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Sigma Xi
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CATEGORY A: BIOLOGICAL ENDPOINTS, BIOMARKERS, SURROGATE MARKERS, AND IMAGING TECHNOLOGIES
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Display contrast changes with viewing-angle in medical AMLCDs: measurement methods and effect on observer detection performance
A. Badano, D. H. Fifadara, CDRH, FDA, Rockville, MD
The diagnostic value of medical imaging modalities that rely on display devices for interpretation, review, consultation or guidance for localization is affected by the display image quality. As a consequence, the assessment of image quality in medical- and consumer-grade display devices has become a key element in the characterization and quality control of local and teleradiology applications of diagnostic modalities. Increasingly for most consumer and high-end imaging applications, the active-matrix liquid-crystal display (AMLCD) is the device technology of choice due to its good image quality, small footprint, and low cost. However, and in spite of recent progress, LCD monitors are not Lambertian, and therefore display contrast varies significantly with viewing-angle. This has generated the need for manufacturers and users to develop methods for the characterization of the angular emission, and to study its implication on diagnostic performance. Currently, the two most widely used methods to measure the angular luminance response of AMLCD are the goniometric and the Fourier-optics methods. We show that while both methods have their advantages, there can be differences between them as large as 38%. The effect of these angular variations on visual detection tasks is then studied by measuring detection performance of human observers. The results prove that changes in display contrast translate into changes in subtle signal detectability.
Metabolomics analysis to find biomarkers of disease state
C. Beecher, Metabolon, Durham (RTP), NC 27713
Background: Metabolomics is an omics-style science that examines biochemical content of a biological sample to ascertain physiological status. When compared to "normal" tissues, disease tissues frequently demonstrate strong, statistically significant perturbations in their biochemical profiles that provide insight into the underlying pathology. We will highlight this approach with work done in collaboration with the NICHD (and for which we received the 2005 March of Dimes Research Award) on premature birth.
Methods: The total biochemical content of a biological sample is determined in order to understand the physiological status of the sample at the time the sample was collected. The detailed chemical measurements are derived through parallel mass spectral analyses of extracts of the biological sample. In this project, the samples consisted of amniotic fluid from women presenting with preterm labor. A statistical analysis revealed several classes of compounds that were found to be predictive of eventual outcome.
Results: The analysis of amniotic fluid of 113 women in pre-term labor revealed several classes of compounds that could serve as biomarkers for the medical outcome. The possible outcomes included premature birth, full term birth, or infection/inflammation. Since each of these outcomes has different medical requirements the ability to predict outcome was quite significant. The final results included many statistical approaches and was determined to have over 88% predictivity and excellent selectivity and sensitivity.
Conclusions: Metabolomic analyses show great promise in biomarker determination. In the case of pre-term labor, the markers discovered were highly predictive and medically significant.
NMR Metabonomics of Urine from Wistar Rats Dosed Separately with Kidney and Liver Toxicants
R. D. Beger1 , L. K. Schnackenberg1 , Y. P. Dragan1 , M. D. Reily2 , D. G. Robertson2 , 1NCTR, FDA, Jefferson, AR, 2Pfizer Global Research & Development, Ann Arbor Laboratories, Ann Arbor, MI
A collaboration between Pfizer and the Center of Metabolomics Research at NCTR was initiated to study the ability of NMR in combination with pattern recognition techniques to rapidly detect toxicity through analysis of urine samples. Single low and high doses of 4-aminophenol (PAP; 15 mg/kg, 150 mg/kg), galactosamine (Gal; 60 mg/kg, 600 mg/kg), and thiacetamide (THI; 20 mg/kg, 200mg/kg) were administered to male Wistar rats in groups of 4 for each toxin and dose level. Further, saline was administered to four rats as the drug vehicle control, for three toxins. Predose urine samples (Day 0) and samples from post-dosing (Day 2 and 3) were collected for each rat in metabolism cages at Pfizer and monitored by 1D 1H NMR at NCTR. Chenomx Eclipse software (Chenomx, Edmonton, Canada) was used to spectrally identify 198 metabolites in the 1D NMR spectra of urine. Partial least squares-discriminant function (PLS-DF) models of liver and kidney toxicity were developed from 8 metabolites. The metabolites used to build both the high and low dose PLS-DF models of toxicity were 3-hydroxyphenylacetate, homocysteine, 2-phosphoglycerate, 5-hydroxylysine, creatine, indole-3-acetate, sarcosine, and myo-inositol. The average of four training models of liver and kidney toxicity built from the high dose data was 98.2%. The average of four training models of liver and kidney toxicity built using the low dose data was 86.2 %.
Serological Attraction of Non-toxin Egg Protein to Staphylococcal Antienterotoxins
R.W. Bennett, CFSAN, FDA, CP, MD
Raw whole liquid and dried eggs which were purported to contain staphylococcal enterotoxin were evaluated by a number of ELISA-based methods. The initial evaluation was to establish whether the purported positive ELISA reactions were as a result of toxin antienterotoxin serological activity. Secondary considerations were to study some basic characteristics of the protein, determine whether the protein occurred in the yolk and/or white portions of the eggs, make differential observations of this protein in fertilized and non-fertilized eggs and to follow the fate of the protein during extended egg incubation at 37°C. The controls used in this study were eggs offered in retail outlets for human consumption. Three ELISA-based methods (manual polyvalent detection system, manual monovalent ELISA and an automated polyvalent enzyme-linked fluorescent immunoassay) were used to investigate this protein in eggs. To determine the part of the egg containing the protein, egg white and yolks were separated and processed separately in the same manner prior to analysis of the extracts for the protein. Generally, the ELISA results were instantaneous and strongly positive reactions with the manual and automated polyvalent systems suggesting non-specific binding of the egg protein to one or more antibodies to the staphylococcal enterotoxins. Further analysis with the monovalent ELISA showed positive reactions when egg extracts were tested separately for enterotoxins A, B, C, D, and E. In that the egg protein reacted with antibody serotypes A - E, separately, it was conclusive evidence that this protein from fertilized eggs had caused false-positive reactions and that the eggs did not, indeed, contain preformed staphylococcal enterotoxin; thereby the eggs could not be deemed to be adulterated.
Graphical User Interface (GUI) for mammography review and CAD development
S. Bhat, S. Paquerault, N. Petrick, CDRH, FDA, Rockville, MD
Introduction: Radiologists have long known that some breast cancers remain undetected on screening mammograms due to technical and human factors. To help reduce the number of missed cancers, researchers and commercial firms have developed computer-aided diagnosis (CAD) systems to aid the radiologists in detecting breast cancer. The FDA has a keen interest in understanding how these devices perform and under what situations they may systematically fail to detect lesions.
Method: In order to assist in our CAD research on these devices, we are developing a GUI for displaying mammograms and CAD results using the cross-platform scripting language Tcl/Tk. The main advantage of this programming language is that it can be ported to just about any UNIX, Windows and Macintosh operating system.
Results: Our proposed GUI has been designed to: 1) allow the user to review mammograms using a configurable display environment that allows the number of displayed images and mammographic views to be customized; 2) display the CAD detected lesions using either simple markers or the computer defined boundaries; and 3) interface directly with our CAD and evaluation software allowing researchers to quickly modify and evaluate new CAD algorithms and algorithm parameters (e.g., the classifiers, filters, and thresholds used by a CAD algorithm)
Conclusions: We have designed a GUI, which can be used both as an image review tool as well as an interface for quickly modifying and evaluating new CAD algorithms. We plan to further extend this CAD tool for 3D CT imaging as well.
Predicting clinical resistance to Gleevec treatment by in vitro applied stable isotope-based dynamic metabolic profiling (SIDMAP)
L. G. Boros, P. W-L. Lee, UCLA, Los Angeles, CA
Purpose: Gleevec has been marketed for the treatment of BCR-ABL+ leukemias after a fast-track approval by the FDA in 2001. In the meantime significant resistance to Gleevec developed partially due to an adaptation of the metabolic network to non-oxidative nucleic acid precursor ribose synthesis in the pentose cycle (Drug Discov. Today Therapeutic Strategies 1:435-443, 2004). Herein we describe the foreseen limitations of Gleevec using stable isotope-based dynamic metabolic profiling as an in vitro tool to indicate drug resistance and treatment failure.
Methods: Lama84 myeloid leukemia cells obtained from Gleevec resistant patients were incubated with the [1, 2-13C2]-D-glucose tracer for 72 hours in the presence of 1000 nM Gleevec and metabolic network fluxes were compared with SIDMAP profiles obtained 5 years ago (J. Biol. Chem. 276:37747-53, 2001).
Results: Lama84 cells obtained 87% of their nucleic acid precursor ribose via the non-oxidative branch of the pentose cycle, while previous SIDMAP studies indicated a dose dependent increase in non-oxidative ribose synthesis by 5.2%, 12.1% and 19.3% in the presence of 100 nM, 500 nM and 2000 nM Gleevec treatments, respectively.
Conclusions: Clinical resistance to Gleevec in leukemic cells highly expressing the intact BCR-ABL tyrosine kinase construct is attributable to the up-regulation of non-oxidative ribose synthesis thereby escaping the drug's strong inhibitory effect on oxidative ribose-phosphate synthesis. SIDMAP studies, which accurately predicted the use of alternative nucleic acid precursor synthesis pathways five years ago, would assist the FDA in the approval of targeted drugs by addressing metabolic adaptation and limitations before human trials begin.
Comparative Genomic Hybridization Using Oligonucleotide Microarrays and Total Genomic DNA
L. Bruhn1 , N. Sampas1 , M. Barrett1 , A. Ben-Dor1 , P. Anderson1 , A. Scheffer1 , P. Tsang1 , C. Gooden2 , R. Walker3 , B. Curry1 , R. Kincaid1 , D. Lipson1 , M. Bittner2 , P. Meltzer3 , Z. Yakhini1 , S. Laderman1 , 1Agilent Technologies, Palo Alto, CA, 2Translational Genomics Research Institute, Phoenix, AZ, 3National Human Genome Research Institute, Bethesda, MD
Array-based comparative genomic hybridization (aCGH) measures copy number variations at multiple loci simultaneously, providing an important tool for studying genomic alterations associated with cancer and developmental disorders as well as germ-line copy number polymorphisms. The broadest utility of aCGH, including both a simplified preparation of targets and hybridization of samples to any array design of interest, requires preserving the greatest possible complexity of targets derived from whole genome samples. Therefore, we developed probe design criteria, assay conditions, and analysis methods that enable 60-mer oligonucleotide microarrays to be used for CGH measurements using total genomic DNA (Barrett, MT, et al., PNAS 101, 17765-17770 (2004). We designed a 60mer oligonucleotide array with 40K probes specifically designed for CGH representing sequences throughout the human genome with a bias for known and predicted gene loci. We tested the performance of this array for reproducibly measuring and mapping losses, and amplification events of varying levels and sizes using both unamplified and phi29 amplified total genomic DNA from a series of model systems. The mean slope of experimental versus theoretical log-ratios for chromosome X probes on this array in XY versus XX hybridizations typically exceeds 0.9, with probe by probe error rates of less than 10% in the separation of their log-ratio distributions. To further validate the performance of this platform we used well characterized cell lines, including diploid cells with partial deletions in chromosome 18q, and diploid and aneuploid tumor cell lines with known amplification and deletion events. We show that the highly processive DNA polymerase phi29 can be used to prepare aCGH templates from as little as 5-10ng of starting material that yield high quality aCGH measurements throughout the genome. Phi29 provides a simplified isothermal method for amplifying limiting material. However, nonspecific DNA fragments of high MW are generated in the absence of sufficient input template. To better ensure reproducible and robust aCGH assay quality, we have developed methods and protocols using the Agilent BioAnalyzer to enable quality control and accurate quantitation for key pre-hybridization steps including: phi29 amplification of genomic samples, restriction digestion of templates and target labeling. We have also developed visualization tools and statistically robust computational tools that take into account the estimated errors on the measured log ratios in mapping aberration boundaries, and for identifying common aberrations across multiple samples. We tested the reproducibility of our platform by using tumor cell line samples including the colon adenocarcinoma cell line HT29 in hybridizations performed in different laboratories (Agilent Labs, NHGRI, TGen). We present results, using these methods, demonstrating that in situ synthesized 60-mer oligonucleotide arrays can reproducibly detect genomic lesions including single copy and homozygous deletions, and variable amplicons throughout the genome using full complexity genomic DNA samples.
Metabolite profiling for biomarker discovery and applications
K. Busch1 , T. Walk1 , B. Bethan1 , V. Haake1 , R. N. Trethewey1 , A. J. Krotzky2 , 1metanomics GmbH & Co. KGaA, Berlin, Germany, 2metanomics GmbH & Co. KGaA & metanomics Health GmbH, Berlin, Germany
Background
Metabolite profiling, the parallel analysis and bioinformatics interpretation of hundreds of metabolites and thousands of metabolome signals is applied to discover novel biomarkers to support all phases of drug development and nutrition.
Methods
metanomics leading metabolite profiling technology platform combines >50 mass spectrometers (GC-MS and LC-MS/MS) with fully integrated powerful data mining systems. This technology is being applied to a variety of sample types like blood, urine and tissues. Each sample analysis reliably covers several hundreds of known and unknown metabolites and yields additional several thousand metabolite signatures. The bioinformatics platform includes a wide range of univariate and multivariate data mining techniques and innovative data bases to discover single and complex biomarkers. The proven technology is being utilized in pre-clinical and clinical studies, accelerates necessary decisions, improves risk management and disease monitoring.
Results
Results are presented from metabolite profiling in blood matrices for mode of action and biomarker discovery in the context of toxicology studies and diabetes research. It is shown that metabolite profiling conclusively differentiates between different drugs, gender responses and between dose groups as well as identifies biomarkers for diseases such as diabetes type 2. The clear identification of key marker metabolites enables interpretation of biological contexts, underlying pathways and mechanisms related to drug effects.
Conclusions
Mass spectrometry based metabolite profiling is a powerful and reliable technology that supports all phases of drug development and toxicology. Very broad and in parallel highly sensitive and precise metabolite analysis enables biomarker discovery and allows direct insights into mode of action, underlying pathways.
