, a human pathogen chosen because it is known to cause dramatic egg spoilage.
Egg MR images, weights, and T2 values (an NMR parameter related to viscosity) were recorded periodically, while changes in yolk and albumin volumes were determined. At one month post-infection, bacterial counts within the infected eggs were enumerated.
MRI images showed dramatic qualitative changes over time. After 12 days of bacterial growth, the yolks appeared flattened. Yolk surfaces later became fenestrated and blebbing was evident. By one month post-infection the yolks had disintegrated and the concentration of A. hydrophila had increased to 107- 109 CFU/egg. Egg mass and T2 values declined over the thirty-day period, but did so equally in treated and control eggs.
In conclusion, A. hydrophila growth dramatically changes egg morphology as visualized by MRI. T2 values decreased over time, but were not affected by the growth of A. hydrophila. MRI may be an appropriate tool for better understanding the sequence of events that occurs in eggs during spoilage from bacterial growth.
C.M.Lathers1, B.Ferguson2, S.Wang2, T.Yuan2, X.C.Zhang2, 1HFV-1, CVM, FDA, Rockville, MD, 2Physics Dept, Rensselaer Polytechnic Institute, Troy, NY
Terahertz (T-rays; 1012Hz or THz waves) electromagnetic radiation, frequency between infrared and microwave bands, monitors leaf moisture, flame chemical elements, skin burn severity, and skin cancer. It has the potential to obtain images of cells and bacteria with micron resolution Biomedical instrumentation requires high dynamic range, fast data acquisition times, and increased sensitivity to detect single-molecule thick layers. A near-field imaging microscope was constructed with micron spatial resolution and high sensitivity to sense, image, and analyze monolayer biomedical samples. T-ray tomography provides 3-D images using computed tomography (T-ray CT). T-ray CT allows 3-D structure imaging, using spectroscopic far-infrared response. T-rays are focused, transmitted through target, and detected. The target is raster scanned and rotated. Hardware extends standard 2-D THz imaging system. Filtered back projection algorithms reconstruct the material complex refractive index. This is repeated for each horizontal cross-section and frequency to build a 3-D spectroscopic image. This method was applied to a turkey femur, soaked in ethanol to remove water moisture. Size and basic bone structure were accurately recovered with ~3 mm resolution. Internal bone features were not reconstructed accurately due to limited resolution and diffraction effects, requiring more work to understand THz radiation interaction with biological molecules, tissues and bone. Opinions are authors’ and not FDA’s.
A.G.Badano1, B.D.Gallas1, K.J.Myers1, A.E.Burgess2, 1CDRH, FDA, Rockville, MD, 22Radiology Department, Brigham and Women's Hospital, Boston, MA
Display devices for medical diagnostic imaging should have a diffuse emission where luminous intensity falls according to Lambert’s law. Actual display devices are never truly Lambertian. In active-matrix liquid crystal displays (AMLCD), the departure from the Lambertian profile depends on the gray level. AMLCDs with complex pixel designs having multiple domains, in-plain switching or vertically-aligned technology are being used in primary and secondary diagnostic workstations. When these displays are viewed from different directions, the detectability of lesions across the screen is not uniform and can be severely reduced along diagonal directions (i.e., in the corners of the screen). Our purpose is to determine the effect of non-uniform changes of the angular emission on the detection of low-contrast signals in noise backgrounds. We used a temporal two-alternative forced choice approach with test images displayed at the center of the screen. A staircase technique was used during the pilot experiment to determine the detection threshold in order to increase the efficiency of our study. By comparing our experimental results with predictions from mathematical observers, we will be able to apply this methodology to assess the magnitude of the viewing angle problem in many different AMLCD technologies without lengthy and costly human observer experiments.
D.E.Godar1, F.Urbach2, F.P.Gasparro3, J.C.van der Leun4, 1CDRH, FDA, Rockville, MD, 2Temple University School of Medicine, Department of Dermatology, Philadelphia, PA, 3Thomas Jefferson University, Department of Dermatology, Philadelphia, PA, 4Ecofys, Utrecht, The Netherlands
Since 1986, people were told they get about 80% of their lifetime UV dose by the age of 18. That belief originated from the mathematical conclusion that diligent use of sunscreens (SPF 15 or higher) during the first 18 years of life would reduce the lifetime incidence of non-melanoma skin cancers by 78%. The data was misconstrued to mean that people also got about 80% of their lifetime dose by 18 (linear relationship). However, those calculations were based on the incidence of non-melanoma skin cancers being related to the square of the UV dose. Careful analysis of UV exposure data shows that American’s actually get less than 25% of their lifetime UV dose by 18. This finding also appears to be true worldwide because Australia, England and the Netherlands report a similar UV exposure pattern. UV initiated damage early in life can be promoted by subsequent exposures to progress into tumors later in life. For example, the non-melanoma skin cancer, squamous cell carcinoma is dependent on the cumulative UV dose. Thus, a better educational approach for reducing skin cancers would be to instruct fair-skinned people to protect themselves from being exposed to too much UV radiation throughout their entire lives.
N.Petrick1, A.G.Badano1, H.P.Chan2, B.Sahiner2, 1CDRH, FDA, Rockville MD, 2University of Michigan, Ann Arbor, MI
We have been investigating whether the addition of a microlens layer between the phosphor and a photodetector can improve light collection in indirect detection x-ray imagers. A simulation package has been developed for estimating optical light collection in these systems. A digital x-ray imager combining an 82-micron-thick Gd2O2S:Tb phosphor screen, a fused silica microlens layer, and a 127-micron pixel pitch photodetector (optical fill factor of 57%) was modeled. The imager had a single microlens matched to each photodetector pixel to refocus the scintillation light produced by the screen. Ray-tracing Monte Carlo simulations of a uniform x-ray source oriented perpendicular to the imager plane were conducted and the distribution of light photons across the surface of the pixelated photodetector was estimated. The light collection varied from 58% to 74% for lens indices of refraction varying from 1.2-1.6. In comparison, only 56% of the light was collected for the same imager configuration without a microlens layer. A 9% increase in light spread was observed with the inclusion of the microlens layer. This study shows that a properly designed microlens layer can produce an imaging system which collects substantially more light from a phosphor screen stimulated by x-rays.
S.M.Comfort, CDER, FDA, Rockville MD 20852
Treatment of status epilepticus usually includes intravenous phosphenytoin (PHT). In the community setting post-seizure dosing of I.V. phosphenytoin is often written as TID instead of QDay. A literature search does not suggest a pharmacological basis for this practice. This paper uses the techniques of modeling and simulation, to determine if there are significant serum concentration differences between these dosing strategies. Thirty (30) healthy subjects were simulated in a 72-Hour, Randomized, Unblinded, Crossover, Pharmacokinetic study of I.V. Phosphenytoin using QDay or TID dosing. The results demonstrate a small difference in average PHT serum concentrations (Mean Diff. = 1.27 mg/L, 95% CI [1.16, 1.37]). Analysis shows this difference is statistically significant (two-tailed paired t-test, p = 0.0001). However, both dosage regimens produce average concentrations within the accepted therapeutic range of 10 — 20 mg/L. In conclusion, TID I.V. dosing does result in slightly higher serum levels than qD dosing. However, the small difference is unlikely to outweigh the risk of possible medication errors and costs associated with more frequent dosing.
T.G.Donkar1, S.M.Madson2, F.M.Khambaty1, 1CFSAN, FDA. College Park, MD, 2ORA, FDA, Denver, CO
FDA has played an active role in PulseNet (National Molecular Subtyping Network for Foodborne Disease Surveillance) since its inception. PulseNet includes CDC, FDA, USDA, all 50 US States, Health Canada, and the UK. It relies on the centralized analyses of bacterial DNA “fingerprint” patterns generated by Pulsed-Field Gel Electrophoresis (PFGE). At CFSAN, analysis of PFGE patterns is conducted on bacteria (E. coli, Listeria, Salmonella spp.) recovered from foods by ORA District Labs. After analysis against the CFSAN database, the patterns are submitted to the National Database at CDC. Here they compared against clinical and environmental patterns submitted by partner labs. CDC notifies appropriate partners when a potential “match” is observed. Results from PulseNet analyses are used to demarcate clusters of linked illnesses and provide supporting evidence connecting an implicated food to cases of illness.
PulseNet has been a valuable tool in assisting FDA compliance programs. Recently, PFGE analysis of L. monocytogenes recovered over a six-month period from ingredients, environmental, and foods from a sushi processing plant showed that the plant had a chronic contamination problem from a single strain. Furthermore, the PFGE pattern of this strain matched that of a clinical isolate, inviting further regulatory scrutiny. In the past, such matches have assisted in establishing previously undetected epidemiological links, hence strengthening the compliance case.
Q.F.Graves, CFSAN, FDA, College Park, MD 20740
The statistical analysis and computer simulation used in providing the supporting information for the FDA decision on the citrus juice HACCP Final Rule, for the microbiological indicator (E. coli) is discussed relative to the effectiveness of the E. coli testing program based on 5 logs reduction in E. coli levels. Various sampling and testing designs were developed to verify compliance with the 5 logs reduction criterion. Consideration were given to distinguishing the “In-control” and “Out of control” situations, while minimizing the number of false alarms, using a moving window approach which also minimizes the time the process is allowed to be out of control.
G.R.Henderson1, K.Kretsinger2, M.E.Frank3, J.D.Park4, M.O.Walderhaug5, B.C.Kovak6, A.M.Fry7, J.W.Rushing8, J.J.Guzewich5, 1FDA, CFSAN, OC, ECRS, College Park, MD, 2CDC, Atlanta GA, 3ORA, Rockville, MD, 4CFSAN, FDA, College Park, MD, 5FDA, CFSAN, College Park, MD, 6ORA, Salisbury, MD, 7CDC, Atlanta, GA, 8Clemson, Univ, Charleston, SC
FDA, CDC and 22 States investigated a Salmonella Newport outbreak From July through of 2002. This outbreak had 404 confirmed cases with 285 cases having the same PFGE pattern. PFGE analyses were performed with two enzymes to verify that the S. Newport isolates had matching patterns. The pattern of the PFGE was very uncommon, at least among previous isolates from cases occurring in the Eastern Part of the United States. This strain is antibiotic sensitive. The epidemiological curve was bimodal with peaks on August 24 and October16, 2002. Epidemiological analysis indicated that tomatoes were the most likely vehicle. CDC and States used a questionnaire to obtain information about consumption of various types of tomatoes from cases and well controls. Tracebacks were conducted on two clusters of cases from the first epidemiological peak and one cluster from the second peak. All tracebacks implicated the same tomato packing shed in the mid-Atlantic Region. Inspections of the packing shed found numerous deviations from Good Agricultural Practices and Good Manufacuting Practices.