Molecular Network Analysis of Metastatic Colorectal Carcinoma Using Reverse Phase Protein Microarray Based Phosphoproteomic Portraits
V. S. Calvert1 , K. M. Sheehan2 , E. Mammano3 , C. Belluco3 , V. E. Espina2 , J. D. Wulfkuhle2 , D. Nitti3 , M. Lise3 , L. Young4 , P. J. Munson4 , L. A. Liotta2 , E. F. Petricoin1 , 1CBER, FDA, Bethesda, MD, 2CCR, NCI, Bethesda, MD, 3University of Padova, Padova, Italy, 4CIT, NIH, Bethesda, MD
Protein kinases represent some of the most important drug targets in medicine today since aberrant kinase activity underpins many disease states. In the past it has been difficult to interpret kinase activity in tissue because extraction of the kinase separates it from surrounding regulatory context. Past attempts at cell network analysis have relied on gene microarray analysis, which does not recapitulate what is occurring at the proteomic level. On the other hand, the record of ongoing kinase activity states and cell signaling processes are imprinted on their phosphorylated protein substrates. An understanding of the pathways responsible for tumor survival are critical for developing novel targeted therapies tailored for the metastatic signature. To begin understanding the critical clinical question of whether and to what extent signaling network changes upon metastasis, we exploited two colorectal cancer related study sets in which patient-matched colorectal cancer specimens along with hepatic and lung metastatic lesions were analyzed by reverse phase protein microarrays. Unsupervised clustering analysis were developed using 40 different phospho-specific signaling endpoints. Phosphoproteomic network analysis was performed to find correlates with the metastatic phenotype compared to the primary portrait. Our results indicate that, unlike analysis of gene microarray data, we observe a significant difference between the metastatic lesion and the patient matched primary tumor at the phosphoproteomic level. This study represents the first reported attempt at the systematic functional mapping of cellular signal networks in the metastatic setting, which will be critical to realize the promise of patient-tailored therapy.
Development of a mammalian retinal ganglion cell model of electrical stimulation.
E. D. Cohen, D. D. Chan, CDRH, FDA, Rockville, MD
Background: Many retinal implants for the blind rely on electrical current stimulation of the retina for eliciting the sensation of vision. These designs use retinal ganglion cells, stimulated either directly or indirectly by electrical current, to relay action potentials to the brain. To evaluate their safety and efficacy, we must understand the effects of electrical current stimulation on different ganglion cell types involved in central vision. A well-studied model is cat X and Y ganglion cells. The light-evoked firing of X-cells is sustained, while firing of Y-cells is transient.
Methods: We are developing a model system for simulating electrical current stimulation of ganglion cells using real physiological and anatomical data. Neuronal models of ganglion cells were generated from dye-filled recorded neurons using a Neurolucida anatomical reconstruction system (Courtesy McBain Lab, NINCDS/NIH), and imported into the modeling program NEURON. A five-channel, voltage-gated conductance model was used. We compared real firing patterns from electrical current-stimulation to the model's firing patterns.
Results: The firing patterns of Y-cells to injected current showed more spike adaptation than X ganglion cells. The total membrane area of dendrites of the two ganglion cell types also differed by approximately 2-fold, with Y-cells having larger areas. The axon diameters of Y-cells were twice that of X-cells, as were cell body diameters.
Conclusions: X and Y ganglion cell types differ in their axon diameters, firing patterns and physical membrane area, which may generate different responses to electrical stimulation in retinal implants.
Automation: An Approach to Standardizing Multi-Functional Cellular Profiling Assays in Clinical Trials
C. Smith, J. Wilkinson, S. D'Costa, E. Rabellino, Custom Biopharma Solutions, Biomedical Research Division, Beckman Coulter Inc.
Background
Functional immune cell-profiling assays have been evaluated as surrogate end points to determine the efficacy of vaccine or therapeutic strategies for clinical trials. However, their utility has not been fully realized due to the variable nature of the assays which is particularly pronounced in T cell functional assays. With a view to reducing variability and standardizing targeted steps of T cell functional assays, an automated methodology for simultaneous staining and analysis of multiple intracellular cytokines and cytotoxicity markers via flow cytometry was developed and validated.
Method
To verify the concept of improved standardization with automation, a modification of available sample preparation instruments that enabled automated pipetting, incubation, staining and analysis of intracellular and surface molecules via flow cytometry was devised. A 5-color flow cytometry assay (2-3 surface markers; 2-3 intracellular cytokines/cytotoxic markers) was developed to characterize the T-helper and T-cytotoxic responses in human peripheral blood mononuclear cells (PBMCs) stimulated in a restricted polyclonal manner by SEB/CD28, and by an antigen-specific peptide pool.
Results and Conclusions
The automated sample staining and analysis greatly improved both inter and intra assay precision while reducing the "hands-on" sample preparation and analysis time. The complex nature of the immune response in PBMC specimens was revealed on evaluation of the cytokine and cytotoxic marker combinations in the context of multiparametric T cell response evaluations. Comparable approaches can be applied to other cellular participants such as Natural Killer T, Natural Killer and Dendritic cells. The use of automation thus provides a greater degree of standardization in complex cell functional assays enabling a correlation of the complex immune response profiles to vaccine efficacy or disease progression without interference due to the variable nature of the manual assays.
A Systems Biology Approach to Biomarker Discovery Utilizing Integrated Cytomic and Proteomic Techniques
S. D'Costa1 , C. Snow1 , E. Betgovargez2 , E. Rabellino1 , M. Simonian2 , 1Custom Biopharma Solutions, Biomedical Research Division, Beckman Coulter Inc., 2Proteome Lab Development, Biomedical Research Division, Beckman Coulter Inc.
Purpose.
A multi-pronged approach needs to be pursued to identify and evaluate relevant biomarkers. This requires that assays that have so far been carried out in isolation, for e.g. gene expression, protein synthesis, phenotype and function, etc. need to be integrated to better understand the "complex" responses that are associated with biomarker discovery. This "systems biology" approach can to some extent be achieved by unified genomic, proteomic and cytomic analyses. In the current study, the authors have attempted such an evaluation by integrating well-known techniques of flow cytometry-based phenotypic analysis and cell sorting with protein fractionation and analysis to identify biomarkers of cellular activation.
Methods.
Peripheral blood mononuclear cells were subjected to "restricted"polyclonal stimulation with staphylococcal enterotoxin B (SEB), fluorescently labeled with antibodies to distinguish activated and non-activated cells and subsequently isolated using flow-based sorting techniques. Lysates of activated and non-activated cells as well as cell-free supernatants were fractionated by two-dimensional gel-free liquid chromatography. Intact proteins were separated by their isoelectric points in the first-dimension and these fractions were further separated by hydrophobicity on a second-dimension. Using powerful and easy to use differential display software a high resolution protein profile of the complex mixture was obtained and analyzed.
Results and Conclusions
Qualitative and quantitative differences in protein profiles in activated and non-activated cells and supernatants could be identified thus allowing for easy discovery of putative protein/peptide biomarkers. The liquid-phase fractions of proteins in the native state, permits direct characterization of the differentially-expressed proteins, and thus accomplishes a thorough identification and analysis of biomarkers for monitoring and measuring cellular functional responses. Further combinations of genomic, proteomic and cytomic profiling will accomplish a unified evaluation of biomarker discovery and validation relevant to this response and other research models
Cloning and Analysis of Mono-Specific Single Chain Fragment Variable (scFv) Fragments that Recognize German Cockroach Allergens Bla g 1, Bla g 2, Bla g 4, and Bla g 5
N. C. DeVore, W. JJ. Finlay, E. N. Dobrovolskaia, J. E. Slater, CBER, FDA, Bethesda, MD
Rationale. Cockroach allergens may be an important cause of inner city asthma.Although many allergens have been standardized, cockroach extract has not. We have shown (Clin. Exp. Allergy 2002; 32:721-727) that there is a wide disparity in potency and allergen content among commercially available cockroach extracts. As part of our efforts to standardize cockroach extracts, we have produced scFvs that recognize Bla g 1, Bla g 2, Bla g 4 and Bla g 5.
Methods. Three 6-month-old female white leghorn chickens were immunized with rBla g 1, rBla g 2, rBla g 4, and rBla g 5 (Indoor Biotechnologies) using Freund's adjuvants. The animals were sacrificed, and RNA was isolated from spleens and bone marrow. Libraries were constructed from amplified cDNA, and scFv recombinant antibodies were produced from the antibody repertoire of these chickens. Phage bearing these scFvs were panned against rBla g 1, rBla g 2, rBla g 4, and rBla g 5. Four clones from each library were identified as recognizing the specific recombinant Bla g.
Results. Anti-Bla g 1, anti-Bla g 2, anti-Bla g 4, and anti-Bla g 5 scFvs specifically recognize rBla g 1, rBla g 2, rBla g 4, and rBla g 5, respectively, by ELISA and Western blot analysis. The antibodies also recognize specific proteins in crude cockroach extracts.
Conclusions. We have generated scFvs that specifically recognize each of the recombinant Bla g proteins. These recombinant antibodies will be useful in developing assays for the standardization of cockroach extracts.
A Low Molecular Weight, Orally Active TpoR Agonist, SB-497115, Does Not Prime Platelets for Activation or Agonist-Induced Aggregation In Vitro
J. Erhardt1 , P. Tapley2 , C. L. Erickson-Miller2 , 1GlaxoSmithKline, King of Prussia, PA, 2GlaxoSmithKline, Collegeville, PA
SB-497115 is a small molecular weight, orally active molecule that requires Tpo receptor (TpoR) expression for activity and activates JAK/STAT signalling in platelets. When administered to healthy male subjects at oral doses of 30 mg and above for 10 days a dose dependent increase in the platelet count was observed. The ability of recombinant Tpo to prime platelets for enhanced agonist-induced aggregation is well-documented, thus this study was undertaken to compare the ability of rhTpo (150 ng/ml) and SB-497115 (0.01-10 uM) to affect platelet activation. Platelets were obtained from healthy human volunteers, following written informed consent, by standard venipuncture technique. Platelet aggregation was induced by a submaximal concentration of adenosine diphosphate (ADP) (1.0 uM) in experiments examining synergy with rhTpo (150ng/mL) or SB-497115 (0.01-10 uM). For examination of potential inhibitory actions of SB-497115 on platelet function, aggregation was induced by ADP (3 uM), collagen (2 ug/mL), or the thrombin receptor activating peptide (TRAP; 20 uM). Pre-treatment of platelet samples with rhTpo, but not SB-497115, potentiated the effects of 1.0 or 1.5 uM ADP. No inhibitory effect on normal ADP, Collagen or TRAP-induced aggregation was seen with either SB-497115 or rhTpo. The expression of platelet P-selectin, an early marker of platelet activation, was analyzed by flow cytometry. Treatment with SB-497115 (0.1 - 10 uM) demonstrated no induction of platelet CD62P expression above background levels, while rhTpo produced a modest but significant and reproducible increase in the percent of CD62P+ platelets. This study demonstrates that the orally active small molecular weight agonist of the Tpo receptor, SB-497115, does not mimic the ability of rhTpo to enhance ADP-induced aggregation or to induce P-selection expression on human platelets. Finally, SB-497115 has no antagonistic effect on ADP-, TRAP-, or collagen-induced platelet aggregation. Thus, SB-497115 is differentiated from rhTpo with respect to priming platelets for activation.
AGE-RELATED DIFFERENCES IN SUSCEPTIBILITY TO TOXIC EFFECTS OF VALPROIC ACID IN SPRAGUE-DAWLEY RATS
P. Espandiari, T. J. Miller, A. Knapton, J. Zhang, J. Hanig, CDER, FDA, Silver Spring, MD
Prediction of adverse drug effects in children is a timely issue driven by recent legislation specifically mandating safe and effective drugs for children. Doses for children are often adult doses normalized to appropriate pediatric parameters in the clinic. They generally do not take cognizance of very special developmental differences that may produce significant vulnerabilities in vital target organs of children that can result in serious toxicities that are atypical in adults. Valproic acid (VPA) has been reported to cause several cases of fatal hepatotoxicity in infants and temporary increases in serum transaminases (~ 40% of patients). In this study, different age groups of male Sprague-Dawley (SD) rats (25, 40, 80 days old) corresponding to human toddlers, young and mature adults were treated with VPA at doses of 120, 360, 500, or 650 mg/kg i.p. injection for 4 days to determine pediatric vs. adult susceptibility. The order of sensitivity to the toxicity of VPA observed in this study was 25>80>40 days. The most dramatic toxic effects were observed in 25 day old rats at 650 mg/kg VPA: 57% survival rate, early elevations in ALT, serious G.I. disturbances, and white blood cell deficiencies. In addition, histopathology at this dose showed focal necrosis and apoptosis in the liver. Decreased serum platelet counts and diminished rate of growth were observed at all doses. In comparison, no lethality occurred in the 40 and 80 day old rat groups, and a decreased growth rate was detected in 40 day rats with all treatments. There was also an absolute weight loss in the highest dose groups at 40 and 80 days as well as the middle dose group at 80 days. This correlated with lowering of serum total protein and white blood cell counts in these age groups. Focal necrotic lesions and apoptotic cell death were observed in the liver of 80 day old rats treated with the two highest dose levels. These data indicate that the pattern of toxicity of VPA in different age groups of SD male rats is quite dissimilar. Further studies will explore these findings and its implications for pediatric drug safety.
Pregabalin: Pharmacokinetics, Pharmacodynamics and Interspecies Scaling
R. Feng, J. Koup, L. Radulovic, H. Bockbrader, P. Worboys, Pfizer
Background: Pregabalin (PGB, Lyrica), a derivative of the inhibitory neurotransmitter γ-aminobutyric acid, is clinically effective in the treatment of neuropathic pain, epilepsy, and anxiety. In current work, PGB pharmacokinetics and pharmacodynamics in preclinical species and interspecies scaling were discussed.
Methods. Plasma and/or tissue concentrations were determined in mice, rats, and monkeys following PGB administration. Pharmacokinetic/pharmacodynamic modeling was performed using NONMEM.
Results. PGB was rapidly absorbed in animals and mainly excreted in urine as unchanged. PGB renal clearance and volume of distribution in humans are well predicted by preclinical species. A minor metabolite in mouse and rat urine was identified as the N-methyl metabolite. In dogs, approximately 45% of the PGB dose was excreted in urine as N-methyl metabolite. No significant inhibition of human CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, or 3A4 was observed in vitro at PGB concentrations up to 1000 µM, suggesting low probability for PGB to elicit drug-drug interactions through inhibition of CYP450 isozymes. PGB exhibited potent activities in rats in the maximum electroshock seizer test for epilepsy, in chemical allodynia models for neuropathic pain, and in Vogel conflict test for anxiety. The estimated EC50 values ranged from 1.9 - 3.7 µg/mL in the rat models, which are consistent with the steady state average PGB concentrations (1.6 - 5.6 µg/mL) in humans at efficacious doses of 150 - 600 mg/day.
Conclusion. The projected human pharmacokinetic parameters and the efficacious concentrations are consistent with clinical data.