A.M.Lando1, A.S.Levy1, F.Aguilar2, 1CFSAN, FDA, College Park, MD 20740, 2CITA Institute, Universidad de Costa Rica, San Jose, Costa Rica
This study compares food handling and risky food consumption results of female food preparers from two surveys on consumer food safety behavior, attitudes, and perceptions: the 1998 FDA/FSIS Food Safety Survey (female subset n=1178) and the 2000 Costa Rican Food Safety Survey (n=1069). Results indicate that Costa Ricans have better hand washing behavior before beginning to cook and after handling raw eggs than their American counterparts, but about the same or slightly worse behavior for washing hands after handling raw meat and raw fish. Respondents in both Costa Rica and the US report low levels of consumption of raw clams and raw oysters. Respondents in Costa Rica were more likely to eat raw ceviche, steak tartare, and foods with raw eggs, but less likely to eat rare hamburgers than respondents in the US.
S.A.Koehler1, C.H.Wecht2, P.L.Schraeder3, C.M.Lathers4, 1Coroner's Office, Allegheny County, Pittsburgh, PA, 2Coroner's Office, Allegheny County, Pittsburgh, PA, 3Albert Einstein Med Ctr and Jefferson Med Coll, Phila, PA, 4CVM, FDA, Rockville, MD
A one-year examination of causes of death among epileptics examined at the Allegheny County Coroners Office, Pennsylvania was done. The incidence of Sudden Unexpected Death in Epilepsy (SUDEP) is not known. SUDEP is a diagnosis of exclusion. Approximation of incidence is ascertained by examination of all deaths examined at Coroner’s/Medical Examiners Offices, although these offices traditionally list such deaths as seizure disorders. Data collected included epidemiological characteristics, forensic features of death, medical history, medications, toxicological analysis, and the cause/manner of death. Among the 1200 autopsied cases twelve cases were identified with a seizure disorder listed on the death certificate (DC). Seizure disorder was the Immediate Cause of Death in 83.3% (9 natural and 1 accident) cases, and secondary to cardiomyopathy in one case and a glial tumor in another. Antiepileptic drugs were detected in 5 cases, but only 1 was within therapeutic range. Coroner’s 2001 data in Allegheny County found 0.75% (9 of 1200) of the autopsy cases met SUDEP criteria. However, the DC listed the death as seizure disorder. These findings support the results of a national survey of Coroners/ME showing a national underutilization of the SUDEP category. Opinions are those of the authors and do not the FDA.
H.Li, Y.Tsong, Quantitative Methods and Research Staff, Office of Biostatistics, CDER/FDA
In designing a parallel group bioequivalence study for topical antifungal products, one problem that the experimenter faces is that whether or not a patient is qualified for the study depends on the results of their baseline culture. It can take almost as much time as the length of the study to have the laboratory results. It is not unusual to find that up to as many as 50% of the patients who received treatment are disqualified during or at the completion of the study. We examined the potential applications of a group sequential method with the number of analyses and information times unfixed. Some of the issues relative to the type I error rate inflation will also be discussed.
P.T.Liu, D.A.Street, CFSAN, FDA
The standard statistical procedures for analyzing a two-way contingency table are not adequate for analyzing the CAERS data due to the “multi-symptom” and “multi-exposure” confounding that is present. “Mutual-exclusivity” among cells, that is, no two cells having any cases in common, is a necessary condition for making any meaningful statistical inferences. In this article we: 1) examine the sampling scheme and table structure, 2) define the sample unit, sample description space, and sample distribution, 3) estimate the number of exposures by the multi-capture-recapture technique, 4) reconstruct and decompose the table into numerous mini-2x2 contingency tables that do satisfy the “mutually-exclusive” requirement, 5) estimate the “odds ratio” for assessing the impact of adverse reaction, and 6) test the significance of the odds ratio and use the corresponding p-value for identifying the most problematic products.
T-H.Ng, CBER, FDA, Rockville MD
In simultaneous testing for non-inferiority and superiority, multiplicity adjustment is not necessary because the type I error rate is controlled as argued by Dunnett and Gent (1996), and Morikawa and Yoshida (1995). However, simultaneous testing for non-inferiority and superiority without any adjustment allows an experimental treatment that is expected to have the same effect as an active control to claim superiority by chance alone without risking a non-inferiority claim. As a result, 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 clams of superiority. This is a concern of simultaneous testing for non-inferiority and superiority in a confirmatory trial.
Disclaimer: These results are those of the author and do not necessarily reflect an official position of the FDA.
G.A.Pennello, CDRH, FDA, Rockville MD
Surprisingly, perhaps, simultaneous tests for non-inferiority and superiority of an experimental treatment relative to a control do not need to be adjusted for multiplicity. Because the two null hypotheses associated with these tests are nested, tests procedure that follow the closed-testing principle for controlling the overall Type I error reduce to simply testing for non-inferiority first, and if accepted, then testing for superiority. Nonetheless, some statisticians are uncomfortable with the lack of multiplicity adjustment. We present several statistical and clinical points of view that indicate that concern over unadjusted, alpha-level simultaneous tests for non-inferiority and superiority may be warranted. In particular, we discuss a Bayesian point of view.
D.R.Tavris1, B.Gallauresi1, B.Lin2, S.E.Rich1, R.D.Shaw2, W.S.Weintraub2, K.Hewitt2, 1American College of Cardiology
Abstract
Over a five year period, the Center for Devices and Radiological Health (CDRH) of the Food and Drug Administration (FDA) received 1880 reports of serious injuries and 36 reports of death associated with hemostasis devices, used to stop femoral artery bleeding following cardiac catheterization. The purpose of this study is to assess the relative risk of serious complications following femoral artery catheterization, by type of device (vs. manual compression controls) and by gender.
Data was obtained from the American College of Cardiology-National Cardiovascular Data RegistryTM, and included information from 214 institutions and 166, 680 cardiac catheterizations performed during the year 2001. Multiple logistic regression, using seven different outcomes, was used to assess the risk associated with type of device and gender, while controlling for demographic and physiologic variables, type of procedure, and several indices of co-morbidity.
Serious adverse events were reported in 1.56% of patients, the most common being bleeding (1.13%). Odds ratios for women were approximately twice as great as for men for five of the seven outcomes.
Data is presented showing apparent protective effects for the hemostasis devices, which varied by type of device (collagen plug vs. suture) and by type of procedure (interventional vs. diagnostic.)
Data is presented showing apparent protective effects for the hemostasis devices, which varied by type of device (collagen plug vs. suture) and by type of procedure (interventional vs. diagnostic.)
Y.Tsong, W.Chen, CDER, FDA, Rockville, MD
Traditionally, the safety of a treatment is measured with the probability of the adverse event of interest. The control-adjusted risk is measured by the difference between the two probabilities of the test and control treatments. On the other hand, efficacy of a test treatment is often measured with continuous or ordinal outcome. In order to assess the risk/benefit of a test treatment, we propose to convert the efficacy measurement in terms of the probability of a benefit with a defined size. Under such benefit definition, as a tool to bridge the risk and benefit comparison. The statistical framework of the probability measurement under normal assumption is presented.
Y.Tsong, M.Shen, W.P.Adams, G.J.Singh, CDER, FDA, Rockville, MD
Equivalence of the particle size distribution (PSD) plays an important part of in-vitro bioequivalence of the generic inhaler drug product. The generic product is determined to be in-vitro bioequivalent to the reference product if the difference in PSD between test and reference products is no larger than the variation among reference products by a regulatory defined equivalence limit when a standard testing equipment is used. A few innovative statistical procedures were proposed. A simulation study was conducted to identify the critical limits of equivalence. A comparison of the procedures in terms of their ability to assess PSD equivalence was conducted with a simulation study.
Y.Tsong, J.J.Shen, S.Wang, CDER, FDA, Rockville, MD
A clinical trial for bioequivalence assessment of a non-systemic generic drug product often consists of two treatment arms (i.e. test and reference treatments) and placebo. In order to make equivalence test between the test and reference treatments meaningful, the experimenter needs to show that both the test and the reference treatments are superior to placebo. The hierarchical ordering of the three tests can be planned in the group sequential design. After showing that placebo is inferior to both treatments during the inner look, it is only ethical to terminate the placebo arm. Power of the bioequivalence test may be improved over regular group sequential test plan using the error spending adaptive plan.
M.A.Zabriski, CVM, FDA, Rockville MD
Studies involving neonates from litter bearing species are problematic in defining individual animals as experimental units because animals are group housed during some phases of the experiment. During acclimation, pre-weaned animals are generally used and must be group housed with their dams. At post-weaning, animals remain group housed with littermates for welfare reasons. The poster will present methods that maintain individual animals as experimental units during the experimental period by considering ‘litters’ as blocking factors. Complete and incomplete blocking methods are used to balance treatment groups and genders within litters during the group housing phase of the experiment.
B.A.Zhen, P.A.Lachenbruch, CBER, FDA, Rockville MD
It has been FDA's position that Congress generally intended to require at least two adeqate and well-controlled studies, each convincing on its own, to establish effectiveness. Nevertheless, FDA has been flexible within the limits by congressional scheme. Under some specific circumstances, a sponsor may propose to conduct a single trial or may be willing to start the second trial only if the results from the first trial are not striking. If different strategies for approval are used, the operational alpha level (maximum chance of approving an ineffective product) may not be controlled at the same level. This report presents a method to calculate the operational alpha level for different strategies and shows that the operational alpha level using some strategies may be inflated compared to the one based on the two-trial convention. This method also permits one to examine the effect on the operational alpha level if different strategies of testing are invoked. It is concluded that consideration of the operational alpha level should be taken into account when selecting different strategies for approval.
K.Koti, G.Gupta, CBER, FDA, Rockville MD
Typically, in randomized, multi-center, placebo-controlled, parallel group design confirmatory clinical trials, the efficacy related data are collected at the baseline and at the end of the treatment phase. Let YB and YEdenote the “efficacy measurements” made at the base line and at the end of the treatment phase, respectively. The difference Y= YB - YE is identified as a primary efficacy endpoint. Data on Yare analyzed to establish efficacy of the product. In some of these investigational and new drug applications submitted to the United States Food and Drug Administration, sponsors propose to use “analysis of covariance” model on the difference Y that includes terms for treatment, center and baseline measurement YB as a covariate. In this article, we put forth arguments against including the baseline measurement YB as a covariate. We discuss the deficiency of such a sponsor proposed model comparing it with several other linear models that could have been considered to analyze the data under consideration. In particular, we point out that the left side of the sponsor proposed model assumes that YB is random while the right side assumes otherwise. This is a conceptual discrepancy and a source of controversy. We claim that the resulting partial F test lacks theoretical justification. Computer does not understand it. The proposed analysis of covariance model invariably yields a lower p-value that is statistically a fake. A simulation study that addresses these issues is presented.