JIFSAN STUDENT PROJECT: DEVELOPMENT OF IMMUNOHISTOCHEMICAL STAINING TECHNIQUES FOR PCNA AND APOPTOSIS TISSUE MARKERS ON RAT TISSUES IN THE CFSAN PATHOLOGY LABORATORY.
M. Ferri1 , S. Francke-Carroll2 , B. Magnuson3 , 1College of Live Sciences, UMD, College Park, MD, 2CFSAN, FDA, College Park, MD, 3Dept. of Nutrition and Food Science, UMD, College Park, MD
Purpose: The purpose of this one-year JIFSAN student project was to establish immunohistochemical (IH) staining capabilities at the CFSAN/Pathology Laboratory (PB).
Background: The markers that were chosen for method development were PCNA (proliferating cell nuclear antigen) and Apoptosis. At the start of this JIFSAN project, the IH capacity of the PB laboratory was limited, consisting of a time intensive, manual staining procedure. IH positive staining antigen could often not be demonstrated because often sub-optimally fixed tissues are submitted to Pathology for IH staining while advanced antigen retrieval procedures were not available. Another problem area was the degree of unspecific background staining common in laboratory rodent tissues due to endogenous biotin.
Methods: Various instruments and reagents were adopted into a standard protocol, including a commercial decloaker device and a semi-automated-stainer. Best results were obtained using a conjugated secondary antibody, which reduced background staining significantly.
Results: During the one-year JIFSAN project, standard operating protocols for IH with PCNA and Apoptosis were developed in the PB laboratory. Both protocols were successfully applied in experimental studies by a collaborating laboratory at the University of Maryland.
Conclusion: The developed protocols will serve as the basis for development of a wide variety of other tissue markers requested by principal investigators for IH staining of experimental studies.
Analysis of Potentially Pathogenic Phenotypes of Cell Membrane Microparticles in Apheresis Platelets
M. P. Gelderman, L. B. Carter, J. Simak, LCH, CBER, FDA, Rockville, MD
Purpose: Cell membrane microparticles (MP) are released into the circulation from stimulated platelets, blood or endothelial cells. Their elevated counts have potential for different pathogenic activities.
Methods: We analyzed MP in apheresis platelet units (APU, n=26) on day 6 of storage using a three-color flow cytometry assay. Plasma from healthy volunteers (HVP, n=12) was used for comparison. To identify the cellular origin of MP, we used monoclonal antibodies to platelets (CD41), WBC (CD45), RBC (CD235a), and endothelial cells (CD105, CD144). Procoagulant phenotypes were also analyzed; MP expressing tissue factor (CD142) and binding of annexin V (AVB).
Results: We found in APU not only a higher CD41+MP count, but also a two-fold increase of CD235a+MP (p<0.001) and a 5x increase of CD45+MP (p=0.018) vs. HVP. In addition, MP of endothelial origin (CD105+CD45-MP) were 3x higher in APU (2790/µL; 840-9870) vs. HVP (930/uL; 710-1930; p=0.002). Surprisingly, the count of CD142+MP (4670/µL; 1830-11150) in APU was 3x higher vs. HVP (1580/µL; 1070-2510; p<0.001). CD142+MP were associated with WBC (20%), platelets (50%), or endothelial cells (20%) antigens. Counts of CD142+AVB+MP were 2080/µL (730-8040) in APU, and 1330/µL (720-1700; p=0.042) in HVP. In addition, the overnight incubation of washed APU MP with HUVEC resulted in a significant increase in cell expression of CD54 and CD142.
Conclusions: We found significantly elevated counts of platelet, WBC, RBC, endothelial cell derived MP, and MP of procoagulant phenotypes in APU when compared to HVP. Further studies to investigate a possible correlation between these findings and APU transfusion related adverse events are warranted.
 | Sigma Xi Outstanding Poster Award - 2005 FDA Science Forum |  |
A Subclinical Renal Injury Model in Rats for Detection of Nephrotoxic Compounds
P. L. Goering1 , E. F. Madden1 , R. P. Brown1 , B. A. Rosenzweig2 , K. L. Thompson2 , 1CDRH, FDA, Silver Spring, MD, 2CDER, FDA, Silver Spring, MD
Background: The objective of this study was to determine if rats with subclinical renal injury (SRI) are more susceptible than healthy control animals to nephrotoxic metals that exert their effect on different nephron segments.
Methods: To induce SRI, rats were dosed with gentamicin (sc) for 3 days. Twenty-four hr after the third injection, SRI- and saline-pretreated rats were injected with a single NOAEL or LOAEL dose of chromium (sc), mercury (iv) or lithium (ip) salts, metals which typically exert effects on proximal convoluted tubules (S1/S2 segments), proximal straight tubules (S3 segment), and distal tubules, respectively. The following biomarkers were evaluated 24 hours after metal injection: blood urea nitrogen (BUN) and creatinine; urinary N-acetyl-ß-glucosaminidase (u-NAG), creatinine, and total protein (u-TP); and kidney levels of kidney injury molecule-1 (KIM-1) mRNA.
Results: BUN levels were increased by 1.5-, 1.4-, 1.7-fold and NAG/Cr levels were elevated by 3.4-, 5.7-, and 2.2-fold in SRI rats challenged with chromium, mercury, and lithium, respectively, compared to control rats challenged with these metals. Kidney levels of KIM-1 mRNA were elevated due to gentamicin alone and elevated to a higher level in SRI rats treated with mercury, but not lithium and chromium.
Conclusions: SRI model rats demonstrated increased susceptibility to the effects of several nephrotoxic metals that target different nephron segments, thereby increasing the utility of the model for preclinical evaluation. U-NAG appeared to be the most sensitive biomarker to these metals, and kidney KIM-1 expression was elevated only in mercury-treated rats but not chromium- or lithium-treated rats.
Development and Validation of a High Content Cell Based Assay Using Imaging Technologies for the Detection of Neutralizing Antibodies to a Therapeutic Protein in Human Serum
J. J. Pennucci, S. J. Swanson, S. Gupta, Amgen Inc.
Purpose: To describe the development and validation of a cell-based bioassay for the detection of neutralizing antibodies to a therapeutic protein (TP) in human serum. The assay endpoint was based on cell imaging using fluorescence-based reagents incorporated into and at the cell surface.
Methods: The assay uses a human epidermoid carcinoma cell line that responds to a growth factor (GF) by signaling through a receptor that has tyrosine kinase activity (RTK) and autophosphorylates upon stimulation. Autophosphorylation is followed by activation and translocation of a transcription factor STAT-1 from the cytoplasm into the nucleus. The TP is an antagonist to the receptor and blocks GF binding resulting in lower RTK activity and STAT-1 translocation. A rabbit polyclonal affinity-purified neutralizing positive control antibody (NAb) to the TP restores GF binding and thus RTK activity and STAT-1 translocation. The cell line's response to the GF and TP was evaluated in the presence and absence of NAb at different concentrations of pooled human serum. TP inhibition curves and NAb titration curves were validated in 10% human serum. To determine the assay cutpoint,100 healthy donor sera (50 males and 50 females) were screened in the assay and the 95% prediction limit of the ratios of donor sera to pooled normal serum for the tested population was calculated. NAb spike-recovery experiments using 125 ng/mL NAb were conducted with 50 healthy donor sera.
Results: 4 ng/mL of GF elicited a robust tyrosine phosphorylation of the receptor and STAT-1 translocation in the cells. TP inhibited the GF-induced response in a dose dependent manner. A concentration of 100ng/mL TP was selected to hold constant in the assay for NAb detection. The limit of detection of the assay was determined to be 125 ng/mL NAb (in undiluted serum) which caused a significant inhibition of the RTK activation and STAT-1 translocation induced by 100 ng/mL TP.
Conclusions: Fluorescence based cell imaging methods for the detection of receptor tyrosine phosphorylation and STAT-1 translocation provided a novel multiplexed endpoint for use in a NAb assay.
The utility of monitoring cardiac troponin T to detect cardiac injury induced by low doses of isoproterenol in rats
E. H. Herman1 , J. Zhang2 , A. Knapton1 , N. Rifai3 , F. Sistare1 , 1DAPR, CDER, FDA, Silver Spring, MD, 2DAPR, CDER, FDA, Silver Sring, MD, 3Children's Hospital, Boston, MA
Backround: Cardiac troponin T (cTnT) is released from damaged myocytes and has been utilized as an biomarker of cardiac injury. In Previous studies high doses (4 to 80 mg/kg) of isoproterenol (Iso) caused acute cardiac lesions and increases in the serum levels of c TnT. The present study explored the utility of monitoring serum cTnT to detect cardiac injury at much lower Iso doses.
Methods: Groups of SD rats (8 wks) were dosed once (sc) with 0.008 to 0.5 mg/kg Iso and euthanized 3 to 48 hours later. Lesion severity was assessed on a scale of 0 to 5 (based on the degree of necrosis and apoptosis, inflammation, edema basement membrane damage and collagen deposition). cTnT levels were determined by Elecsys STAT immunoassay.
Results: cTnT levels were elevated in all animals (mean 0.20 to 0.28 ug/ml) 3 hours after administration of 0.008, 0.016, 0.032 or 0.064 mg/kg Iso but minimal cardiac lesions (severity score=1) were found in only 3 of 19 rats given the 0.032 and 0.064 mg/kg doses. However, by 6 hours cardiac lesions (severity score=1) together with increases in cTnT (0.13-0.18 ug/ml) were found in 20 of 22 animals given these same 2 Iso doses. Doses of 0.125, 0.250 or 0.50 mg/kg Iso caused both significant increases in in cTnT levels and marked myocardial alterations that reached a peak 3 and 6 hours post dosing. At 3 hours mean severity scores of 3.4, 4.2 and 5 and mean cTnT levels of 2.7, 6.8 and 13.6 ug/ml were observed in rats given 0.125, 0.25 and 0.50 mg/kg Iso respectively. The magnitude of myocardial alterations induced by these doses of Iso declined between 12 and 48 hours post treatment (mean lesion severity was 1.6-1.7 and cTnT levels were 0.06-0.20 ug/ml at 48 hours).
Conclusions: These results indicate that very small single doses of Iso are capable of causing time-and dose-dependent myocardial alterations and these alterations are associated with detectable increases in serum levels of cTnT.
Resolution of clofibrate efficacy and toxicity in rats using a combined LC/GC/MS metabolomics biomarker approach
A. Berger, I. Shah, A. J. Higgins, Icoria, Inc., Research Triangle Park, NC
Background: Clofibrate is a hypolipidemic drug (PPARα agonist) that has also been shown to produce hepatotoxicity. Our aim was to determine if the two actions could be distinguished using metabolomics.
Methods: Clofibrate or vehicle was administered orally (0, 50, 250 mg/kg/d) to groups of 6 rats and serum and livers were collected 6 and 24 h after either a single or 14 daily doses. Global biochemical profiles were determined by LC/MS and GC/MS.
Results: There was a dose and time-related increase in liver weights and mitotic figures, but no changes in serum enzymes. Recovery of biochemical profiles from clofibrate exposure occurred at 24 h, and was more pronounced after 14 days, suggesting adaptation. Stepwise regression was used to determine components contributing most to liver hypertrophy; and these components were putatively identified and mapped to pathways, highlighting potential mechanisms involved in liver hypertrophy. When combined with GC/MS profiles, with NIST 2002 library matching, we detected components and pathways related to clofibrate efficacy, such as fatty acid oxidation. Other changes observed were in amino acid and polyamine metabolism, pathways that might relate more to toxicity.
Conclusions: Global LC/GC/MS metabolomics, in combination with multivariate statistics, detected sets of liver biomarkers that showed adaptive changes, and that were associated with both efficacy (lipid metabolism) and toxicity (liver hypertrophy). This demonstrates the potential for a functional metabolomics approach to separate on-target and off-target drug effects.
Supported in part by NIEHS SBIR Award# 291200445524C and NIST ATP Award# 7UNANB2H3009
Cdc42 as a potential therapeutic target for the reduction of cellular proliferation and migration as measured in MDA-MB-231 breast cancer cells
D. S. Hirsch, Y. Shen, W. J. Wu, OBP, DMA, CDER, FDA, Bethesda, MD 20892
Background: High proliferative index and invasive capacity are two indicators of poor prognosis in breast cancer. Cdc42, a member of the Ras superfamily of small GTPases, is a GTP binding protein that regulates cell proliferation and migration. Several studies indicate that increased Cdc42 protein levels are found in breast cancer tissue as compared to normal breast tissue. The biological significance of increased Cdc42 protein levels is not known. We tested the hypothesis that reduction of Cdc42 protein levels in an aggressive breast cancer cell line, MDA-MB-231, would inhibit cellular processes critical to the breast cancer progression.
Methods:
A series of breast cancer cell lines were screened for Cdc42 protein levels by western blot. Activated Cdc42 protein levels were determined using GST-p21 binding domain assay, an affinity chromatography assay that specifically detects activated Cdc42 protein levels. Migration was studied using boyden chamber and wound healing assays.
Results:
Higher levels of activated Cdc42 were found in MDA-MB-231 cells, an aggressive cell line, as compared to a less aggressive breast cancer cell line known as MCF7. Cdc42-specific siRNA reduced Cdc42 protein levels by greater than 50% in MDA-MB-231 cells. siRNA-mediated reduction in Cdc42 protein levels resulted in reduced proliferation and cell migration in MDA-MB-231 cells.
Conclusions:
Cdc42 is a critical regulator of both cell proliferation and cell migration. Targeted introduction of Cdc42-specific siRNA to cancer cells may offer a novel therapeutic approach to inhibit these processes. Future studies are aimed at determining the suitability of Cdc42 as a therapeutic target.
How Biomarkers can Improve Clinical Drug Development?
P. R. Jadhav1 , M. U. Mehta2 , J. V.S. Gobburu2 , 1CDER, FDA, Rockville, MD and MCV/VCU, Richmond, VA, 2CDER, FDA, Rockville, MD
Purpose. To provide an overview of the utilities of biomarkers to streamline important decisions during drug development supported by relevant examples.
Methods. Recently, use of biomarkers has acquired a lot of attention for its utility in drug development process. A critical literature review was undertaken to evaluate uses of biomarkers towards rational drug development.