Key words and Phrases: GLM procedure; Type III sum of squares; Partial F-test; p-value culture; Kefauver-Harris Amendment.
M.Desai1, A.Pinto2, A.Adigun2, J.Hilligoss2, S.H.Haidar3, N.Chang1, B.Rappaport1, S.M.Huang1, J.C.Gorski2, S.D.Hall2, 1CDER, FDA, Rockville MD, 2Indiana University, Indianapolis, IN, 3CDER, FDA, Rockville M
The pharmacokinetics and QT interval pharmacodynamics of 3 intravenous bolus doses (0.625 mg, 2.5 mg, 5.0 mg) of droperidol were studied in a 4 period, randomized, single blind, placebo controlled, cross-over clinical trial in healthy subjects. Eight subjects were enrolled and exposed to one or more doses for a total of 15 exposures (excluding placebo). The study was stopped because of moderate to severe neuropsychiatric side effects experienced by the volunteers. The mean systemic clearance + SD was 0.6 + 0.1 L/min and did not vary between the 3 doses. Within the first 5 minutes of drug administration there was a dose dependant increase in the mean heart rate change from baseline of 0.5 + 6.3, 8.6 + 6.9, 13.3 + 9.1, and 16.2 + 7.3 beats per minute on placebo and the 3 doses of drug respectively. Although the study was under-powered, there was a trend toward a dose dependant increase in the mean maximal QTc interval change from baseline (placebo subtracted) of 1, 13, and 30 milliseconds on the 3 doses respectively. Most remarkable were two outliers with QTc.changes from baseline of 77 and 79 msec on 2.5 and 5 mg doses of droperidol respectively compared to a maximal QTc change from baseline on placebo of 38 msec.
Acknowledgements: Collins JM, Jenkins JK, Honig PK, Hung HMJ, Kweder SL, Lee IP, Lesko LJ, Machado SG, Malinowski H, McCormick C, Pollock M, Sevka M, Temple R, Throckmorton DC.
L.X.Yu1, A.B.Straugh2, P.J.Faustino3, A.B.Ciavarella3, E.B.Asafu-Adjaye3, L.J.Lesko4, D.P.Conner4, A.S.Hussain4, 1Office of Generic Drugs, Rockville, MD 20855, 2College of Pharmacy, University of Tennessee, 874 Union Ave, Memphis, TN 38163, 3Division of Product Quality Research, Kensington, MD 20895, 4Office of Pharmaceutical Science, Rockville, MD 20897
These studies were designed to test the regulatory hypothesis that highly soluble and highly permeable drugs (BCS Class I) are bioequivalent under fed conditions. Metoprolol and propranolol as well as a BCS Class III drug, hydrochlorothiazide, were selected as model drugs. The relative bioavailability of two FDA approved (Orange Book AB rating) solid oral dosage forms of metoprolol, and propranolol/hydrochlorothiazide (combination tablets) were evaluated in human volunteers under fed conditions using a two-way crossover design. Eighteen subjects completed the metoprolol study while 17 subjects completed the propranolol/hydrochlorothiazide combination tablet study. The plasma concentration of metoprolol, propranolol, and hydrochlorothiazide were determined using validated bioanalytical hplc methods. In the metoprolol study, the 90 percent confidence intervals of Cmax and AUCinf were 98-118 and 92-124, respectively. For propranolol the 90 percent confidence intervals of both Cmax and AUCinf were 91-121 and 91-120, and for hydrochlorothiazide, a BCS Class III drug, the 90 percent confidence intervals for both Cmax and AUCinf were 96-107 and 96-106, respectively. These study results support the hypothesis that BCS Class I drug are likely to be bioequivalent under fed conditions. In addition, BCS Class III drugs, have the potential to be bioequivalent under fed conditions.
Board T-03 Studies in Pregnant Women: Pharmacokinetic/Pharmacodynamic and Fetal Safety
D.Kennedy, K.Uhl, K.Chapman, M.A.Miller, FDA, Rockville, MD
Pregnant women are usually excluded from clinical trials unless the product is being developed for a condition unique to pregnancy. Pregnancy may change the absorption, distribution and activity of many drugs. Information on the effects of drug during pregnancy is rarely available. Recent changes in the human subject protection regulations have eliminated the need for paternal consent for most clinical studies involving pregnant women but pregnant women are still classified as a vulnerable population. To address the fetal safety concerns, FDA has funded research to assess fetal outcomes in pregnant women exposed to amoxicillin, doxycycline, ciprofloxacin, or azithromycin, utilizing Tennessee’s automated Medicaid data system. To collect data on dosing, FDA has funded PK/PD studies with antihypertensives and antimicrobials. Either longitudinal or population PK designs are used to study PK/PD during the 2nd, and 3rd trimester. As a control, PK/PD parameters in the postpartum period are measured. These studies will help eliminate the barriers to studying drug use in pregnant women by demonstrating that is this possible to conduct such studies ethically and economically.
M.G.Paule1, R.L.Baldwin2, R.A.Flake2, D.J.Blake2, M.C.Edwards2, C.R.Feild2, J.B.Meaux2, J.J.Chelonis2, 1NCTR, FDA, Jefferson, AR, 2Arkansas Children's Hospital, Little Rock, AR
An automated system was used in children (n = 720) 5 to 13 years old to quantitate time estimation performance. The temporal response differentiation (TRD) task required subjects to hold a response lever down for at least 10 but no more than 14 seconds. Nickels were delivered for each correct response. Evidence of timing ability (a peak in the percentage of lever holds occurring in the reinforced window of ~30%) was noted in children as young as 6 years old, after which correct responding increased with age to ~65% at age 13. There were no sex differences in TRD task performance. Children with higher IQ scores made more correct lever holds. The performance of young children with below average IQ was more variable than that of children with above average IQ. For children with ADHD (n =17), TRD task performance improved significantly when they were tested within 2 hr of taking their prescribed dose of methylphenidate (MPH). Percent lever holds between 10 and 12 sec increased; % short (2-4 sec) lever holds, % burst responses (rapid lever presses) and response variability decreased. These data demonstrate that the TRD task provides useful measures of cognitive function in children and that MPH enhances timing precision in children with ADHD. (Supported by NCTR and Arkansas Children’s Hospital)
L.F.Buhse1, R.E.Kolinski1, B.J.Westenberger1, A.M.Wokovich1, C.W.Chen2, S.Turujman2, M.G.Basak2, G.L.Kang3, 1DPA, OPS, CDER, FDA, St. Louis, MO, 2ONDC, OPS, CDER, FDA Rockville, MD, 3OGD, OPS, CDER, FDA, Rockville, MD
The existing classification of dosage forms for topical drugs needs to be re-examined to ensure that definitions for different dosage forms are consistent and that dosage forms can be distinguished from each other. Current definitions of lotions, gels, creams, and ointments vary depending on literature source, market history, traditional use or application type. The purpose of this study was to obtain a scientifically based systematic classification of dosage forms for topical drugs. This study was limited to lotions, gels, creams and ointments administered to the outer surface of the body.
A variety of prescription and over the counter lotions, gels, creams, and ointments were evaluated using viscosity, loss on drying (LOD), surface tension, specific gravity, appearance, water solubility, water absorption and composition. Viscosity was the most discriminating property separating creams and lotions. LOD and composition separated ointments and creams. Composition and appearance separated gels from the other dosage forms.
J.Derbis, T.Toigo, J.Woods, B.Evelyn, D.Banks, OC, FDA, Rockville MD
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 NIH, through its National Library of Medicine along with the FDA and others, developed the Clinical Trials Data Bank, known as ClinicalTrials.gov. The FDA guidance, Information Program on Clinical Trials for Serious or Life-Threatening Diseases and Conditions, issued on March 18, 2002. It recommends to industry procedures for submitting protocol information to ClinicalTrials.gov through a Web-based protocol registration system (PRS). FDA is completing a program to educate sponsors about Section 113 and to measure the number of protocols available through ClinicalTrials.gov. Letters were mailed to sponsors of more than 2, 000 INDs in CBER and CDER. The letter informed sponsors about the guidance document and the PRS. Since publication of FDA’s guidance, 731 non-government sponsored clinical trials have been submitted to ClinicalTrials.gov through the PRS.
Y.Yang1, L.X.Yu2, P.J.Faustino1, D.A.Volpe1, C.D.Ellison1, R.C.Lyon1, 1Division of Product Quality Research, Kensington, MD 20895, 2Office of Generic Drugs, Rockville, MD 20855
In this study the permeability and solubility of several beta-blockers were determined; a correlation between drug permeability in vitro and the extent of intestinal absorption in humans was established, and a classification was made according to the Biopharmaceutics Classification System (BCS). Apparent permeability coefficients (Papp) of the model drugs (acebutolol, atenolol, labetalol, metoprolol, nadolol, sotalol, and timolol) were measured using the Caco-2 cell line. The solubilities of these model drugs were determined at 37oC over a pH range of 1.0 to 7.5. All samples were measured by high-performance liquid chromatography (HPLC) assays validated according to USP validation methods. Data obtained from the permeability and solubility studies were utilized in classifying the drugs into high/low solubility and high/low permeability groups. The permeability coefficients ranged from 1.0 × 10-7 to 4.8 × 10-5 cm/sec, and a good correlation was observed between the permeability coefficients in Caco-2 cells and the extent of intestinal absorption in humans with the exception of sotalol. The study results show that labetolol, metoprolol, sotalol and timolol are classified as BCS Class I drugs and may be candidates for a biowaiver.
M.Katzper, FDA, Rockville, MD
In looking at pain alleviation claims it is of interest to know what degree of pain is expected to be alleviated. Furthermore, it is of interest to know the percentage of people that achieve a given level of relief. There are a variety of methods and metrics devised to find out how much pain a person experiences. Therein lies a difficulty. If the effects of two drugs are measured differently, how can we tell which one is better? FDA has received applications where multiple measures of the same pain entity were taken. This provides an opportunity to inspect and compare the different measures in the same individual. We have done so for a number of the most common metrics such as the four point categorical scale, the 100 mm visual analog scale and the WOMAC (Western Ontario MacMaster Questionnaire ) and its pain subscale. There is a discrepancy between measures for individuals. Changes in pain, giving pain alleviation percentages also differ. However, linear relationships between some measures can be established for group averages.
A.S.Raw1, L.X.Yu1, M.S.Furness1, K.P.Woodland-Outlaw1, N.E.Nashed1, E.Ramos1, S.Miller2, R.C.Adams1, F.Fang1, R.M.Patel1, F.O.Holcombe1, Y.Chiu2, A.S.Hussain3, 1CDER-OGD, FDA, Rockville MD, 2CDER-ONDC, FDA, Rockville, 3CDER-OPS, FDA, Rockville
Pharmaceutical polymorphic solids of the same chemical compound differ in internal solid-state structure and therefore possess different chemical and physical properties. The differing physical and chemical attributes of the various polymorphic forms, may in turn, affect drug product manufacturability and drug product quality/performance such as stability, dissolution, and bioavailability. Based upon these considerations, a scientific perspective is provided regarding the relevance of pharmaceutical solid polymorphism to the determination of drug substance "sameness" in Abbreviated New Drug Applications (ANDAs). It is scientifically concluded that pharmaceutical solid polymorphism has no relevance per se in the determination of drug substance "sameness". In addition, three decision trees were developed based on concepts from the ICH Guideline Q6A and the Biopharmaceutical Classification System, which provide a conceptual framework for evaluating when and how polymorphs of drug substances should be monitored and controlled in ANDA submissions.
D.G.Roman, W.J.Rodriguez, R.Roberts, D.Murphy, CDER, FDA, Rockville MD
We conducted a retrospective analysis of pediatric additions to the drug labeling for new drug applications approved in the Division of Metabolic and Endocrine Drug Products of the FDA following the implementation of the pediatric rule (length of period studied: 2.4 years). Nine products with pediatric safety and efficacy data in the drug label were identified. Three drug labels (2 thyroid hormones and a multivitamin product) contain data from published literature [“505 (b)(2) applications”]. Six drug labels [two insulin analogs and four growth hormone (GH) products] reflect data obtained in pediatric efficacy studies completed as part of the respective drug development programs. These studies lasted between 7 months (an insulin analogue) and 2 years (a GH product), enrolled between 50 (a GH) and 463 (an insulin analogue) patients, and included both efficacy and safety information. In addition, the following were added to the drug label: four new pediatric indications [age-specific type 1 diabetes (2 insulin analogues), Prader Willi Syndrome (GH), small for gestational age children over 2 years of age (GH)], one new dose regimen, and pharmacodynamic data. For only one drug product the pediatric studies were the result of the Pediatric Exclusivity legislation.
H.R.Sauberman1, J.M.Cooper2, P.A.Bernhardt2, A.Pinkos2, 1ODE, FDA, Rockville, MD, 2OIVD, FDA, Rockville, MD
Manufacturer submissions to the FDA for invasive glucose measurement devices are generally 510(K) premarket notification applications. In this process, a manufacturer is asked to show that a device is "substantially equivalent" (SE) to a predicate device that has been legally approved for marketing. The concept of "substantial equivalence" will be presented in terms of the type of information needed to support an SE determination. Non-invasive glucose measuring devices, on the other hand, are treated differently from a regulatory perspective in that they are considered to be high risk medical devices that require a pre-market approval (PMA) application. The PMA process calls for scientific data from laboratory and clinical studies. This data is reviewed for substantiation of the claims for intended use. The PMA process is described in the second part of the presentation as it is applicable to non-invasive devices.
S.R.Ahmad1, A.Trontell2, J.G.Beitz3, 1Division of Drug Risk Evaluation, Office of Drug Safety, CDER, FDA, Rockville, MD, 2DSRCS, ODS, CDER, FDA, 3DDRE, ODS, CDER, FDA
Background: In the United States, acetaminophen (paracetamol) is one of the most popular and safe drugs when used within the recommended dose range. However, acetaminophen is also one of the leading causes of poisoning exposures in the U.S. In 1999, the overall number of calls to poison control centers concerning acetaminophen exposures was 108, 102.
Objectives: The objective of this review is to determine the extent of acetaminophen-related poisoning in the U.S.
Methods: The Toxic Exposure Surveillance System or TESS is a poisoning surveillance database maintained by the American Association of Poison Control Centers (AAPCC) in cooperation with the majority of U.S. poison control centers. A review of the AAPCC's annual reports from 1995 to 1999 was done to determine the extent of poisoning in association with exposure to acetaminophen products.
Results: In the five-year review period, the overall number of calls to poison control centers concerning acetaminophen exposures declined slightly but the number of fatalities increased by nearly 100% from 76 in 1995 to 141 in 1999. Overall, acetaminophen related fatalities represented 16% of the total 873 fatalities that were reported in the TESS database in 1999. Approximately 50% of acetaminophen-associated fatalities occurred in individuals who took single-ingredient acetaminophen products which are available over-the-counter (OTC). The remaining fatalities occurred in individuals who took acetaminophen combination products including butalbital, or diphenhydramine, or aspirin or aspirin/caffeine, or narcotics namely hydrocodone, oxycodone, codeine or propoxyphene. Most of these combination products are prescription products. Of the 141 fatalities in association with acetaminophen reported to TESS in 1999, fifty-five percent or 77 of the cases had suicidal intent, and 8% were unintentional. Other or unknown reasons accounted for the remaining 37% of the cases. Overall, 37% of all acetaminophen exposures documented by TESS in 1999 occurred in children under 6 years old, 22% in children and adolescents between the ages of 6 and 19 years old, and 38% in adults over 19 years. Age was unknown in 3% of the acetaminophen exposures. Thirty-four percent of children up to the age of 19 years were taking adult formulations of acetaminophen. At least 22% of children under 6 years of age used adult formulations.
Conclusions: Acetaminophen remains an important cause of morbidity and mortality in the U.S.
S.L.Brown, M.H.Reid, H.J.Duggirala, FDA, Rockville
A silicone adjustable gastric banding system was approved by the FDA in June, 2001. In a surgical procedure, the band is wrapped around the upper part of the stomach, creating a pouch that can hold only a small amount of food. The band may be adjusted by injecting or removing saline via a port that is attached to the inflatable band with a catheter (tube). This device is used for weight reduction for severely obese patients in whom other methods of weight reduction have failed. We reviewed and characterized the adverse event reports on the silicone adjustable gastric banding system during the first 14 months after approval. The FDA received 556 reports of adverse events related to the use of adjustable silicone gastric bands. Serious injuries reported for the device included eight cases of band erosion into the stomach, one with migration into the small bowel; eight cases of heartburn, reflux, regurgitation and/or vomiting; one case of “strangulation” of the stomach and subsequent bleeding; and one case due to gastric outlet obstruction secondary to band slippage. However, the overwhelming majority of reports (89.7%) received were for leaks at or near the port. A description of the reports and their significance are presented.
S.L.Brown, J.F.Todd, H.D.Luu, FDA, Rockville MD
Women with breast implants are at the same risk for breast cancer as other women and are urged to undergo screening mammography. FDA regulates both breast implants and mammography quality. We were recently alerted to the issue of breast implant rupture during mammography by a letter from a consumer. We searched the Manufacturer and User Facility Device Experience (MAUDE) database for adverse events in women with breast implants during mammography. MAUDE includes mandatory and voluntary reports submitted to the FDA on adverse events associated with the use of medical devices. Our preliminary review of reports on silicone gel breast implants, saline breast implants, and mammography equipment identified 41 reports that mentioned breast implant rupture during mammography. These events were reported to the FDA between 1992 and 2002. We review these reports and discuss characteristics of implants that may increase risk of rupture during mammography.
S.J.Casper, J.Lyndly, P.Jung, K.Cheeseman, M.Kerr, A.Santiago, B.Dunston, C.Burgess, J.Kennedy, J.Weaver, J.Kelly, D.Rogan, K.Falci, FDA/CFSAN/OSAS
Post-market surveillance of products regulated by the Center for Food Safety and Applied Nutrition (CFSAN) is an important responsibility for the Center. The collection of voluntarily submitted adverse event reports or complaints of CFSAN regulated products is used for this purpose. Previously each report would go to its respective office within the agency and would be unknown to other offices, and additionally these offices used several methodologies in order to compile, review and evaluate these reports, including, for example, various databases, or paper reports.
After an internal review of this system, CFSAN became aware of the need to develop a centralized system to monitor all adverse event reports and consumer complaints associated with a CFSAN regulated product. The CFSAN Adverse Event Reporting System (CAERS) is the solution that combines all reports involving CFSAN products into a single database. Users will be able to access the database, run queries, and generate reports on products, outcomes, illnesses, locations, and trends just to name a few possibilities. Its versatility comes from the ability to access cross-office information from a single source in a very short time. There will still be limitations but the end result will greatly enhance the work, efficiency and timeliness of adverse event collection and reporting.
A.C.Mackey, A.Brinker, J.G.Beitz, S.Kress, CDER, FDA, Rockville, MD 20856
Alosetron is a 5-HT-3 receptor antagonist marketed in the U.S. between Feb 2000 and Nov 2000 for the treatment of women with diarrhea-predominant irritable bowel syndrome. The marketing of alosetron was suspended by its sponsor in Nov 2000. We review case counts, case definitions, and clinical attributes of three unique medical entities collected in review of domestic adverse event reports for alosetron as presented before an FDA Advisory Committee in April 2002. We report inclusion of 84 cases of ischemic colitis, 6 cases of small bowel ischemia, and 113 cases of serious complications of constipation. This series of 203 total cases includes 7 reports of death and 49 cases of surgery. These data do not reveal any potential risk factors; they do demonstrate the spectrum of events that have been reported to the FDA. We will continue to monitor these events closely now that alosetron has been reintroduced in the US market.