Results. A wide variety of biomarkers are employed in the early drug development to screen compounds, in the diagnosis of patients and for follow- up of drug effects routinely. Additionally, biomarkers such as blood pressure, cholesterol, testosterone levels, tumor size and rate of gastrointestinal polyp formation have been the basis of drug approvals. As long as a biomarker has a reasonable mechanistic basis, there are several important contributions of biomarker( s) towards improving the efficiency of drug development, irrespective of whether the relationship with the clinical outcome( s) is formally established or not. Typically, biomarkers can aid in
Conclusions. Selection of the dose range and doses for further investigation in the pivotal trials is the single most important use of biomarkers. Utility of biomarker knowledge in identifying patients with important differences and in making various regulatory decisions was also reviewed. Better appreciation of the underlying mechanism of disease and drug action, routine collection of biomarker data and sophisticated analyses as early as possible in drug development might lead to optimal drug therapy and more efficient use of available resources.
Vaccinia Immune Globulin Treatment in a Mouse Model of Progressive Vaccinia
M. C. Kennedy1 , N. Eller1 , J. Manischewitz2 , H. Golding2 , D. E. Scott1 , 1Div of Hematology, OBRR, 2Div of Viral Products, OVRR
Progressive vaccinia is a severe life threatening complication of that can occur following the administration of the Dryvax smallpox vaccine. Vaccinia Immune Globulin (VIG) is a high titer anti-vaccinia virus product produced from immunized donors that is the only currently licensed product for the treatment of this disease. In order to evaluate the efficacy of VIG we have developed a murine progressive vaccinia model using dermal scarification with Dryvax virus in severe combine immunodeficient mice (SCID). This model displays a number of clinical characteristics seen in human progressive vaccinia such as progressive expansion of the primary lesion, escar formation, followed by lethal systemic spread of the infection. In our hands this model has shown a highly reproducible survival curves allowing us to test different post-exposure prophylaxis VIG treatment modalities. Results: We have found that VIG treatments given at 48 hours post-scarification is capable producing healing of the primary vaccinial lesion in 50% of SCID mice challenged with 106 virus/mouse, 67% of mice challenged with 105 virus and 89% of mice challenged with 104 virus. All mice treated with VIG showed a significant prolongation of survival time compared to virus challenged mice receiving no treatment. Also, this VIG treatment regime gives significant numbers of SCID mice surviving without any apparent disease symptoms for as long as four months at the lower virus challenge levels of 105 and 104 virus per mouse. We believe that the data generated by these studies is directly relevant to VIGIV therapy in human progressive vaccinia.
Characterization of a recombinant Interleukin-13 (IL-13) Receptor alpha 2 extracellular domain in blocking biological activities of IL-13
M. Kioi, S. Seetharam, N. Talwar, R. K. Puri, CBER, FDA, Bethesda, MD
Background: Understanding the mechanism of action of targeted therapeutics is critical for optimal development of biological products. Our group is involved in the identification of tumor-associated proteins to elucidate the mechanism of action and develop novel therapeutic approaches. IL-13 is a key cytokine involved in allergic diseases, inflammation, and cancer. IL-13 receptor alpha 2 (IL-13Rα2) is known to be a high affinity receptor, which is overexpressed in cancer cells and many inflammatory conditions. To characterize the biological significance of IL-13Rα2, we generated a soluble form of IL-13Rα2 extracellular domain (ECDα2).
Methods: We developed several techniques for expression and purification of ECDα2 protein including Escherichia coli and mammalian system. After purification, biological activities of ECDα2 protein were determined.
Results: First few attempts for expression of soluble fusion ECDα2 and stabilization of this protein after refolding failed. We later succeeded in expressing ECDα2 protein with high yield in 293FT cells. ECDα2 was mainly secreted as a monomer and heavily glycosylated. The purified ECDα2 inhibited IL-13-induced TF-1 (erythroleukemia) cell proliferation and protein synthesis, IL-13 binding on IL-13R-expressing cells, and IL-13-induced STAT6 phosphorylation. However, IL-4-induced proliferation and STAT6 phosphorylation were not affected.
Conclusions: These results indicate that ECDα2 protein is difficult to express and purify due to eight cysteine residues within the molecule. In addition, IL-13Rα2 is not involved in IL-4 signal cascade, and thus, this protein may be useful in blocking IL-13-specific effects for a variety of conditions.
Metabolic health status by metabonomics
D. Jacobs, Z. Ramadan, A. Fuerholz, S. Kochhar, Nestlé Research Center, PO BOX 44, Vers-chez-les-Blanc, CH-1000 Lausanne-26, Switzerland
Metabolites are products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes. The impact of metabonomics in food industry is potentially widespread. The technology can be applied to the following areas: identification of biomarkers leading to proprietary actives, taste and flavor chemistry, clinical trial efficacy and safety, and development of robust scientific concepts for nutritional claims. Identification of validated biomarkers holds a tremendous potential to substantiate specific claims. The primary objective of the study was to define metabolite profiles of urine, plasma and saliva in general human population and search for phenotypic (gender, age etc.) and for lifestyle (sports, smoking etc.) specific patterns.
A total of 150 healthy adults (about 50 % females and 50 % males) were recruited at Nestlé Research Center, Lausanne to donate one-time sampling of fasting plasma, urine and saliva. The subjects completed their habitual lifestyle questionnaire and one dimensional 1H NMR spectra were recorded on each of the samples, and analyzed by multivariate statistical methods. Mathematical models were developed in an attempt to understand inter-individual metabolome variations. The data show how metabonomic study could lead to understanding of mechanism underpinning the effect of specific diet and other parameters on the individual health status. A major strength of the new -omic technology is that it is minimally invasive, and can thus easily be applied to study subtle variations in human metabolic health status and metabolism's dynamic relationship to environment that includes typical lifestyle pattern including diets etc. In conclusion, the metabonomic approach employing 1H NMR of biological fluids (plasma, urine, saliva, etc.) examines comprehensive biochemical profiles of low-molecular-weight metabolites in various biofluids that are modulated in response to various habitual lifestyle stimuli. The study provides solid base to define inter-individual metabolite profile variability, and to construct multivariate boundaries of normality.
Development of a multivariate statistical model for nephrotoxicity biomarker identification based on urinary 1H-NMR spectral pattern
M. Linder1 , I. Gustafsson1 , J. Lindberg1 , R. Torgrip1 , P. Ciaccio2 , S. Emeigh Hart2 , G. Betton3 , I. D. Wilson3 , E. Lenz3 , I. Schuppe-Koistinen1 , 1AstraZeneca R&D Södertälje, Sweden, 2AstraZeneca R&D Wilmington, DE, 3AstraZeneca R&D Alderley Park, UK
Methods
Male Han Wistar rats were treated with 3 compounds (mefenamic acid, N-phenylanthranylic acid and indomethacin) inducing renal papillary necrosis (RPN) and 3 compounds inducing renal cortical injury (gentamicin, cyclosporine and mercuric chloride). Approximately 1500 1H-NMR spectra were acquired on a 600 MHz NMR spectrometer. Spectral peaks were aligned using an in house alignment software and individual multivariate models were developed for each study. For biomarker identification all 6 studies were combined in one multivariate statistical model using Partial Least Squares- Discriminant Analysis.
Results
The model compounds for RPN induced different degrees of papillary toxicity. All compounds changed the endogenous metabolite pattern of rat urine as the treatment groups cluster separately from controls in each study. A combined multivariate model was developed including 1H-NMR spectra from animals treated with cortical toxins. Based on this model, potential biomarkers for RPN were identified including increased levels of glycolate, urocanate and phenylalanine as well as decreased levels of ornithine, betaine and choline.
Conclusion
Regiospecific renal damage, such as RPN, is difficult to monitor and predict. Using NMR-based metabolite profiling kidney toxicity-specific pattern were identified. A number of metabolites correlating with RPN have been tentatively assigned. These potential biomarkers for RPN require further validation.
| Sigma Xi Outstanding Poster Award - 2005 FDA Science Forum |
Effects of Implantable Cardioverter-Defibrillator (ICD) Shocks upon Human Blood
V. Krauthamer1 , R. Malinauskas1 , E. Mitrojorgji1 , M. S. Stratton1 , K. Higginson1 , J. Zivny2 , J. Vostal2 , 1CDRH, FDA, Rockville, MD, 2CBER, FDA, Bethesda, MD
The 100,000 patients annually undergoing ICD implantation with electrical defibrillation threshold testing have a relatively high risk of thrombo-embolic events (5%). It is unknown if the electric current of the ICD shocks causes platelet activation and hemolysis. Human blood from healthy individuals was shocked in vitro in electroporation vials using a defibrillator. Electroporation vials are ideal for studying blood damage because they accommodate small sample volumes, and have parallel electrode plates that provide a uniform current density. We applied energy levels from 0 - 35J, which generate up to twice the maximum equivalent current densities as used in commercial transvenous defibrillator leads. Blood temperature was measured to separate thermal from electrical effects. Platelet activation was evaluated by measuring expression of P-selectin on the surface of activated platelets using flow cytometry. Red blood cell (RBC) damage was assessed using blood smears, measuring hemoglobin released into plasma, and with a blood cell counter. Increased levels of hemolysis were noticeable at 8J (current density of 2.5 A/cm2), while platelet activation was increased only at 35J (6.7 A/cm2) in whole blood (but not in platelet-rich plasma). Alterations in RBC morphology, platelet count, mean RBC volume, and RBC distribution width occurred at the higher energy levels. Our preliminary findings support the results of a clinical study that threshold testing of ICDs is not associated with platelet activation. In vitro testing using electroporation vials may provide a new method to evaluate blood damage caused by electrical stimulation from medical devices.
A novel in vitro system for the evaluation of human drug adverse effects: The integrated discrete multiorgan co-culture (IdMOC) system
A.P. Li, Advanced Pharmaceutical Sciences Inc.
Primary cell culture systems, especially human cell system such as human hepatocytes, are now routinely used to evaluate drug effects, including efficacy, metabolism, and toxicity. Using a cell type derived from an organ (e.g., hepatocytes from a liver), one can obtain experimental system pertaining to the properties of the chosen organ. However, a drug administered to a whole organism will be subjected to multiple organ interactions. The drug may be metabolized by multiple organs, and the parent drug and its metabolites may be multiple organ effects. Further, metabolites from one organ (e.g. liver) may cause specific effects on cells of another organ (e.g. heart). Therefore, while the conventional single-organ in vitro system allows one to assess drug properties pertaining to a single organ (e.g. hepatic metabolism or hepatotoxicity), accurate prediction of in vivo effects requires an experimental system with multiple organ interactions as the whole organism in vivo. The IdMOC system consists of wells-in a well: multiple cell types can be cultured discretely in the inner wells, and when the cells are ready for integration, the wells are flooded with medium so that they are now connected by a common medium, akin to the blood connecting the organs in a human body. An application of IdMOC is demonstrated with tamoxifen, an anticancer agent for the treatment of estrogen-dependent breast cancer. Tamoxifen was evaluated for its cytotoxic effects on the following human cell types: MCF-7 breast adenocarcinoma cells, hepatocytes, renal proximal tubule epithelial cells, aortic endothelial cells, small airway epithelial cells, and astrocytes, representing the target cancer cells, and the normal organs liver, kidney, vascular system, lung, and central nervous system, respectively. Tamoxifen was found to be highly cytotoxic towards the MCF-7 cells, and expressed difffertial cytotoxicity towards the various normal cells. The results are similar to the known anticancer efficacy and normal organ toxicity of tamoxifen. The IdMOC represents an improved in vitro experimental system to assess human drug properties, including toxicity, metabolism, and drug distribution.
Testing biomaterials for stimulation of major inflammatory pathways
D. B. Lyle, J. C. Shallcross, V. M. Hitchins, J. J. Langone, CDRH, FDA, Silver Spring MD 20903
Background: Reactions to biomaterials present in implantable combination devices may include inflammation, such as mediated by macrophages, that may harm patient tissue or potentially interfere with proper function of the device. One of the substances produced by activated macrophages is nitric oxide (NO).
Methods: RAW 264.7 cells were grown in culture and treated at various times with drugs/reagents. Supernatants were assayed for nitric oxide via an increase in the fluorescence of the dye 2,3-diaminonapththalene (DAN) to nitrite.
Results: DAN allowed for detection of a very early (24 hours or less) initial response in nanomolar range of the inflammatory reaction of RAW 264.7 to E. coli 026:B6 lipopolysaccharide (LPS). By looking at early time points (20 and 24 hrs), an interferon-gamma augmentation of the LPS effect was readily discerned. The full interferon-only effect was studied by taking readings at 48 hrs or later. The cells produced a robust response of nitric oxide to uM zymosan comparable to that of nM LPS, which also was augmented by Interferon. Macrophage activation through the main inflammatory signaling pathway (nuclear factor-kappa beta) was confirmed in this system by parthenolide inhibition of zymosan and LPS stimulation. Interferon, which signals by a different pathway (the JAK pathway), was only partially inhibited by parthenolide.
Conclusions: A robust response of NO production by the murine macrophage cell line RAW 264.7 may serve as a convenient platform to confirm and identify cellular mechanisms and pathways to determine safety of implanted devices.
An In Vitro Test for Inflammatory Potential of Hyaluronic Acid Preparations
D. B. Lyle1 , J. C. Shallcross1 , V. M. Hitchins1 , C. N. Durfor2 , J. J. Langone1 , 1CDRH, FDA, Silver Spring, MD 20903, 2CDRH, FDA, Rockville, MD 20850
Background: Hyaluronic acid (HA) is a natural component of the body's extra-cellular matrix, contributing to the viscosity of skin. HA derived from different sources is used clinically as a tissue-filler to smooth skin wrinkles, and injected as a lubricant to treat the joint pain of osteoarthritis. Adverse events from injection of HA preparations have been reported, which may be inflammatory in nature and involve macrophages.
Methods: RAW 264.7, a well-characterized murine macrophage cell line, was treated at various times with HA preparations derived from bacterial, animal, and human sources (purchased from Sigma) and control reagents. Supernatants were assayed for nitric oxide (NO), an indicator of macrophage activation/inflammation, via an increase in the fluorescence of 2,3-diaminonapththalene (DAN) to nitrite. Endotoxin concentrations in HA were assayed using a Limulus amebocyte lysate (LAL) chromogenic kit (BioWhittaker).
Results: HA preparations directly stimulated NO production by RAW 264.7 cells. The NO stimulation was attributable to endotoxin contamination, as determined by the LAL assay. In 10 mg/ml HA preparations endotoxin ranged from 0.5 ng/ml to 15 µg/ml. "Cryptic" responses were revealed by simultaneous treatment with mouse interferon-gamma.
Conclusions: Inflammatory reactions attributed to HA preparations may be due not to the native molecule itself, but to possible endotoxin contamination. Ascertaining the potential of HA preparations to induce macrophage inflammation is an important preclinical and batch-certification test. These results were obtained with HA preparations not approved for clinical use. The validity of these results with HA products approved by the FDA for clinical use remains to be explored.