R.A.Bright, D.E.Dwyer, CDRH, Rockville, MD
Although the incidence of tampon-related Toxic Shock Syndrome (TSS) has apparently decreased since the early 1980s, TSS and other tampon problems continue to be reported. FDA received 404 reports of 412 events during 8/1/1996 — 9/1/2002; 123 were for TSS (including 7 deaths), 34 for local infection, and 198 for tampon breakage. Emergency room care was specified for 31. The National Electronic Injury Surveillance System (NEISS) for 7/1/1999-6/30/2000 was used to estimate national rates of tampon-related emergency room visits: 67 emergency room visits were related to tampons (national estimate: 2100, with 95% confidence interval (CI) of 1100 to 3100). Cause-specific national estimates were 18 visits for TSS, 227 for pain or infection, and 2014 (CI: 1402-2626) for tampon removal, specifically because it was “stuck” for 1589 (CI: 1113-2065). For both data sources, the rates were highest for women in their teens and twenties. Comparing data from FDA and NEISS showed underreporting to FDA by 30 to 400 fold, depending on the tampon problem being reported. Tampon retention is a surprisingly frequent problem that might increase the probability of TSS.
R.A.Bright1, S.Anderson2, T.Frost2, P.Wiessner3, J.Orme2, T.Clemmer2, R.S.Evans2, M.H.Samore3, 1CDRH, FDA, Rockville, MD, 2LDS Hospital, Salt Lake City, UT, 3Univ. of Utah, Salt Lake City, UT
Problems associated with medical devices but not related to alarms were studied in 2 intensive care units (Shock-Trauma ICU and Medical-Surgical ICU) within a tertiary referral hospital. A nurse native to each unit and anthropologically-trained in observational techniques directly observed 5 rooms during each of 104 six-hour shifts (4 randomly selected shifts/ week) over a 6 month period during FY2002. Problems occurring in non-observation rooms and during non-observation times were identified by soliciting verbal reports from nursing staff and reviewing charts. Of 131 problems, 34% were adverse device events and 66% had potential for patient harm. Rates of problems/ occupied bed-day were 100 - 300 fold greater by observational methods than chart review and verbal solicitation combined. Problems encompassed >20 categories of devices; no single category accounted for >13% of problems. Grouped according to device purpose, 12 problems were associated with devices used to infuse critical medication, 13 with the patient monitoring system (Marquette), 29 with other monitors of physiologic function, 17 with devices necessary for sustaining critical function (e.g., ventilators), and 60 with devices that had non-critical functions. Rates of medical device problems in 2 ICUs detected by observational methods were much higher than those uncovered by other means.
M.H.Samore1, S.Anderson2, T.Frost2, P.Wiessner1, J.Orme2, T.Clemmer2, R.S.Evans2, R.A.Bright3, 1Univ. of Utah, Salt Lake City, UT, 2LDS Hospital, Salt Lake City, UT, 3CDRH, FDA, Rockville, MD
Problems associated with medical device alarms were studied in 2 intensive care units (Shock-Trauma ICU and Medical-Surgical ICU) within a tertiary referral hospital. A nurse native to each unit and anthropologically-trained in observational techniques directly observed 5 rooms during each of 104 six-hour shifts (4 randomly selected shifts/ week) over a 6 month period during FY2002. Poisson-method 95% confidence intervals around incidence rates were calculated. Of 81 alarm problems observed in the monitored rooms, 40 resulted in patient harm. Forty-five problems were related to respiration, oxygenation, or ventilation monitoring, 22 to blood pressure, heart rate, or arrhythmia monitoring, 9 to an infusion pump, and 1 to a feeding pump. Incidence rates per 100 occupied observed bed-days for no-harm and patient harm problems (with 95% confidence intervals) in the STICU were, respectively, 20 (12-31) and 35 (24-49). The corresponding incidence rates for the MSICU were 20 (13-31) and 6 (2-12). Most (79%) of the problems were attributed to a combination of user error and device malfunction or flawed alarm design. None of the problems were documented in the medical record or in incident reports. These rates of alarm-related device problems in two ICUs indicate an understudied important threat to patient safety.
R.P.Brown1, M.E.Stratmeyer1, L.A.Alonge2, P.J.Phillips3, 1Office of Surveillance and Biometrics, 2Office of Device Evaluation, CDRH, FDA, Rockville, MD 20852
Science-based regulation is an integral part of the Total Product Life Cycle (TPLC) process for medical devices. The use of a science-based approach to support regulatory decision-making and risk communication activities in CDRH is illustrated by the use of the results of the safety assessment of di-2-ethylhexyl phthalate (DEHP) released from polyvinyl chloride (PVC) medical devices to support the development of a public health notification and a draft guidance document. The CDRH Office of Science and Technology (OST) recently conducted a safety assessment of DEHP released from PVC devices (http://www.fda.gov/cdrh/ost/dehp-pvc.pdf and concluded that male neonates undergoing certain medical procedures represent a patient population at increased risk for developing adverse effects following exposure to DEHP-containing medical devices. Based on the results of the safety assessment, the CDRH Office of Surveillance and Biometrics (OSB) issued a Public Health Notification (http://www.fda.gov/cdrh/safety/dehp.html) providing guidance to health care providers on means to limit exposure of this patient population to DEHP. In addition, the CDRH Office of Device Evaluation (ODE) issued a draft guidance (http://www.fda.gov/cdrh/ode/guidance/1407.html) for the purpose of soliciting public comment on measures that manufacturers of DEHP-containing PVC devices can take to reduce patient exposure to DEHP. This process illustrates the successful interaction among CDRH Offices to address a public health issue.
R.P.Brown, M.E.Stratmeyer, Office of Science and Technology, CDRH, FDA, Rockville, MD 20852
Toxicity data from parenteral routes of exposure are not readily available for most compounds released from medical device materials. In the absence of data from clinically relevant routes of exposure, various route-to-route extrapolation approaches, including pharmacokinetic modeling, can be considered to obtain equivalent dose estimates necessary to derive parenteral Tolerable Intake (TI) values from oral toxicity data. However, these approaches can be data and resource intensive, and, as a practical matter, pharmacokinetic data have not been obtained for many compounds released from medical device materials. As an alternative, conversion factors (CF) have been empirically derived from a distribution of oral/parenteral LD50 ratios to facilitate the derivation of interim parenteral TI values from oral toxicity data. Based on the 90th percentile of the distribution of oral/parenteral LD50 values, it appears reasonable to use a CF of 10 when estimating an equivalent intraperitoneal or subcutaneous dose from an acute oral dose and a CF of 30 when extrapolating between acute oral and intravenous data. Limited validation of this proposed approach with long-term parenteral data suggests that this default methodology provides a reasonable approximation of parenteral NOAEL values; however, TI values derived using this approach should be considered to be interim until route-specific data are obtained.
C.E.Eirkson, FDA
The FDA Center for Veterinary Medicine, along with other government and industry members of the Veterinary International Committee on Harmonization (VICH), is developing procedures for an environmental risk assessment for veterinary medicinal products. Members of the VICH include representatives from Japan, European Commission, Australia/New Zealand, Canada and the US. This report provides an update on the development of the second phase of risk assessment guidance. The first phase of the guidance became final in March 2001 as CVM guidance number 89 (http://www.fda.gov/cvm/guidance/guide89.PDF). This first phase addresses environmental risk based primarily on limited environmental exposures. The second phase of the risk assessment guidance has been under development since October 1998. This phase consists of tiers A and B which contain specific testing and decision criteria using a risk quotient method. The risk quotient method is a deterministic approach which incorporates estimates of the environmental concentration (exposure) and no-effects concentration (effects) to estimate a relative environmental risk. The risk assessment guidance is under consideration for adoption by the harmonizing committee of the VICH.
R.K.Elespuru, R.Garrett-Young, CDRH, FDA, Rockville MD
Understanding specific causes of cancer is invaluable in the design of strategies for risk analysis of new products, and for developing preventive health measures for minimizing disease incidence. Increasing evidence indicates that mutant p53 gene function is a central factor in human cancer development. Can this information be used to develop better predictive tests for cancer risk? Can new technologies update pre-clinical screening tests for FDA regulated products that were developed 30 years ago? As part of an exploration of the potential of p53 mutant analysis in prospective cancer risk assessment, we examined the IARC TP53 data base. New searching tools and accumulated data (now ~18, 000 entries) in the database provide a “molecular epidemiology” approach to cancer causation. Squamous carcinoma of the esophagus met our criteria for examination, including defined pathology, significant incidence in different geographical or cultural regions, and identifiable risk factors (smoking and alcohol). The molecular analysis provided new information on a tobacco-specific carcinogen, and agents related to esophageal cancer in China and Iran. This information is useful in assessing the extent of the link between external causative agents and p53 mutations found in tumors. Examination of product interactions with the p53 gene in vitro may provide a new approach to pre-clinical safety assessment.
S.H.Haidar, J.J.Zhang, S.G.Machado, S.B.Johnson, FDA, Rockville
Two groups of patients with ischemic heart disease were evaluated for circadian patterns in the QT and QTc (QT corrected for heart rate) interval, inter- and intra-subject variability, and commonly used heart rate correction formulae. The first group, consisting of 45 patients, were on medication not known to affect the QT interval. The second group, also 45 patients, were on anti-arrhythmics and other drugs known to cause QT prolongation. Electrocardiogram data were collected continuously for both groups using a Holter Monitor. QT data were analyzed using Bazett’s, Fridericia’s, and a third correction formula. Bazett’s formula appeared to provide the best correction in the group with no QT altering drugs. This was unexpected, given that the average heart rate was greater than 80 beats per minute (BPM), and Bazett’s equation is known to show bias (over-correct) at heart rates exceeding 60 BPM. A circadian pattern of prolonged QT and QTc interval was detected in both groups, with a peak occurring in early afternoon. This increase averaged about 15 milliseconds in the corrected QT interval.
*Data for this project were provided by the Ischemia Research and Education Foundation (IREF).
Board W-08 Did Drugs Withdrawn in Recent Years Have Greater Risk in Women? Findings From Postmarketing Reporting of Adverse Events Data
M.C.Chen1, S.M.Huang2, J.Beitz1, R.Temple3, 1Office of Drug Safety, , 2Office of Clinical Pharmacology and Biopharmaceutics, , 3Office of Medical Policy, CDER, FDA, Rockville, MD
A review of 12 drugs withdrawn from the US market between 1997 and 2001 indicates that serious drug interactions and risk of QT prolongation and Torsades de Pointes (TdP), were among the critical factors leading to withdrawal of half of them. A gender analysis of the relevant post-marketing adverse event reports received by the FDA showed that many of these drugs had higher reporting in females than males, but in most cases this appeared to reflect more use in women and the cases/use ratios seemed generally similar for most adverse events. The exceptions seemed to be mibefradil, a CYP3A inhibitor, for all ventricular arrhythmias, and astemizole, where TdP and QT prolongation occurred far more commonly in women.
M.E.Kraeling, J.J.Yourick, R.L.Bronaugh, Office of Cosmetics and Colors, CFSAN, FDA, Laurel, MD 20708
Diethanolamine (DEA) is contained in pharmaceuticals and various cosmetic and personal care products. The National Toxicology Program (NTP) raised concerns about the safety of DEA. Therefore, studies were conducted to measure the extent of DEA absorption in human skin relevant to exposures from consumer products. DEA penetration was determined from three different product classes: shampoos, hair dyes and body lotions. Two commercial products from each class were spiked with [14C]DEA and applied to excised viable and non-viable human skin in flow-through diffusion cells. Products remained on the skin for 5 min, 30 min and 24 hours for shampoos, hair dyes and body lotions, respectively. At the end of 24 hours, most of the DEA that penetrated was found in skin with only small amounts absorbed into the receptor fluid: 0.08%, 0.09% and 0.9% for shampoos, hair dyes and body lotions, respectively. In 72-hour studies, only small amounts of additional DEA were absorbed into the receptor fluid. In repeat dose studies with a lotion, DEA appeared to accumulate in skin (29.2%) with very little diffusing out into the receptor fluid. Therefore, skin levels of DEA should not be included in estimates of systemic absorption used in exposure assessments.
C.M.Lathers1, S.A.Koehler2, C.W.Wecht2, P.L.Schraeder3, 1HFV-1, FDA, Rockville, MD, 2Office of the Coroner, Allegheny County, Pittsburg, PA, 3Albert Einstein Med Ctr, Philadelphia, PA
2001 records from the Allegheny County Coroner’s Office, Pittsburgh, PA, jurisdiction of ~1.3 million people, were reviewed to evaluate SUDEP incidence, postmortem findings, and AED levels. Of 6000 cases investigated, more than1200 autopsies were done, including 12 with seizure disorder. Toxicological screens were done on blood, bile, urine and eye fluids. Eight were on phenytoin; no AED level was detected in 3; 4 had subtherapeutic AED levels and only 1 had therapeutic levels. Of these 8, 2 were also on neurontin. In 1, both phenytoin and neurontin levels were 0 and in 1 the phenytoin was subtherapeutic and neurontin level was 0. One victim on phenytoin was also taking valproic acid and both levels were subtheraputic. One victim with subtherapeutic phenytoin had subtherapeutic lamotrigine. Only 1 of 12 SUDEP had therapeutic levels of phenytoin and carbamazepine. 1 victim was prescribed clonazepam with 0 level; 3 of 12 SUDEP had no known AED prescribed and none detected. In summary, AED levels at autopsy were 0or subtherapeutic in 11 of 12 SUDEP. Lack of AED levels appears to be a risk factor for SUDEP Patient compliance is important to prevent SUDEP. Opinions are authors and not policy of the FDA.
M.Bonner1, C.M.Lathers2, 1OPP, OPPTS, EPA Washington, DC, 2CVM, FDA, Rockville, MD
Risk assessment is the method of systematically identifying and assessing factors that influence the probability and consequences of a negative event occurring. Risk assessment provides the basis for the primary prevention of harm, in particular to identified vulnerable populations, and assists in prioritize of programs and techniques used to protect health concerns on Earth. Space travel involves acute and chronic hazard sources of risk that are varied and include risks from contamination, biological agents, and physiological imbalances. NASA conducted the first risk assessment to address questions of accidental transfer of Earth microbes to Mars during the first unmanned flights. This risk methodology directly benefited all living on Earth as the methodology was expanded to address many questions related to human and veterinary public health. Both risk assessment and risk communication programs were developed by NASA to address this program. The primary innovation of this approach is now widely used in risk assessment to determine probabilities of initiating events and to then assess probabilities conditional on the initiating event for subsequent events leading to the adverse outcome of concern. Risk assessment methodologies allow for the effective recognition and control of current and future risk for protection of the Earth and humanity. Opinions are authors and not policy of FDA.
B.L.Parsons, F.A.Beland, L.S.Von Tungeln, R.R.Delongchamp, P.Fu, R.H.Heflich, NCTR, FDA, Jefferson, AR
Allele-specific Competitive Blocker-PCR (ACB-PCR) is a sensitive, quantitative method for measuring mutations important for carcinogenesis. ACB-PCR, which can detect mutant fractions as low as 10-5, was used to measure the frequency of H-ras codon 61 CAA to AAA mutation in B6C3F1 mouse liver DNA. Mutant frequencies, at eight months post-treatment, were determined for mice receiving 0.3 mmol of 4-aminobiphenyl or the DMSO vehicle as newborns. Three independent analyses were performed on the twelve mice in each group, with concurrent analysis of mutant fraction standards. Both the prevalence and frequencies of mutation were higher in treated animals than in controls. The mutation was detected in 66.7% of 4-ABP-treated mice and 50% of DMSO-treated mice. The average mutant fractions detected in treated and control mice were 45.1 x 10-5 and 1.5 x 10-5, respectively. Measurements of 4-ABP-treated mice showed greater variability than those of control mice, consistent with chemical-induced clonal amplification of initiated cells. This mutational response can be correlated with tumor outcome. By 12 months, tumors develop in 79% and 8.3% of B6C3F1 mice treated as newborns with 4-ABP and DMSO, respectively. These results support the idea that oncomutation frequency is a useful metric for estimating cancer risk.
J.J.Yourick1, A.Marks2, R.Y.Lochhead2, M.E.Kraeling1, R.L.Bronaugh1, 1OCAC, CFSAN, FDA, Laurel, MD, 2Dept. of Polymer Science, Univ. of Southern Mississippi, Hattiesburg, MS
Diethanolamine (DEA) functions in cosmetics as an emulsifier, thickener, wetting agent, detergent, and foam booster. Unreacted DEA concentrations in DEA condensate cosmetic raw materials can reach 20%. [14C]DEA skin absorption was measured using a spiked consumer lotion applied at 3.0 mg/cm2. DEA was applied to skin in diffusion cells for 24 hr, then the skin was washed. Absorption was measured for up to 72 hr using flow-through diffusion cells. In rat skin, the percentage of applied dose absorbed over 24 hr was 1.4 % with approximately 4% in skin. In 72 hr studies, the percentage of applied dose absorbed was 1.6% with approximately 4% in skin. At 24 hr, there was 1.9 percent of the applied dose remaining in both the stratum corneum and the viable epidermis/dermis. DEA skin levels did not decrease over 72 hr. Little DEA found in skin at 24 hr diffused through the skin into the receptor fluid. Therefore, DEA skin levels should not be considered when estimating DEA exposure.
M.Cisar, J.Whitley, M.E.Haffner, OPD
2003 marks the 20th anniversary of the Orphan Drug Act. Although each ‘orphan’ disease affects less than 200, 000 patients in the U.S., an estimated 25, 000, 000 Americans suffer from orphan diseases. The Office of Orphan Products Development (OPD) provides several economic incentives including marketing exclusivity, tax credits, and grants to facilitate development of qualified products to treat rare diseases. To date, OPD has facilitated the approval of 238 drugs and biological products. The OPD grants program is the largest source of extramural clinical research grants in the FDA. With an annual budget of $13.3 million, OPD grants often bridge the critical gaps between basic research, clinical development, and marketing approval. OPD funds clinical studies of drugs, biologicals, devices, and medical foods for treating rare diseases. OPD has funded over 400 clinical studies resulting in 29 new product approvals.
N.Alderson, S.F.Ali, S.Bond, B.Coles, A.Debrabant, P.J.Faustino, M.Grant, A.Khan, M.Kulka, A.Lucas, D.Momcilovic, T.Patterson, C.Rosebraugh, M.Spence, S.Stern, J.Ward, G.Wood, Office of the Commissioner, FDA, 5600 Fishers Lane, Rockville, MD 20857
The Committee for the Advancement of FDA Science (CAFDAS) is a chartered, internal advisory committee to the Commissioner, Associate Commissioner for Science, and the Senior Science Council. Designed to address FDA-wide science issues from a working-level scientist perspective independently of center or discipline, CAFDAS is composed of two representatives appointed from each Center and ORA to serve three-year terms. The primary objective of CAFDAS is to provide a forum for working level scientists to advise and assist the Commissioner in promoting an increase in the overall effectiveness and productivity of FDA science. Most importantly however, CAFDAS is a conduit for ideas and concerns from working-level scientists to senior FDA management concerning the state of science within the FDA. This activity includes providing constructive guidance and solutions to scientific challenges the agency currently faces as well as to future workforce needs. In the past CAFDAS has addressed such topics as leveraging, debated the need for the implementation of mini-sabbatical programs, and has been a strong advocate of quality-of-work life issues. Recently, CAFDAS was instrumental in the evaluation and selection of OSHC intra-agency research grants. The committee’s focus for the coming year is to provide FDA scientists with information on funding sources available to agency scientists and the resources necessary to compete for such funding.
N.Alderson, S.F.Ali, S.Bond, B.Coles, A.Debrabant, P.J.Faustino, M.Grant, A.Khan, M.Kulka, A.Lucas, D.Momcilovic, T.Patterson, C.Rosebaugh, M.Spence, S.Stern, J.Ward, G.Wood, Office of the Commissioner, FDA, 5600 Fishers Lane, Rockville, MD 20857
The Committee for the Advancement of FDA Science (CAFDAS) focus for the coming year is to provide FDA scientists with information on funding sources available to agency scientists and the resources necessary to compete for such funding. CAFDAS has compiled a series of documents that identify resources and procedures to use within FDA’s leveraging program. This single information resource is designed to give all FDA scientists, both research and regulatory, a starting point for pursuing external resources and partnerships ranging from Memoranda of Understanding (MOUs) to Cooperative Research and Development Agreements (CRADAs). These resources provide the FDA scientist with information about leveraging activities, experiences of others that have developed leveraged projects, and some of the pitfalls in establishing such collaborative efforts. This information is linked to the work of the FDA Leveraging Taskforce (http://intranet.fda.gov/leveraging). At this site, you will find a wealth of information to guide you in your search to seek out collaborative research partners and research projects. It is our goal that through this Series on Leveraging Techniques, CAFDAS can effectively serve FDA scientists in identifying and pursuing leveraging options. If you would like to learn more about CAFDAS, please check out our website located at SCIENCE FIRST
H.S.Ko, CBER, FDA, Rockville, MD
Many biologic products derived from fractionation of blood are subject to labeling requirements for prescription drugs. Because of their unique source material, they may pose special challenges in pregnancy labeling. A review was conducted in the approved package inserts of 37 marketed blood-derived biologic products (BDBP), randomly selected on the basis of availability in the PDR or from the manufacturer. These included coagulation factors, immune globulins, specific antibodies, albumin, and enzyme inhibitors. All 37 products are labeled under Pregnancy Category C with language pertaining to lack of animal reproduction studies. Some Pregnancy subsections include wording regarding use in nursing women, while one actually contains data from reproductive toxicity studies in animals. Two mention lack of harm to fetus, but one does not recommend use in pregnant women. Pregnancy information is also found in other parts of the label, including Information for Patients (5), Dosage and Administration (3), Precautions (2), Clinical Pharmacology (1) and Laboratory Tests (1). In addition to the potential effects of active ingredients, the risks of BDBP to the fetus may come from transmissible pathogens or methods used to inactivate them. These additional issues have to be considered in the light of usage for different indications, at different stages of pregnancy, or with inadvertent exposure. More useful labeling of these important products would provide appropriate risk and benefit information for the management of the pregnant woman.