Age-related changes in intestinal crypt density may contribute to increased sensitivity of soy-fed female rats to acute toxicity of azoxymethane
A. Tracy1 , S. Francke-Carroll2 , B. A. Magnuson1 , 1University of Maryland, 2FDA
Soy isoflavone consumption by the public has significantly increased due to popularity of supplements and foods with added isoflavone for possible hormonal, cancer and heart disease-preventive properties. During one of our feeding studies for colon cancer prevention, we observed acute intestinal toxicity specifically in older female rats fed a soy-supplemented diet in response to treatment with the well-established colon carcinogen azoxymethane (AOM). Investigating possible mechanisms for this observation, we focused on two parameters: a) the expression of CYP2E1, an important enzyme responsible for metabolism of toxins and carcinogens, and b) on differences of the intestinal crypt density in older versus younger rats. We preformed real time PCR to quantify CYP2E1 in liver tissue. We assessed the intestinal crypt density stereometrically by applying a standardized square grid to standardized digital photographs of young, middle-aged and old rat colons that were sectioned in a standardized manner. The number of crypts crossing defined grid lines at defined points was calculated for each colon section from an individual rat and the average number of crypts/line was computed for each animal.There was no change in the gene expression of CYP2E1 due to either diet or age. Both age and diet affected colonic crypt density. Soy-fed rats had a statistically significant higher crypt density than the control rats, and young rats had a significantly higher crypt density than the mature and old rats (p<0.001). These findings suggest that age as well as a soy-supplemented diet decreases the colon crypt density, and thereby increasing susceptibility to acute toxicity following carcinogen injection.
Effect of age on azoxymethane-induced colonic apoptosis in rats
Y. J. Kwon1 , S. Francke-Carroll2 , B. A. Magnuson1 , 1University of Maryland, 2Pathology Branch
Aging processes may alter the regulation of apoptosis. This study investigated the effect of age on the time course of azoxymethane (AOM)-induced apoptosis in the colon. Young (6 week), middle-aged (12 month), and old (21 month) F344 male rats were treated with single s.c. injections of either saline or AOM (15mg/kg) and 0 (saline-treated), 4, 8, 16, or 24 hours later incidence of apoptosis (total stained cells/colon crypt) was estimated by TUNEL assay in the distal colon. In all age groups, apoptosis rarely occurred at the 0 hr time point and the apoptotic peak occurred at the 8 hr time point. After reaching the peak, apoptotic incidence decreased and there was no significant difference (p>0.05) between 0 hr and 24 hr time points in young and middle-aged rats compared to a significantly higher incidence in old animals. Old rats showed the highest and most rapid increase of apoptosis followed by middle-aged rats: old rats showed significantly higher apoptosis (4.1 ± 0.88 apoptotic cells/crypt) compared to young (1.13 ± 0.25) and middle-aged animals (1.73 ± 0.40) at the 8 hr time point. Apoptosis in old rats was also significantly higher (2.35 ± 0.63) than that of young rats (0.76 ± 0.18) at 16 hr. However, the magnitude of age-related difference in response to apoptosis decreased with time (significant age ´ time interaction): at the 24 hour time point, apoptosis was not significantly different among different ages of rats. The induction of apoptosis is greater and occurs for a longer time period in old animals. This age-related difference may be at least partially responsible for our previous finding of age-related difference in the inhibition of colon carcinogenesis by dietary interventions.
Quantitative morphometric analysis (QMA) of prion protein (PrPSc) for improved diagnosis of Transmissible Spongiform Encephalopathies (TSEs)
O. Maximova, R. Taffs, K. Pomeroy, D. McMahon, P. Piccardo, D. Asher, FDA
Background: TSEs are commonly diagnosed by histopathological examination and immunohistochemical (IHC) detection of disease-associated prion protein (PrPSc) in infected tissues. The traditional evaluation of IHC staining in tissues is based on visual inspection of sections and then grading by an arbitrarily chosen semi-quantitative scale. This approach is limited by observer subjectivity and bias, often yielding inconsistent and variable results. Our goal was to develop an objective method to facilitate reliable diagnosis of TSEs using image analysis of PrPSc immunostaining.
Methods: Paraffin-embedded sections from brains of hamsters infected with the 263K strain of scrapie were used to standardize detection of PrPSc by IHC. Commercial image analysis software (MetaMorph™) was then used to quantify PrPSc immunostaining.
Results: We first identified a "negative-chromaticity subdomain"-a range of hue, saturation, and intensity (HSI) that included all light from negative-control tissue sections. A chromaticity subdomain for positive PrPSc immunostaining was then defined by subtracting negative hue values from those of the entire visible light spectrum. This method allowed unambiguous detection of even minimal amounts of PrPSc in infected tissues. Performance of QMA was evaluated by comparing visual scores of distribution and intensity of PrPSc immunostaining with QMA data. QMA results correlated very well with traditional scores.
Conclusions: QMA of PrPSc provides a straightforward, reliable and objective measure of immunostaining that might aid in the diagnosis of difficult cases of TSE and offers a potentially powerful tool for many other applications requiring quantitative evaluation of immunohistochemical staining.
In Silico Discovery of Bridging Renal Biomarkers
D. Mendrick1 , M. Lawton2 , 1Gene Logic Inc., Gaithersburg, MD 20879, 2Pfizer Global Research & Development, Groton/New London Laboratories, Pfizer Inc, Groton, CT 06340
Background:The need for bridging biomarkers to predict nephrotoxicity and to follow its resolution in preclinical species and in humans continues to be of major interest to the FDA and pharmaceutical companies. Gene Logic has compiled large reference databases of gene expression data from normal and toxicant-treated rat tissues and from normal and diseased human tissues. This offers the possibility for in silico data mining to identify potential bridging biomarkers that might be useful for toxicity applications and/or disease diagnosis
Methods: Gene expression data generated on Affymetrix GeneChip® microarrays (RG_U34A, RAE 230 2.0, HG-U133) were evaluated. Examination was conducted on normal rat and human tissues to examine each gene's tissue distribution as this is important particularly if one chooses to monitor its encoded protein or peptides in urine or blood. To focus on the identification of predictive (leading) biomarkers, analysis was conducted on 1) toxicant- and vehicle-treated rat kidney tissue and 2) normal and diseased human renal tissue.
Results: Initial choices of genes focused on those reported in the public domain to be potential nephrotoxicity biomarkers including kidney injury molecule-1 (KIM-1,Havcr1) and cysteine rich protein 61 (CYR61). The analysis then was expanded to discover new potential biomarkers.
Conclusions: Large reference databases of normal and phenotypically altered tissue (caused by human disease conditions and experimental studies in preclinical species) afford the opportunity for in silico data mining to discover potential bridging biomarkers. Additional "wet lab work" will be needed to validate such markers.
Phenotyping Escherichia coli O157:H7
A. Mukherjee, K. L. McCutchan, E. W. Brown, J. E. LeClerc, T. A. Cebula, CFSAN, FDA, Laurel, MD
Background: As more genome sequences of microbial strains become available, it is becoming evident that variability among closely-related strains is prevalent. In order to investigate whether these variations are manifested in their phenotypes, a high throughput phenotypic microarray system capable of identifying more than 1200 phenotypes is being used to analyze a diverse collection of E. coli O157:H7 isolates.
Methods: Bacterial cells were inoculated into twenty 96-well plates containing various substrates: such as different carbon and nitrogen sources; varying conditions like pH and ionic environments; and various antibiotics. The plates were incubated at 37°C for 48 h. All wells contain a reducible tetrazolium dye, which, upon cell growth and respiration, forms color. The intensity of the color is recorded every 15 minute and analyzed.
Results: Eighty strains of E. coli O157:H7 have been analyzed by phenotypic microarray. Cladistic analysis using the phenotypic data grouped the strains into clades; phenotypes were also used to resolve clades of O157:H7 strains based on molecular markers. Additionally, we observed that O157:H7 strains can utilize sucrose but not D-serine as a carbon source, whereas most K-12 strains utilize D-serine but not sucrose. PCR analysis was used to confirm variability in the relevant chromosomal regions of these strains.
Conclusion: Cladistic analysis has enabled us to assign signature phenotypic changes that uniquely define clades or 'bins' of O157:H7. We are exploiting the ability of O157:H7 strains to utilize sucrose and not D-serine as a carbon source in an effort to distinguish O157:H7 from other pathogenic E. coli strains.
Candidate Cross-Species Biomarkers of Fibrosis and Hepatitis
M.S. Orr, Gene Logic Inc.
Background: There is a need in the toxicology field to develop diagnostic biomarkers that are relevant to multiple species such as rat, dog, and human. Genomics provides an opportunity to discover novel cross-species biomarkers for identifying phenotypes such as hepatitis and fibrosis, especially if the biomarkers are tissue specific secreted proteins that can be monitored in the blood.
Methods: Microarray analysis using Affyemtrix gene chips® was utilized to identify dysregulated secreted genes in human or rat samples. Comparisons of the dysregulated genes identified in either the human or rat studies were performed in order to identify candidate cross-species diagnostic markers of hepatitis and fibrosis.
Results: Microarray analysis identified 80 differentially expressed genes that coded for secreted proteins in human cirrhotic/fibrosis tissue samples and many of the genes were liver specific based on the gene expression profile across a panel of normal human tissues. Interestingly, APOA1 was one of the 80 secreted proteins identified by this genomic analysis and it is currently utilized as a biomarker for the early detection of fibrosis. In addition, Fetuin-B (FETUB), a secreted protein that can be monitored in serum, was identified as a novel candidate cross-species biomarker of fibrosis, as it is dysregulated in both the human hepatitis/fibrosis samples and rat DMN treated liver samples.
Conclusions: This study illustrates a genomic approach for identifying candidate cross-species biomarkers of cirrhosis/fibrosis for humans and rats. The study identified a previously known biomarker of fibrosis (APOA1) and a novel candidate biomarker of fibrosis, FETUB.
Elevated Circulating Endothelial Microparticles in Acute Stroke Patients: A Correlation with Brain Lesion Volume and Outcome
J. Simak1 , M. P. Gelderman1 , H. Yu2 , V. Wright2 , N. Alberts-Grill2 , J. T. Stranix2 , A. E. Baird2 , 1CBER, FDA, Rockville, MD, 2NINDS, NIH, Bethesda, MD
Purpose: Elevated endothelial cell membrane microparticles (EC MP) in blood have been demonstrated in various diseases with a vascular injury component. The aim of this study was to investigate if circulating EC MP show a relationship with outcome after acute stroke and with the ischemic brain lesion volume measured by magnetic resonance diffusion-weighted imaging (DWI).
Methods: EC MP were analyzed in the blood of 42 acute stroke patients (AS): 20 patients with National Institutes of Health Stroke Scale (NIHSS) scores < 5 were classified as mild stroke (MS) (median NIHSS= 2; 25th-75th%: 0-2), while the other 22 patients with NIHSS >/= 5 (NIHSS=12; 6-21) were classified as moderate to severe stroke (SS). Peripheral venous blood samples were collected at a median time of 36 hours after the onset of clinical symptoms. Blood samples of 23 age matched control volunteers (CTRL) were used for comparison. EC MP were identified by antibodies to EC antigen CD105 (endoglin) and the highly specific CD144 (VE-cadherin) using a three-color flow cytometry assay. Platelet, white, and red blood cell MP were identified using cell specific antibodies to CD41a, CD45, and CD235a, respectively. Lesion volume was measured on magnetic resonance diffusion-weighted imaging (DWI) and clinical outcome was based on the Rankin score at hospital discharge.
Results: Plasma counts of CD105+CD41a-CD45- EC MP were elevated in SS (median: 840/µL; 25th-75th%: 565-1079/µL) as compared to CTRL (415/µL; 201-624/µL; p=0.014). Moreover, CD105+CD144+ EC MP were elevated in SS (261/µL; 137-433/µL) when compared to MS (154/µL; 99-182/µL; p=0.031) or the CTRL group (140/µL; 79-247/µL; p=0.031). Interestingly, CD105+CD41a-CD45- EC MP, but not CD105+CD144+ EC MP, exhibited in AS group a correlation (p=0.005; r=0.45) with brain lesion volume on DWI. In addition, CD105+CD144+ EC MP in the admission samples correlated (p=0.0007; r=0.54) with the Rankin disability score in the AS group at hospital discharge as did CD105+CD41a-CD45- EC MP (p=0.007; r=0.44).
Conclusion: Specific phenotypes of EC MP in the plasma samples of stroke patients were associated with stroke severity, ischemic lesion volume and clinical outcome. Analysis of EC MP in peripheral blood of stroke patients could be of diagnostic and prognostic use.
Effects of the Anesthetic, Ketamine, in Developing Rat and Monkey Forebrain Cultures
W. Slikker, Jr.1 , C. Hotchkiss2 , N. Sadovova3 , R. Devine3 , X. Fu4 , A. Scallet1 , J. Hanig5 , C. Wang1 , 1NCTR/Neurotoxicology, 2Bionetics Corporation, 3Toxicologic Pathology Associates, 4NCTR/Biochemical Toxicology, 5CDER/FDA
Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, has been used as a general pediatric anesthetic for surgical procedures in infants. Recent data from developing rats suggest that anesthetic drugsmaycause dose-dependent neurodegeneration.
Purpose: To compare ketamine-induced developmental neurotoxicity using rhesus monkey (postnatal day (PND) 3) frontal cortical and rodent (PND 7) forebrain culture and also to determine if dysregulation of NMDA receptor subunits promotes ketamine-induced apoptosis.
Methods: Primary brain cell cultures were incubated for 24 hrs with 1, 10 or 20 µM ketamine alone or co-incubated with ketamine and NR1 antisense oligonucleotides (2 µM). After washout of ketamine, cultures were kept in serum and glutamate-containing medium for 24 hrs.
Results: Ketamine (10 and 20 µM) caused a marked reduction in immunostaining for PSA-NCAM, a substantial increase in DNA fragmentation as measured by cell death ELISA, increased TUNEL-positive cells, and a reduction in MTT metabolism. NR1 antisense protected the neurons from ketamine-induced degeneration. Western analysis showed that neurotoxic concentrations of ketamine resulted in a decrease in PSA-NCAM expression. NR1 antisense prevented this effect of ketamine, suggesting that ketamine-induced cell death is associated with a compensatory up-regulation of the NMDA receptor.
Conclusions: NR1 offers neuroprotection from enhanced degeneration in vitro, and forebrain cell death induced by ketamine negatively impacts cortical synaptogenesis. Based on these in vitro results from developing brain tissue, cultured monkey forebrain is just as sensitive to the apoptotic effects of ketamine as the cultured rodent forebrain. Supported by NCTR/CDER/FDA, NTP and NICHD.