D.Batson1, D.Beranek2, P.Bianchi3, P.Bourkland4, H.Dallas5, C.Leggett6, W.Long5, D.Murphy7, J.Olson3, P.Spitzig3, 1CVM, Laurel, MD, 2NCTR, Jefferson, AR, 3CDRH, Rockville, MD, 4CDER, Rockville, MD, 5CFSAN, College Park, MD, 6ORA, Rockville, MD, 7CBER, Rockville, MD
The Leveraging Points of Contact (POCs) continue to represent their Centers fostering leveraging across the Agency and especially with outside partners. Leveraging partners include the regulated industry, Federal, state and local regulatory agencies, academia and consumer groups. Projects have been educational, scientific, inspectional and compliance oriented. A website has been established as an agency-wide tracking system for collaborative projects ( http://lat.intranet.fda.gov:591/leveragingresources/ ) and a related database of opportunities for grants and other innovative funding sources directed at the Agency’s scientists ( http://lat.intranet.fda.gov:591 ). The Leveraging POCs share best practices, use of legal mechanisms, and other resources as needed. They work to encourage the use of leveraging among FDA staff and provide advice and counsel to those involved in implementing leveraging activities.
E-mail Address: OCLeveragingContactsinFDA@oc.fda.gov.
D.C.Roselle, L.X.Yu, FDA, Rockville MD 20855
The Office of Generic Drugs (OGD) recently established the OGD Education and Training Committee to promote science education and communication, and to enhance professional development. The Committee consists of members from all Office Divisions, the Immediate Office, and IT, and represents the different scientific disciplines within OGD. Committee members function as project managers for the individual and ongoing educational offerings that they organize. This arrangement provides OGD with a wide variety of scientific educational opportunities that traverse scientific disciplines and that facilitates scientific communication between OGD Divisions. The committee organizes numerous scientific seminars each year that are presented by speakers from OGD, CDER, FDA, the Federal Government, Industry, and Academia. Many ongoing programs have been established and are presented by OGD Review Scientists or Pharmacists. They include monthly or bi-monthly Round Table Discussions, Brown Bags, and Generic Drug Review Open Forums; colleagues from other offices within CDER are often invited and participate. OGD has also recently established a Regulatory Science Training Series. This program offers full or multi-day symposia on scientific issues of current interest and high priority to OGD. Recent symposia include programs on Polymorphism in Drug Substance and Drug Product, Controlled Release of Solid Oral Dosage Forms, and Injectables.
L.A.Verrill, FDA
The Consumer Studies Team - in the Division of Marketing Studies (DMS) in the Office of Scientific Analysis and Support (OSAS) in CFSAN - is spotlighted. Consumer Studies is an internationally recognized, full-service, social science research team. Team members include professionals in psychology, sociology, economics, and policy studies, all with extensive experience in social science research methods. Responding to specific requests from program offices, the team conceives, designs, and administers consumer oriented research, often conducting literature reviews, focus groups, consumer surveys, mall-intercept studies, and experimental research methods. New methods include Internet panel research and experimental auction methods. Close consultation with the program offices ensures that their consumer information needs are met. Research results are provided in the form of briefings, memos, short papers, or full-scale reports which include both text and graphics.
R.A.Benner1, W.F.Staruszkiewicz1, W.S.Otwell2, 1OS, WSL, FDA, Laurel, MD, 2University of Florida, Gainesville, FL
Putrescine, cadaverine, and indole production capabilities of bacteria isolated from wild-caught and aquacultured Penaeid shrimp were evaluated. The numbers and types of microorganisms responsible for the production of putrescine, cadaverine, and indole in wild-caught and aquacultured shrimp decomposed at temperatures ranging from 0-36C increased with decomposition temperature and time. Vibrio spp. were more prominent in aquacultured Nicaraguan shrimp (L. vannamei) than in wild-caught domestic shrimp (L. setiferus and L. brasiliensis) decomposed at temperatures ranging from 0-36C. The only amine-producing (putrescine) microorganism isolated from wild-caught and aquacultured shrimp at all temperatures of decomposition (0, 12, 24, and 36C) was Shewanella putrefaciens. Based on putrescine production by S. putrefaciens at 0 and 12C and putrescine production by S. putrefaciens, Vibrio spp., and Morganella morganii at 24 and 36C, putrescine demonstrated the most microbiological potential to be an effective chemical indicator of decomposition in wild-caught and aquacultured Penaeid shrimp.
V.M.Chenault1, A.A.Cotterell2, C.P.Schnupp2, 1CDRH, FDA, Rockville MD, 2USUHS, Bethesda MD
Approximately 16 million Americans have diabetes mellitus (DM) with concomitant heart disease, stroke, blindness, kidney disease and increased risk of amputation. The incidence of diabetes and obesity among Americans has risen sharply in the past decade, putting millions more Americans at higher risk for medical complications. Diabetes is one of the diseases targeted by HHS Secretary Tommy G. Thompson under the department’s initiative “Steps to a Healthier US”. More and improved animal models are needed to study the pathogenesis of diabetes, provide information to improve diagnostic device development and thus ameliorate or prevent illness. Psammomys obesus (sand rat), a rodent in the gerbil family, is native to the sub-Saharan desert and Northern Africa. Upon eating a high energy diet, the sand rat (SR) develops clinical symptoms consistent with DM and as such, is a good research model. However, it is not easily bred in captivity. CDRH/FDA has the only viable, pathogen-free, breeding colony in the US. This colony is currently used in many research initiatives at FDA, other government agencies and universities. The research goal was to study environmental enrichment aids to improve colony health, development and breeding. Use of social interaction and treat administration positively impacted SR research subjects.
R.M.Fahmy1, W.G.Marnane1, D.M.Bensely1, A.Tatavarti2, K.Tata3, D.Jappar2, G.R.Hollenbeck2, S.W.Hoag2, 1Universirt of Maryland
Purpose: Process analytical technology is an exciting new technique that could replace the laboratory batch testing done today with online in-process testing, which in theory, will allow the lots to be released immediately upon the completion of manufacturing. While the studies have done to date have shown that NIR has great potential, the robustness, accuracyand precision of NIR still need to be better charactrized. Method: 8 different formulas contain sulfamethazine, corn starch, and mg-stearate to make the tablets, sulfamethazine and corn starch were granulated using high sheer technique.The tablets were compressed on a Stocks B2 rotary tablet pressrunning at 30 rpm.Each sample was scanned in reflactance mode by NIR. Tha data were analyzed using the Vision software (FOSS NIR System Inc., Silver Spring, MD). Conclusion: Our preliminary results suggest that NIR is comparable to the traditional analytical technique. However, sampling strategies must be reexamined.
H.A.Hahm1, L.L.Augsburger2, 1Office of Generic Drugs, CDER, FDA, Rockville MD 20855, 2University of Maryland Baltimore, Baltimore MD 21201
A novel automatic disintegration tester (ADT) was designed and constructed to give a digital output of disintegration time and tablet thickness. A central composite experiment design was used to test the effects of Hubersorb® 250 NF (calcium silicate), Explotab®, DiPac®/Xylitab® as fillers, and compression pressure on DT50 (time required for tablet to disintegrate to half its original thickness), tensile strength, friability, expansion coefficient (n), and expansion rate constant (k). The values for n and k were calculated using an equation reinterpreted from Caramella et al. which could categorize the tablet disintegration mechanism as diffusion-controlled (n<1) or interfacial-controlled (n>1). The optimal levels for achieving the shortest DT50 were 60% Hubersorb®, 15% Explotab®, q.s. Xylitab®, and 40MPa compression pressure. Using this optimal formulation, when only the Hubersorb® level was varied from 0 to 85.5% the n value increased from 0.33 to 1.4, indicating an alteration in tablet disintegration mechanism. When the disintegrant type was varied (from Explotab® to AcDiSol®, Primojel®, or Polyplasdone® XL, both the values for n and k changed. Polyplasdone® XL’s DT50 and n were significantly affected by moisture. The novel ADT was a powerful tool in determining the disintegration efficiencies and disintegration mechanisms of rapidly disintegrating tablets.
A.Kornhauser1, R.R.Wei2, J.Z.Beer3, S.G.Coelho3, S.Y.Miller3, B.Z.Zmudska3, V.J.Hearing4, 1OCAC FDA College Park MD, 2FDA College Park, 3FDA Rockville
Hydroxyacids are organic carboxylic acids classified into the alpha and beta types according to their molecular structure. Both AHAs and BHAs are widely used as active ingredients in drugs and cosmetics. Their use has dramatically increased in the last decade. Topical treatments can alter the optical properties of skin either by reducing the differences in the refractive indices at the interfaces (oils), or by removal of the superficial (desquamating) cell layers (exfoliative agents). These possibilities can be investigated by applying the products to normal human skin for a specified period of time and then estimating the degree of direct UVR damage to epidermal keratinocytes through specific biomarkers, following a single UV challenge. Three test products are being investigated in this study: glycolic acid, salicylic acid, and vehicle. The products resemble a standard cosmetic formulation. In this study, the backs of 20 Caucasian subjects are being treated 5 days weekly, for four weeks, with either 10% glycolic acid (pH 3.5), 2.0% salicylic acid (pH 3.5) or placebo in a randomized double-blinded study. Sites within each area are then being exposed to one minimal erythema dose of solar simulated light. Erythema is evaluated both visually and instrumentally. DNA damage and the number of sunburn cells are determined from specimen obtained from shave biopsies by histologic methods. Presently, data obtained from five subjects are being analyzed. This study will contribute data needed for risk evaluation of topically applied agents in combination with sunlight.