A REEVALUATION OF ANNEXIN I EXPRESSION IN NORMAL BREAST AND BREAST CARCINOMA
R. Speer1 , J. D. Wulfkuhle1 , V. S. Calvert1 , M. Raffeld2 , T. A. Braunschweig3 , S. M. Hewitt3 , L. A. Liotta1 , E. F. Petricoin1 , 1NCI/FDA Clinical Proteomics Program, Bethesda, MD, 2Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, 3Advanced Technology Center, National Cancer Institute, NIH, Bethesda, MD
Purpose: To reevaluate the annexin I expression in normal breast and breast carcinoma.
Experimental Procedures: We employed laser capture microdissection in order to select pure cell subpopulations of interest from heterogeneous frozen tissue sections of a human tissue set comprised of normal breast tissue, DCIS and breast cancer. The annexin I expression was analyzed using reverse phase protein microarray and Western immunoblotting. Tissue microarray staining and immunohistochemistry were used to verify the results and to localize the annexin I expression more specifically.
Results: Proteomic analysis of human breast tissue indicates the loss of annexin I expression during tumorigenesis. Lysates of 16 normal breast tissue samples, 1 DCIS sample and 45 breast cancer samples (partly matched) were analyzed by reverse phase protein microarray, showing dramatically decreased annexin I levels in cancer compared to normal breast tissue. Western blotting of 5 normal and 5 breast cancer lysates confirmed these findings. Immunohistochemical staining of both frozen breast sections and paraffin embedded tissue microarray supports the findings of a downregulation of annexin I during tumorigenesis.
Conclusions: Biological insights into the role that the annexin protein family plays in normal and aberrant cellular function may implicate this molecule as key mediator of cell growth and cell cycle modulation. We were able to show the downregulation of annexin I during tumorigenesis of breast cancer. This finding may reveal an important early event during carcinogenesis, namely that the annexin I down-regulation may contribute to this loss of structure and enable the cancer cells to invade adjacent local tissue structures, culminating in distant metastases.
Risk of Local Adverse Events Following Cardiac Catheterization by Hemostasis Device and Gender
D. R. Tavris1 , S. Deh2 , B. Albrecht-Gallauresi1 , R. E. Shaw3 , R. G. Brindis4 , W. S. Weintraub5 , K. Mitchell2 , 1FDA, 2American College of Cardiology, 3San Francisco Heart Institute, 4Sam Framcoscp Kaiser Hospital, 5Emory Center for Outcomes Research
Background:
Reports to the FDA of local complications resulting in serious injuries and deaths associated with the use of hemostasis devices following cardiac catheterization provided the impetus for this study of risk factors from a national cardiac database.
Methods:
Data was obtained from the American College of Cardiology-National Cardiovascular Data RegistryTM, updated to include supplemental information specifically for this research. It included information from 59 institutions and 13,878 cardiac catheterizations (diagnostic and therapeutic) performed during October-December of 2003. Multiple logistic regression, using ten different outcomes, was used to assess the risk associated with type of device and gender, while controlling for demographic, physiologic, and procedure related variables, and several indices of co-morbidity.
Results:
Serious adverse events were reported in 3.54 % of patients, the most common being hematoma (2.00 %).
In the multivariate analysis, factors that were positively associated with 'any vascular complication' included female gender (OR=1.69, p<.0001), sheath size (OR=1.24, p=.005), activated clotting time (ACT) (OR=1.02, p<.0001), renal failure (OR=1.85, p=.006), and emergency indication for the procedure (OR=1.59, p=.001). Compared with manual compression controls, there was one hemostasis device that demonstrated an association with 'any vascular complication' (OR=2.42[1.41-4.16], p=.001).
Conclusions:
Women have almost twice the risk of men for most local complications following cardiac catheterization. Other risk factors included sheath size, ACT, renal failure, emergency indication for the procedure, and one type of hemostasis device. Possible reasons for these associations (including unmeasured confounding variables) will be discussed.
Neuroimaging Studies in CNS Drugs Approved from 1995 to 2004
R. S. Uppoor1 , S. U. Yasuda1 , P. Mummaneni1 , E. Cooper2 , H. Pien2 , G. Sorenson2 , J. M. Collins1 , M. U. Mehta1 , 1CDER, FDA, Rockville, MD, 2Harvard Medical School, Boston
BACKGROUND/AIMS: The objective of this project is to evaluate the use of the imaging modalities in development of Neuropharmacological drugs. Neuroimaging (NI) has the potential to link pharmacokinetics (PK) with pharmacodynamics (PD) and could therefore be an important aid in understanding mechanisms of action and adverse effects of drugs, as well as in identifying mechanisms for variability in drug response. Imaging can be an effective tool in optimizing Neuropharmacological drug development.
METHODS: NDAs approved from 1995 to 2004 in the Division of Neuropharmacological Drug Products were evaluated using a survey type approach. Information collected included the stated purpose of imaging study, PK, imaging, and PD results, and whether the study was used to support dosing or the proposed indication. Evaluation included review of the NDA submission, the final label, the FDA reviews and published literature.
RESULTS: There were 106 NDAs approved in the Neuropharmacology division. Fifteen of these NDAs included NI studies. Five of these NDAs had receptor occupancy studies and 2 were used to support the pharmacology/proof of concept. The receptor occupancy results are in reasonable agreement with the final selected doses/dose regimen.
CONCLUSIONS: A database capturing characteristics of NI studies in drug development has been initiated. Future efforts will focus on identifying those NI characteristics in the literature and in IND/NDAs that can be used to optimize drug development.
Mechanistic studies of the photocytotoxicity elicited by tattoo inks and component pigments
W.G. Wamer, CFSAN, FDA, College Park, MD
Background: A number of anecdotal reports suggest that tattooed skin can exhibit increased sensitivity to the sun. There is a need for systematic examination of tattoo inks and pigments for photoactivity.
Methods: Using an in vitro assay, 65 tattoo inks and 9 pigments were screened for photocytotoxicity. Three pigments and 3 tattoo inks were subsequently selected for additional mechanistic studies. To determine the wavelength dependence for photocytotoxicity, human skin fibroblasts, pre-treated overnight with tattoo inks or pigments, were irradiated with UVA light (320-400 nm) combined with visible light (400-800 nm) or with visible light alone. Photocytotoxicity was assessed as inhibition of colony growth. To gain insight into the mechanism of photocytotoxicity, we examined the connection between photocytotoxicity and oxidative damage, measured as lipid peroxidation.
Results: A significant number of tattoo inks and 3 pigments (Pigment Red 122, Pigment Red 170 and titanium dioxide) were photocytotoxic. The photocytotoxicity of titanium dioxide was fully dependent on the inclusion of UVA light during irradiation. The photocytotoxicity of Pigment Red 122 and Pigment Red 170 was partially reduced by filtering out UVA light. The photocytotoxicity of inks containing these three pigments showed a similar dependence on irradiation with UVA light. Titanium dioxide and Pigment Red 170 elicited lipid peroxidation concomitant with phototcytotoxicity.
Conclusions: These results suggest that sunlight may play a role in adverse reactions to tattoos. The roles of UVA light and oxidative damage need to be further investigated.
Application of A Model-Based Analysis to Improve the Drug Development Plan for A Life-Threatening Disease
Y. Wang, R. Powell, N. Beasley, P. Marroum, J. V.S. Gobburu, CDER, FDA, Rockville, MD
Background: Drug X was designed to delay the time to a disease event. A retrospective subgroup analysis of a failed pivotal trial (A) identified a responder population for the intent-to-treat patients in a second pivotal trial (B), which also failed. The purpose of the analysis was to explore the potential reasons for the failure of the two trials and provide guidance for future trials.
Methods: A time-dependent biomarker-disease event relationship was explored in both trials by the Cox proportional hazard model. The effect of drug X on biomarker level was analyzed in both trials by ANCOVA with baseline biomarker level as the covariate. Based on the Cox regression model, a simulation was performed to investigate the required biomarker level reduction to show significant clinical benefit.
Results: Biomarker level reduction was found to be correlated with a reduced risk of having the disease event. Drug X was shown to be able to lower the biomarker level significantly compared to placebo. The time-to-event analysis in both trials, however, failed to show the clinical benefit of drug X. The simulation results indicated that biomarker level reduction was not sufficient to show the expected clinical benefit given the sample size studied.
Conclusion: The analysis showed greater biomarker level reduction was needed to show the clinical benefit of drug X. To maximize the probability of success, higher doses should be studied in future trials given the observed dose-response relationship for the biomarker in dose-ranging studies. The sponsor accepted the recommendation and is studying higher doses.
Expression of hypoxia inducible factor (HIF-1a), erythropoietin (EPO) and heme oxygenase in guinea pig (Cavia porcellus) after exchange transfusion with hemoglobin-based blood substitutes.
L. Yeh, P. Buehler, N. Tayebi, A. I. Alayash, CBER, FDA, Bethesda, MD
Purpose: Expression of many mammalian genes is regulated by oxygen (O2) tension. This study was designed to test the concept that administration of cell free Hb-based oxygen carrier (OxyglobinTM) and its various oxidative and oxygenation states can be correlated to the expression of HIF-1α, a global transcriptional factor and other hypoxia sensitive genes such as erythropoietin (EPO), and heme oxygenase (HO-1) in a model of exchange transfusion.
Methods: We performed 50% and 75% blood substitute- OxyglobinTM exchange transfusion in guinea pig (N = 20). Animal without treatment served as baseline control. We excercised the kidney from these guinea pigs at specific time period (up to 72 hr) and organs were frozen in liquid nitrogen. Total RNA was extracted using Trizol. The cDNA for HIF-1α,EPO and HO-1 were generated by reverse transcription with primer pairs designed and synthesized based on the sequences of human in the GenBank. By semiquantitative RT-PCR, mRNA of HIF-1αwere detected using nested PCR technique.The genes were amplified using PCR and the products were separated using 1.5% agarose gel. Expression of G3DPH mRNA by a similar RT-PCR procedure was used as a positive control. The oxygenation and redox states of the Hb in sera obtained from animal at different time points were monitored by spectrophotometry.
Results: Ferrous Hb monitored during transfusion decline as a function of time and RBCs levels in animals. This was coupled with a proportional elevation of the ferric non-functional ferric form of Hb, indicating loss of the oxygen carrying capacity of Hb. Since only partial cDNA of HIF-1α in guinea pig was available in GenBank, the RT-PCR product of HIF-1α was identified and confirmed by restriction enzyme digestion as well as sequencing. Furthermore, the sequenced gene product of HIF-1α from the partial cDNA, was compared with known human HIF-1α sequence with 94% homology. The expression of HIF-1αwas increased three-folds over the control (Sham) after 4 hours of exchange transfusion and up to 72 hr. The levels of both EPO as well as HO-1 mRNAs were also elevated in the kidney tissues of these animals.
Conclusions: We demonstrate for the first time that a redox active "oxygen carrier" crosstalks with an "oxygen sensor" in a model of exchange transfusion. We also demonstrate that HIF-1α activates the transcription of genes whose proteins mediate adaptive responses to hypoxia, including EPO and HO-1.
Estimating the Uncertainty in the Estimated Mean Area Under the ROC Curve of a Multi-Feature Classifier
W. A.. Yousef1 , R. F. Wagner2 , M. H.. Loew3 , 1CDRH, FDA, Medical Device Fellows Program, Rockville, MD and George Washington University, Washington, D.C., 2CDRH, FDA, Rockville, MD, 3George Washington University, Washington, D.C.
This poster considers the problem of binary classification using any number of biomarkers and its assessment in a distribution-free approach. We estimate the area under the ROC curve of a classifier using estimators based on bootstrap methods of Efron and Tibshirani. We then use the method of the influence function to estimate the uncertainty of a particular estimate. Monte Carlo trials show that small-sample estimates can be obtained with little bias.
RhoGDI Protects Cancer Cells against Drug-induced Apoptosis
B. Zhang, Y. Zhang, E. Shacter, FDA, Bethesda, MD
Rho GDP Dissociation Inhibitor (RhoGDI) plays an essential role in control of a variety of cellular functions through interaction with Rho family GTPases including Rac1, Cdc42, and RhoA. RhoGDI is frequently overexpressed in different types of human tumors and chemoresistant cancer cell lines, raising the possibility that RhoGDI might play a role in the development of drug-resistance of cancer cells. Here, we found that overexpression of RhoGDI in MDA-MB-231 human breast cancer cells and JLP-119 lymphoma cells strongly inhibited apoptosis induced by different chemotherapeutic agents. Using DNA vector-mediated RNA interference, we were able to establish a stable MDA-MB-231 breast cancer cell line expressing siRNA targeting RhoGDI, rendering specific and persistent suppression of RhoGDI protein expression. These RhoGDI knockdown cells are significantly more susceptible to drug-induced apoptosis. Furthermore, we confirm that the observed knockdown phenotype is the result of silencing of the intended target by using a rescue plasmid to restore RhoGDI expression and drug-resistance. Taken together, these data suggest that RhoGDI may function as an antiapoptotic molecule that mediates cellular resistance to chemotherapy. In addition, we provide evidence that RhoGDI is a potent inhibitor of caspase-mediated cleavage of Rac1 GTPase. These results add a novel role for RhoGDI, showing that it shields Rac GTPase from proteolytic cleavage. Apoptotic cleavage of Rac1 has been shown to be required for maximal apoptosis to occur in response to cytotoxic drugs. Thus, RhoGDI-mediated inhibition of apoptosis may be partly through protection of Rac1 GTPase from caspase cleavage.
EXAMINATION OF THE VASCULAR INJURY IN SPRAGUE-DAWLEY (SD) RATS INDUCED BY THE PHOSPHODIESTERASE (PDE) IV INHIBITOR SCH 534385: A COMPARISION WITH SCH 351591.
J. Zhang1 , E. H. Herman1 , A. Knapton1 , T. J. Miller1 , P. Espandiari1 , R. Snyder2 , J. Hanig1 , J. L. Weaver1 , 1CDER, FDA, Silver Spring, MD, 2Schering-Plough Research Institute
Our previous study showed that the PDE IV inhibitor SCH 351591 induced vascular injury in SD rats and suggested that a panel of serum proteins could potentially serve as biomarkers for detecting and monitoring vascular lesions (Toxicologist 78: 375, 2004). The present study was begun to determine whether the structurally distinct PDE IV inhibitor SCH 534385 would induce similar vascular injury and, if so, whether these same biomarkers would again be observed . SD rats were given SCH 534385 (20 or 40 mg/kg/day for 3 days) by gavage. Microscopic evaluation of tissues obtained 24 h after the final dose revealed dose-dependent vascular injury. Arterial hemorrhage and necrosis, periarterial inflammation, and microvascular injury (fibrin insudation and fibrin exudation) were found in the mesentery, pancreas, kidney, liver and small intestine. Activation of immune system cells (endothelial cells, mast cells, and macrophages) and proliferation of fibroblasts were also noted at sites of inflammation. Lymphocyte numbers were markedly decreased in splenic and thymic cortex. In peripheral blood, granulocytes were elevated and lymphocytes decreased. SCH 534385 induced increases in the serum proteins alpha-1-acid glycoprotein, haptoglobin, GRO/CINC-1 (homolog of human interleukin-8), vascular endothelial growth factor, and tissue inhibitor of metalloprotease-1. No significant changes were found in C-reactive protein. The pathological changes in the splanchnic vasculature, elevations in serum proteins, and alterations in leukocyte numbers in rats treated with this compound were similar to those seen in rats given SCH 351591. The data show that two structurally different PDE IV inhibitors, SCH 351591 and SCH 534385, induced similar vascular injury and comparable changes in biomarkers. Studies are underway to further characterize the etiology of vascular damage induced by PDE IV inhibitors and the clinical utility of serum biomarkers.
Bioinformatics Analysis of Possible Biomarkerfor the Evaluation of Biologics Product Quality
B. G. Zaslavsky1 , J. Han2 , J. Tiwari3 , R. K. Puri1 , 1CBER, FDA, Rockville, MD, 2CBER, FDA, Bethesda, MD, 3BER, FDA, Rockville, MD
To develop biomarkers of cellular quality, we utilized DNA microarray technology to determine gene expression during various growth conditions of 293 human embryonic kidney (HEK) cell line. These cells were cultured under different confluence states (40%, 90%, and over confluence), RNA isolated and labeled targets were hybridized with high quality ~10K cDNA microarrays. The image was captured by Axon scanner and data analyzed by GenePix software. The data were further analyzed by various tools available at NIH Center for Information Technology microarray database.
The logistic regression method of SAS software was applied to verify the genes identified by gene expression profiles. Two-group classification (over confluence vs. 90% confluence) was used to select the subset of differentially expressed genes. Because the number of studied genes was too large relative to the number of cases available, the leave-one-out cross-validation procedure was used to estimate the subset of most informative genes. For each leave-one-out training set a different logistic classifier was built; therefore the estimated error rate applies to the procedure used to build the classifier. The normalization procedures were performed in the preparation of highly processed raw data for statistical analysis. In order to evaluate the robustness or stability of the logistic classifier we created two different sets of data applying different normalization procedures to the set of raw data. We used one of the normalized datasets as training set for the logistic predictor and another dataset as the test sets. Application of the logistic predictor to the test datasets confirmed the robustness of the predictor to the normalization procedures. This predictor accurately predicted the class membership on the basis of the expression level of key genes. These analyses identified gene clusters that may serve as biomarker for assessment of the quality of 293 cell substrates in different confluence states.
An Analysis of Genetic Toxicity, Reproductive and Developmental Toxicity, and Carcinogenicity Data: I. Identification of Carcinogens Using Surrogate Endpoints
E. J. Matthews, N. L. Kruhlak, R. D. Benz, J. F. Contrera, US Food and Drug Administration, Center for Drug Evaluation and Research (HFD-901), 5600 Fishers Lane, Rockville, MD 20857
A retrospective analysis of standard genetic toxicity (genetox) tests, reproductive and developmental toxicity (reprotox) studies, and rodent carcinogenicity bioassays (rcbioassay) was performed by the FDA Center for Drug Evaluation and Research (CDER) Informatics and Computational Safety and Analysis Staff (ICSAS) to identify the genetox and reprotox endpoints whose results best correlate with rcbioassay observations. Criteria for a good surrogate for predicting carcinogenicity was high (>73%) positive predictive value, specificity, and correlation indicator (the average of the former two parameters), and low (<25%) false positives. The goal was to expand upon the evidence of a correlation between Salmonella t. mutagenicity and carcinogenicity test results established by the National Toxicology Program. ICSAS compiled genetox, reprotox, and rcbioassay databases on 5938, 2115, and 1488 chemicals, respectively; 1112 of the chemicals have both genetox and rcbioassay data and 721 chemicals have both reprotox and rcbioassay data. This study revealed that 11 genetox endpoint results provided a good correlation with rcbioassay findings: 8 gene mutation tests, 2 in vivo clastogenicity tests, and the unscheduled DNA synthesis assay. In addition, the results of 3 endocrine organ reprotox studies correlated with detection of carcinogens. In contrast, the results of many other genetox and reprotox tests were not successful by our criteria in predicting carcinogenicity.
An Analysis of Genetic Toxicity, Reproductive and Developmental Toxicity, and Carcinogenicity Data: II. Identification of Genotoxicants Using In Silico Methods
E. J. Matthews, N. L. Kruhlak, R. D. Benz, J. F. Contrera, US Food and Drug Administration, Center for Drug Evaluation and Research (HFD-901), 5600 Fishers Lane, Rockville, MD 20857
This investigation is part of program to develop in silico methods to provide decision support information for US Food and Drug Administration (FDA) regulatory and research activities. A retrospective analysis of standard genetic toxicity (genetox) tests, and reproductive and developmental toxicity (reprotox) studies was performed by the FDA Center for Drug Evaluation and Research (CDER) Informatics and Computational Safety and Analysis Staff (ICSAS) to create genetox and reprotox endpoint modules using MC4PC computational toxicology software program (MultiCASE, Inc.) that can provide good in silico quantitative structure activity relationship (QSAR) predictions for those endpoints. The modules created correspond to the 14 genetox and endocrine organ reprotox endpoints that produced results that correlated with those of carcinogenicity studies. Of these, the plant mutation data were not suitable for QSAR modeling. The Salmonella mutation module had a sensitivity of 70.1%, a specificity of 89.9%, a positive predictivity of 85.1%, a correlation indicator (the average of specificity and positive predictivity) of 87.5%, and a false positive rate of 10.1% for this same endpoint. The other 12 QSAR modules also exhibited a high correlation indicator (>75%) and low false positive rate (<15%) for their respective laboratory findings. In addition, for 1112 chemicals having both rcbioassay data and also genetox data at at least some of the 10 endpoints, experimental results, and, where these were lacking, in silico predictions for genetox and reprotox data were pooled to determine cumulative predictions of carcinogenicity.
Beyond the LFER Paradigm: Harnessing Atomic Descriptors and Artificial Neural Networks to Predict pKa
R. Fraczkiewicz, G. Fraczkiewicz, B. Steere, M. B. Bolger, Simulations Plus
A vast array of chemical and biological properties of molecules strongly depend on ionization in water - the fundamental solvent in living systems. Consequently, the knowledge and understanding of ionization constants (pKa) is of chief importance to the pharmaceutical and environmental industries. The cheapest and easiest way of obtaining pKa for new molecules is in silico prediction. Almost all computational methods for this purpose are based on a perturbational approach utilizing the Linear Free Energy Relationships (LFER) of Hammett and Taft. An alternative route to pKa prediction, successful in the area of QSAR/QSPR, involves a direct, non-linear correlation between the observed property, pKa, and calculated descriptors. However, unlike straigtforward molecular property-molecular descriptors relationships (e.g., log P, solubility), the localized nature of ionization requires creation of a new class of atomic descriptors. Another serious problem: unlike measured log P or solubility which, essentially, describe single reactions, the measured pKas of a polyprotic molecule are a net effect of (sometimes) very large and complex networks of microscopic equilibria. We have solved both problems and will present the resulting model of pKa prediction.
Development of a Structure-Searchable Subchronic Studies Database for Use in Predictive Toxicology Modeling
C. Nelson, J. Mayer, K. Arvidson, A. McDougal, E. Lee, M. Twaroski, CFSAN, FDA, College Park, MD
Background: Toxicology data are being compiled to create comprehensive, structure-searchable databases with the goal of improving regulatory safety reviews and developing better toxicology guidance. FDA/OFAS is exploring the use of predictive toxicology as a tool in making safety decisions, based on chemical structure, when little or no toxicity data are available.
Methods: Subchronic study information and FDA reviewer conclusions were extracted from toxicology memoranda and original study reports stored in microfiche, paper copies, and electronic files.Electronic dictionaries in the form of pull-down menus were used to standardize language when possible. An MS Access platform based on FDA/CFSAN's Redbook 2000 was developed in order to facilitate data entry and to allow for export of the data into other software programs. The subchronic database was imported into software that allows more comprehensive data visualization and structure searching capabilities.
Results: Subchronic studies submitted to FDA/CFSAN in support of submissions were data mined from multiple formats and consolidated into a structurally searchable platform. This platform allows for efficient data management and identification of similar compounds to compare safety data, such as target organ toxicity.
Conclusions: The FDA is organizing its toxicity data from multiple formats into structure-searchable databases. For new chemicals with limited toxicological data, searching for chemicals with similar structures and substructures will aid in identifying appropriate analogs for structure-activity relationship (SAR) analysis. The compilation of this subchronic database will also be useful in building models that can more efficiently predict toxicity in chronic studies, as well as aid in the design of those studies.
Does the lack of genetic diversity in animal models currently used for safety testing put the public at risk?
S. H. Nye1 , N. V. Cozzi1 , J. Baye1 , D. Evans1 , Y. Evrard1 , S. Korb1 , H. Vernon1 , A. Wittenburg1 , M. Hessner2 , X. Wang2 , H. J. Jacob2 , R. J. Roman2 , 1PhysioGenix, Inc., Milwaukee, WI, 2Medical College of Wisconsin, Milwaukee, WI
Purpose: Safety testing in Pharma is currently done by using inbred or outbred rat strains. Inbred strains offer reproducibility but have limited predictive value to other rat strains let alone humans. Outbred strains lack the perceived degree of genetic diversity and test populations cannot be reproduced in reasonable sample sizes. To address this, PhysioGenix developed genetically diverse PharmGenix rat panels that capture 82% of the commercially available genetic diversity in the rat and are faithfully reproduced in a controlled fashion.
Methods: PharmGenix rats were tested for sensitivity to the toxic effects of the antibiotic gentamicin, the analgesic acetaminophen and the Alzheimer's drug tacrine. Biomarkers in urine and blood were analyzed, organ pathology was assessed and gene expression by microarray analysis was performed.
Results: The differential response exhibited by PharmGenix rat strains revealed genetic components underlie toxicity to gentamicin. PharmGenix rats also responded differentially to acetaminophen, but the pattern of toxicity was dependent on the target organs (liver, kidney). Tacrine elevated serum transaminases in three PharmGenix strains, but hepatotoxicity was missed by the Sprague-Dawley and F344. For mechanistic studies, knowing that PharmGenix rats respond differently to drugs enables 20-fold enrichment in finding relevant susceptibility and resistance genes by microarray and this number is further refined when combining haplotype information.
Conclusions: Genetic background has a large impact on the response to known renal and liver toxicants and suggests that results of efficacy or safety testing performed in any single strain of rats will not predict the range of responses expected in humans.
Comparison of Felbamate and Fluorofelbamate Intermediary Metabolism by Human Liver S9 Fractions Leading to Reactive Metabolites
R. J. Parker1 , N. R. Hartman1 , B. A. Roecklein2 , H. Mortko2 , H. J. Kupferberg3 , J. Stables4 , J. M. Strong1 , 1FDA, 2MedPointe Pharmaceuticals, NJ, 3Consultant, Potomac MD, 4NIH / NINDS
Background: Previous studies have demonstrated that metabolism of felbamate (FBM) in humans and animals generates a reactive intermediate ATPAL which may be responsible for the toxicities observed with this drug. Formation of ATPAL from its immediate precursor requires loss of a 2-position hydrogen and it has been postulated that substitution of this atom with fluorine would prevent the formation of ATPAL. Based on this hypothesis fluorofelbamate (F-FBM) was synthesized and is presently undergoing drug development.
Methods: We compared the metabolism of selected metabolic precursors of FBM and F-FBM in human liver S9 fractions using glutathione (GSH) as a trapping agent for any reactive metabolites formed.
Results: When incubated with human liver S9 fraction, the FBM precursor MCF was oxidized to CCMF and further oxidized to CPPA. In contrast, the F-FBM precursor F-MCF was stable and no metabolites were observed. When CCMF was incubated with human liver S9, both oxidation to CPPA and reduction to MCF were observed and a new atropic acid GSH adduct was identified. When F-CCMF was incubated under the same conditions, both reduced and oxidized metabolites, F-MCF and F-CPPA, were identified.
Conclusions: Our results support the hypothesis that F-FBM and F-CCMF are not metabolized by human S9 fraction in vitro to the known FBM reactive metabolite ATPAL.
The teratogen, valproic acid, alters homeobox gene expression during retinoic acid-induced nerve cell differentiation
D. Reese1 , M. Ramos-Valle2 , J. Breger3 , 1CFSAN, OARSA, DMB, Laurel, MD 20708, 2CFSAN, OARSA, DTNPS, Laurel, MD 20708, 3JIFSAN Student Intern, University of Maryland
Background: A short-term gene expression assay is being developed for detecting potential teratogenic agents in food and dietary supplements. This assay monitors an agent's ability to alter the expression of a key class of developmental control genes, homeobox (Hbox) genes, during retinoic acid (RA)-induced nerve cell (NC) differentiation in P19 mouse stem cells. Known teratogens are being evaluated in this system to assess its effectiveness in "flagging" potential teratogens. Valproic acid (VPA), used to treat seizures and bipolar disorder in humans and a potent teratogen, causing neural tube and limb defects, lower IQs, and developmental delays, was evaluated.
Methods: Hbox gene expression was monitored in P19-cell embryoid bodies exposed 4 days to RA or RA+VPA. Hbox-containing genes were selectively amplified from total RNA by RT-PCR and detected on an oligonucleotide array.
Results: VPA selectively upregulated a subset of homeobox genes (Hoxa9, Hoxc8, Hoxc9, Hoxd8, Hoxd9) that are not expressed during NC differentiation in this system. Upregulation of these genes, which are expressed in the lumbar region of the developing embryo, the site of spina bifida, was dose dependent and occurred at concentrations generated in the blood by therapeutic doses of the drug. Although NC differentiation was observed in valproate-treated cultures, these cultures had a significant population of proliferating/undifferentiated cells not seen in control cultures.
Conclusions: Agents that selectively alter the normal expression of key developmental control genes are potential teratogens and candidates for further evaluation in animal bioassays. Valproate would have been "flagged" by this assay for further evaluation.