J.Lozier1, M.Kraman2, T.Gridley1, B.McCright2, 1The Jackson Laboratory, Bar Harbor, Maine 04609, 2DCGT/OCTGT, Bethesda, MD 20892
Notch signaling is a highly conserved intercellular signaling pathway used by multicellular animals to specify cell fate decisions during the formation of complex structures. To study the role of Notch2 in vivo, a targeted mutation was introduced into mice. Due to unexpected alternative splicing around the neomycin resistance gene this allele of Notch2, Notch2del1, turned out to function as a hypomorphic allele. Mice homozygous for the Notch2del1 allele display developmental defects in the kidney glomerulus, the intrahepatic bile ducts, and the heart. Mice heterozygous for both Notch2 and a potential ligand, Jag1, exhibit most of the phenotypes associated with Alagille’s syndrome, a human disease associated with mutations in Jag1. To further study the role of Notch2 in regulating epithelial cell development during mammalian organogenesis, we have introduced conditional null and conditional gain of function alleles of Notch2 into mice. These tools will help determine the requirements for Notch2 in pluripotent epithelial cell populations in a number of organs including the kidney, liver, and heart. The identification, characterization, and isolation of Notch2 expressing cells in these organs may enable novel tissue engineering projects targeted at replacing damaged human tissues.
R.J.Garcia1, R.Reimschuessel2, 1University of Maryland, MEES, 2Office of Research, CVM, FDA, Laurel, MD
Three recent factors have resulted in a need to understand the dynamics of oxytetracycline hydrochloride (OTC) in aquatic environments: the discovery of pharmaceuticals in sewage, surface, ground and drinking water supplies; the general intensification of aquaculture production methods; and the rising interest in using aquaculture effluent waters to grow vegetables, spices, and algae for human and domestic animal consumption. Since most OTC is excreted as biologically active molecules, concentrations of OTC found in aquaculture effluents could be absorbed by submerged aquatic plants, green algae, and cyanobacteria and affect their development, growth and metabolism. We examined the responses of one of our model algae, Chlamydomonas reinhardtii, to OTC 0.01 to 1000 ug/mL. Preliminary fluorescence microscopy results show OTC is absorbed by C. reinhardtii cells. Additionally, C. reinhardtii were assayed in serial dilutions of OTC and Sueoka’s High Salt Media (SHS) to measure changes in oxygen production. Controls included a known toxic dose of OTC (1 mg/mL), plain SHS with and without algae, 100 mM sodium sulfite (reducing agent), and serial dilutions of SHS plus OTC without algae. Results of bioassays with BD FalconTM Oxygen Biosensor Systems showed a dramatic increase of oxygen in both the algae and the cell free systems. The production of oxygen in the cell free systems may be due to photo-degradation of OTC, which undergoes oxidative degradation generating reactive oxygen species (ROS). This generation of ROS by OTC may cause cell injury and could have biological implications for primary production in integrated aquaculture systems.
A.Ritzert, CFSAN, College Park, MD
The diagnoses of Bovine Spongiform Encephalopathy (BSE) in cattle in 1986 was followed a decade later by the discovery that prions from infected cattle can cross the species barrier and convert normal prions in the human brain to a newly diagnosed version of Creutzfeldt-Jakob Disease. Variant Creutzfeldt-Jakob disease (vCJD) differs from the other forms of CJD not only in the way the disease is transmitted or metastasizes in victims, but also in some important disease characteristics, such as age of the victim at diagnosis, length of life after diagnosis, and mental capability. As vCJD is a recently diagnosed form of Transmissible Spongiform Encephalopathy (TSE), the social costs of the disease have mostly remained uncalculated. Estimating the monetary cost of this devastating disease poses many problems for economists. This research demonstrates one method to estimate prospective costs of early mortality, patient symptoms, and healthcare for vCJD patients in varying stages of this fatal disease.
Ali M. Ardekani1, Vince A. Fusaro2, Sally Ross2, Ben A. Hitt3, Eugene Herman4, Jun Zhang4, Alan Knapton4, Nader Rifai5, Steven E Lipshultz6, Frank D. Sistare4, L.A. Liotta2, E. F. Petricoin III1, 1FDA-NCI Clinical Proteomics Program, Dept. of Therapeutic Proteins/CBER, Food and Drug Administration, Bethesda, MD, 2FDA-NCI Clinical Proteomics Program, Laboratory of Pathology, DCR, NCI, NIH, Bethesda, MD, 3Correlogic Systems, Inc. Bethesda, MD, 4CDER, FDA, Laurel, MD, 5Clinical Chemistry, Children's Hospital, Boston, MA; 6University of Rochester Medical Center, Rochester, NY
Introduction: Doxorubicin is a highly effective antineoplastic agent of the anthracycline family that has been in clinical use for many years. Various types of cardiotoxicity have been demonstrated to be associated with its clinical use and chronic toxicity is the most common form of doxorubicin-induced cardiac toxicity. Currently, a limited number of serum assays involving the screening of troponin isoforms are used to detect cardiotoxicity in patients undergoing chemotherapy treatment with doxorubicin and no clinical diagnostic biomarker is available to detect cardiotoxicity at the early stages of treatment before heart damage occurs. Methods and Results: Proteomic spectra were generated by mass spectroscopy (surface enhanced laser desorption) and proteomic pattern analysis using an artificial intelligence based computer algorithm was performed on 105 serum samples from 20 children with leukemia who were on anthracycline treatment. These samples were serially taken and Troponin T measurement was obtained. A total of 76 samples were determined to be negative (troponin score below 0.01) and 29 positive. For proteomic analysis a training set comprised of 53 samples (38 negative and 15 positive) was selected randomly and analyzed by our bioinformatics tool. A pattern was found that correctly classified the entire training set. Eight clusters containing either positive or negative patients were formed using the combined relative intensities from six ion values. The remaining 52 samples were analyzed as a masked set for testing and validation (38 negative and 14 positive in Troponin T measurements). Using this proteomic pattern, a specificity of 90.2% and sensitivity of 90.9% was obtained. Using the same methodology we also analyzed the proteomic patterns in 98 SHR rat serums that were treated weekly for 10-12 weeks with either vehicle or doxorubicin. Based on histopathology studies and Troponin T measurements, 79 samples were negative and 19 positive. For proteomic analysis a training set comprised of 48 samples (40 negative and 8 positive) was selected randomly and analyzed and a set of clusters were found which correctly classified the entire training set. From the 50 samples used as a test set, 39 samples were accurately predicted as negative and 11 samples with confirmed cardiotoxicity were predicted as positive, giving a specificity and sensitivity of 100%. Conclusion: These results indicate that the combination of mass spectrometry generated data and proteomic pattern diagnostics are potentially powerful tools to discriminate between normal and disease states in a small amount of serum. Furthermore, obtaining more than 90% specificity and sensitivity is indicative of applicability of these technologies in early detection of drug-induced toxicities in either patient-tailored therapies and/or pharmacology/toxicology studies in animal models.
Sharon Miller1, Sergio Coelho1, Paul Howard2, Andrija Kornhauser3, Rong Rong Wei3 and Janusz Beer1, 1Center for Devices and Radiological Health, Rockville, MD 20850, 2National Center for Toxicological Research, Little Rock, AR 72079, 3Center for Food Safety and Applied Nutrition, College Park, MD 20740
Exposure to UV radiation is known to damage DNA and other components of human skin. It is generally known that such damage can lead to health problems such as skin cancer. However, this does not deter approximately 1,000,000 people per day in the US from seeking a tan through the use of artificial UV sources, i.e., sunlamps. Many more people (exact number unknown) seek to obtain a tan from the sun. The cumulative dose of UV that can be attained through the regular use of sunlamps exceeds typical cumulative doses that most people (indoor workers) could receive from the sun. The most common reason that people state for seeking a tan is to improve their appearance. Additionally, there have been recent reports in the news about the positive health effects of UV exposure. This includes increased Vitamin D production in the skin which can reduce the risk of osteoporosis and perhaps even reduce the risk of some cancers. It should be pointed out that the so-called "benefits" of tanning are controversial and no consensus has been reached amongst the experts. It is important for people who seek a tan through UV exposure to be aware of both the risks and potential benefits. FDA plays an important role in protecting the sunlamp user by requiring manufacturers of these devices to adhere to certain standards of safety.
The FDA Photosciences Network
Each year over 1 million new skin cancers are diagnosed in the U.S. of which about 20,000 are fatal. Some of these cancers may be caused by the use of FDA regulated products such as medical devices, sunlamps, cosmetics and photosensitizing drugs, or prevented by the use of effective sunscreens. For this reason, FDA has established a Facility to conduct human photosciences research. The results of this research support development of national and international standards and guidance documents. This research also supports risk analyses of both pre- and post-market products that are regulated by FDA.
The Facility consists of a small clinic with dedicated space for photography, UV exposure, non-invasive measurements of skin and tissue sampling/processing. Currently, the 3 ongoing projects involve CDRH, CFSAN, NCTR and NCI and concern testing of UV response in different racial/ethnic groups, artificial tanning, and interactions of UV and cosmetics.
The Facility is well-equipped and available for FDA researchers from all centers for studies on FDA-regulated UV-emitting, UV-transmitting and UV-modifying products on human skin.
R.D.Beger, K.J.Holm, D.A.Buzatu, J.O.Lay Jr., J.P.Freeman, J.G.Wilkes, D.W.Miller, NCTR, FDA
A spectroscopic data-activity relationship (SDAR) model based on 1D 13C NMR spectral data were developed for 108 compounds. NMR data were used as fingerprints to reflect the electronic and structural characteristics of the compounds. The SDAR model segregated the 108 compounds into 20 strong, 15 medium and 73 weak relative binding classifications. The SDAR model produced a q12 of 0.75. Simulated 2D 13C-13C COSY NMR spectral data were used to develop a quantitative model for 130 compounds. Using the NMR spectral assignments for predicted carbon chemical shifts to identify nearest neighboring atoms formed the 2D COSY spectra. We call the use of such patterns for model building comparative structural connectivity spectra analysis (CoSCoSA). A CoSCoSA model achieved an r2 of 0.83 and a q262 of 0.75, and when used to predict 27 external compounds, qpred2=0.60. The averaged, combined predictions from both CoSCoSA and CoMFA models of internal and external data sets produced more rugged correlations with strong binding estrogenic compounds than individually. The ease of use, accuracy, and speed of SDAR modeling may facilitate high throughput virtual drug discovery and toxicological prediction. ACD Labs has licensed SDAR from NIH and plans to offer a software package soon.