Metabonomics: Bringing Safety Assessment into Early Drug Discovery
D. G. Robertson1 , L. Egnash2 , D. Wells1 , L. Robosky2 , M. Manning2 , M. D. Reily2 , J. M. Stanislawski1 , K. Datta1 , 1Department of Worldwide Safety Sciences, Pfizer Global R&D, Ann Arbor, 2Department of Discovery Technologies, Pfizer Global R&D, Ann Arbor
PURPOSE: Metabonomics has significant potential for bringing safety assessment early into the drug discovery process. Development of a candidate compound was hampered by sporadic and unexplained mortality.
METHODS: A rat in vivo toxicity study with a metabonomic component was conducted to evaluate the phenomenon. Twenty-four rats were divided into 4 dose groups (3/sex/dose) and administered 0, 100, 300 or 1000 mg/kg Compound X, once daily for 7 days. Urine was collected for metabonomic profiling. Routine hematology, clinical chemistry, gross pathology and limited histopathology were evaluated at termination.
RESULTS: All 300 and 1000 mg/kg animals died or were sacrificed in moribund condition by Day 3. A single 100 mg/kg female died on Day 6. Traditional toxicity endpoints, (toxicokinetics, clinical and microscopic pathology) were uninformative as to cause of death. Metabonomics evaluation identified a profound glucosuria accompanied by increased urinary ketone bodies and creatine that was completely reversed by Day 7, despite continued dosing. A second study was conducted at doses of 0, 30, 100 and 300 mg/kg (6 males/group) with more comprehensive serum and urine collection. The second study confirmed the findings of the first study, established the time-course of effects and identified transient decreases in serum triglycerides and elevations in serum glucose, insulin and corticosterone as concurrent findings.
CONCLUSIONS: Metabonomics evaluation identified heretofore-unknown in vivo biochemical sequelae of Compound X treatment that may have implications in understanding the pharmacology of the compound, as well as providing a simple biomarker (urine or serum glucose) and an approach for screening molecules in this class.
The Prediction of Maximum Recommended Therapeutic Dose (MRTD) with a Neural Network Ensemble Model
B. Steere1 , R. Fraczkiewicz1 , E. J. Matthews2 , R. D. Benz2 , N. L. Kruhlak2 , J. F. Contrera2 , 1Simulations Plus, Inc., Lancaster, CA 93534, 2ICSAS, OPS, CDER, FDA, Rockville, MD 20852
Background: MRTD is correlated to the toxic dose threshold in humans. The prediction of toxicity has been identified as a priority of the Critical Path Initiatives.
Methods: The ICSAS group reviewed MRTD data for orally-delivered drugs from clinical trials reports. The structures and MRTD values for 1,220 non-proprietary drugs were collated in a publicly-available database. 1,039 of the structures in this database were used by the Simulations-Plus group to generate molecular descriptors. The data was divided into training and test sets, then selected descriptors were combined with the MRTD values to build an ensemble of 32 neural network models.
Results: The output of the model was binned into categories to characterize the compounds in a 115-member sequestered test set as "Active", "Inactive", or "Inconclusive". The optimal neural net architecture had 5 hidden neurons and employed 40 descriptors. The ensemble correctly identified the inactive compounds 76% of the time, and misidentified them as active 14% of the time. Conversely, the model correctly identified the active compounds 67% of the time and misidentified them as inactive 10% of the time. Performance was consistently better for inactive compounds (high MRTD) than active ones (low MRTD).
Conclusions: The model identifies general chemical features that high-MRTD drugs often share such as low aromaticity, hydrophilicity, and the absence of chemical groups such as phenols and halogens rather than specific atomic arrangements that trigger toxic mechanisms. Even so, the model has sufficient resolution for application in early drug discovery to enrich libraries towards the selection of safer compounds.
MONITORING TIME-DEPENDENT EMERGENCE OF DRUG TOXICITY: USE OF A SPREADSHEET PRESENTATION IN TOXICITY MANAGEMENT
K. M. Wu, J. G. Farrelly, DAVDP, CDER, FDA
Detecting the emergence of drug toxicity is governed by time-dependent and dose-dependent factors, which form the basis of approaches to safety evaluation of a new drug. The approaches employ different durations and doses of drug exposures to subjects in product development. The time-dependent aspect of toxicity progression has not attracted as much attention as dose-dependency characteristics of a toxicity. We present here a spreadsheet tracking system for monitoring time-dependent emergence of toxicity. In the spreadsheet, specific toxicity is labeled as the row's heading whereas the time dimension is proportionally represented by successive columns on the y-axis. Using this format, a toxicity that occurs in one study duration (e.g., 3-month) and not in another (e.g., 2-week) can be tracked/highlighted by a milestone/signal at that time point (3-month). Auxiliary information such as threshold dose/NOEL/AUC, number/% of subjects affected regarding this toxicity could be provided at this time point to facilitate an overview. An overall toxicity profile, including that explored in different species, can be organized together by combing all equivalent data, as presented above, in a single spreadsheet. Questions such as what duration is required for a toxicity to emerge, at what exposure level or dose, and whether the toxicity is a cross-species phenomenon, can be answered from this presentation. It is concluded that by using this approach, the tracking mechanism could provide a comparative, comprehensive but rather concise presentation of a drug's toxicity profile and would significantly facilitate the Agency's secondary and tertiary review processes during drug development.
Review Tool for Pharmacogenomcis Data Submission: ArrayTrack
W. Tong1 , H. Fang2 , S. C. Harris2 , X. Cao2 , H. Sun2 , L. Shi1 , F. Qian2 , R. G. Perkins2 , D. A. Casciano1 , F. M. Goodsaid3 , F. W. Frueh3 , 1NCTR, FDA, Jefferson, AR, 2Z-Tech at NCTR, Jefferson, AR, 3CDER, FDA, Rockville, MD
DNA microarray is a key technology in pharmaco- and toxicogenomics, a field that has been identified in the FDA Critical Path document as a major opportunity for advancing medical product development and personalized medicine. It is expected that the regulation of microarray-based medical diagnostics and the review of microarray data submitted as part of an IND or NDA will become an essential regulatory responsibility for the FDA. A single microarray experiment generates a large volume of data. The management, analysis and interpretation of this vast amount of data are the most critical components in realizing the value of the technology. Many solutions are available, but, unfortunately, most of them, if not all, deal with these components separately, posing a great challenge for reviewers to efficiently handle microarray data. ArrayTrack integrates these three essential components into a single application with a user-friendly and intuitive interface, and thus provides a single solution for reviewers to analyze microarray data, interpret the information and verify the results submitted by sponsors. In addition, ArrayTrack provides the capability to develop an aggregate genomics knowledge base to support scientifically sound future regulatory policies. ArrayTrack has three integrated components: 1) a database (MicroarrayDB) that stores microarray experiment information according to the MIAME guideline; 2) tools (TOOL) that operate on experimental and public data for knowledge discovery; and 3) libraries (LIB) that contain functional data from public databases. Using ArrayTrack, the user can select an analysis method from the TOOL and apply it to selected microarray data stored in the MicroarrayDB; the analysis results are dynamically linked to the LIB for subsequent individual gene analysis, pathway- and Gene Ontology-based analysis and orthologos analysis. Currently, ArrayTrack is being integrated and further refined at the FDA as a review tool for pharmaco- and toxicogenomic data submission.
| Clear Science Communication Award - 2005 FDA Science Forum |
Plasma and/or Urine Penicillin Concentrations Predict Kidney Tissue Tolerance Level
O. A. Chiesa1 , D. N. Heller1 , M. Smith1 , R. Condon2 , J. Von Bredow1 , M. Thomas1 , 1OR, Laurel, MD, 2RJC Associates, MD
Background: Currently the concentration of penicillin in the kidney tissue of steers treated with penicillin is used to determine the acceptability of the drug treated steer carcass for human food consumption. If the penicillin concentration in the kidney tissue is greater than a tolerance level of 50 ppb, the entire steer carcass will be condemned and discarded. We sought to establish a correlation between the kidney tissue drug residue concentration and an easily accessible biological fluid, such as plasma or urine, to permit a pre-slaughter estimate to be made of the level of drug residue in the kidney tissue.
Methods: Steers were treated with an approved intramuscular dose of penicillin. Blood and urine samples were collected at defined intervals. Kidney tissue samples were obtained through laparoscopic surgery and at slaughter. Penicillin levels in plasma, urine and kidney tissue were determined by LC/MSMS
Results: Log-linear plots of penicillin levels in plasma, urine and kidney tissue demonstrated parallel trends over the drug residue elimination period of 3 to 48 hours. The kidney tissue level of 50 ppb penicillin correlates with measurable levels of penicillin in the plasma and in the urine.
Conclusion: A tissue residue-biological fluid correlation for penicillin predicts that a plasma concentration of less than 0.4 ppb and/or a urine concentration of less than 140 ppb may be used with 95% confidence that the kidney penicillin concentration will be below tolerance in 99% of penicillin treated steers. If the penicillin concentrations in the plasma or the urine correlate with a penicillin concentration in the kidney that is greater than the tolerance level, additional drug elimination time will be required before the animal may be slaughtered.
Dose-Dependent Transitions in Mechanisms of Toxicity
N. G. Doerrer1 , W. Slikker, Jr.2 , K. Wallace3 , M. E. Andersen4 , M. S. Bogdanffy5 , J. S. Bus6 , S. D. Cohen7 , R. B. Conolly4 , R. M. David8 , D. C. Dorman4 , D. W. Gaylor9 , D. Hattis10 , J. M. Rogers11 , R. W. Setzer11 , J. A. Swenberg12 , 1ILSI Health and Environmental Sciences Institute, 2NCTR, FDA, Jefferson, AR, 3University of Minnesota, Minneapolis, MN, 4CIIT Centers for Health Research, Research Triangle Park, NC, 5Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, 6Dow Chemical Company, Midland, MI, 7Massachusetts College of Pharmacy, Worcester, MA, 8Eastman Kodak Company, Rochester, NY, 9Gaylor and Associates, LLC, Eureka Springs, AR, 10Clark University, Worcester, MA, 11US EPA National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC, 12University of North Carolina, Chapel Hill, NC
Analysis of dose-response curves for many agents demonstrates that multiple mechanisms of chemical toxicity exist. For example, critical, limiting steps in any given mechanistic pathway may become overwhelmed with increasing exposures, signaling the emergence of new modalities of toxic tissue injury at higher doses. Kinetic- and/or dynamic-mediated responses may be altered in a non-linear manner with increasing dose. Case studies show that, as the dose of an agent increases, dose-dependent transitions such as receptor interactions, altered homeostasis, and saturation of pharmacokinetic and defense or repair mechanisms can and do occur. The ILSI Health and Environmental Sciences Institute (HESI), in partnership with FDA and others, undertook a project to explore dose-dependent transitions in mechanisms of toxicity. At a workshop co-sponsored by HESI and its partners, invited scientists from government, academia, industry, and research institutions evaluated principles and lessons learned from case studies for which dose-dependent transitions in mechanisms of toxicity can be demonstrated. Two scientific papers were published in Toxicology and Applied Pharmacology in late 2004. These papers summarize the HESI project, describe workshop results, and include discussion of the case studies and methods to incorporate these considerations into the risk assessment process. Because there are many examples of deviations from a linear relationship as one explores the full range of the dose-response curve, characterization and consideration of dose-dependent transitions in mechanisms of toxicity is a means to integrating the "best science" into the decision-making process.
Generic drug products demonstrate small differences in bioavailability relative to the brand name counterparts
P. E. Nwakama1 , S. H. Haidar1 , Y. Yang2 , L. X. Yu1 , 1OGD, Rockville, MD, 2OTR, Silver Spring, MD
Background: Bioequivalence (BE) studies are conducted "...to ensure therapeutic equivalence between a pharmaceutically equivalent test (T) drug product and a reference (R) listed drug."The current confidence interval limits of 80 -125% for Cmax and AUC ratios were established based on a survey of physicians which determined that a 20% difference in dose would not be clinically significant for most drugs. The objective of this study was to evaluate performance of the BE criteria, by looking at average differences in exposure (and Cmax) between reference listed drugs (RLD) and their generic equivalents.
Methods: BE studies submitted to the Office of Generic Drugs from 1996 to 2003 were evaluated. With few exceptions, data used for this study came from single-dose, fasting BE studies of approved oral drug products. Statistical analysis was performed on differences in exposure and rate of absorption between the RLD and its generic equivalent using point estimates for AUC and Cmax, respectively.
Results: A total of 1159 BE studies were examined; the sample size of each study ranged from 12 to 127 subjects. Mean difference in point estimates (T - R) for AUC0-T was 3.09%, and 3.01% for AUC0-inf. Mean difference for Cmax was 4.46%.
Conclusion: The results of this study show that generic products have averaged less than 4% difference in body exposure relative to the RLD, although the statistical criteria may allow differences of 20%. This demonstrates an important element of the high quality of generic products that have been approved over the past several years.
The Prediction of Human Pharmacokinetics at the Therapeutic Dose from Low Sub-Therapeutic Doses in Human.
C. L. Holliman, L. Buchholtz, J. McFadden, G. Pace, Pfizer Inc, Ann Arbor, MI, 48105
Background: Ultimately human pharmacokinetics can only be determined by human dosing. Low sub-therapeutic dosing is safe for the subject and can be used to predict human pharmacokinetics at the therapeutic dose. When the ionization efficiency of the compound is high, these studies can be supported with conventional LC/MS/MS.
Methods: Low sub-therapeutic studies are supported using API4000 LC/MS/MS with assays that achieve at least a 20-pg/mL limit of quantitation. The analytes are extracted from large plasma volumes by solid-phase extraction. Selectivity is a significant issue due to the relatively large concentrations of endogenous species co-extracted.
Results: Six case studies are presented. In three studies the strategy was tested in cynomolgus monkeys. In two studies the therapeutic pharmacokinetics are accurately predicted by the very low dose. The exposure was not dose-normalized in the third case. Two more case studies are presented where very low dose legs were added to human Phase I trials. In one case the low dose Cmax was not dose-normalized and all PK parameters were statistically different but in the other case the very low dose accurately predicted PK parameters and bi-phasic clearance. In the final case study three development candidates are separately dosed at very low levels to determine the candidate with a desirable half-life. The observed half-lives spanned a range of 10-64 hours. From this data an appropriate candidate was selected for development.
Conclusions: Very low dose studies can be viable strategy to predict or complement the prediction of pharmacokinetics at the therapeutic dose.
Do Men and Women Differ in Proximal Small Intestinal CYP3A Or P-Glycoprotein Expression?
M. F. Paine1 , S. S. Ludington1 , M. L. Chen2 , P. W. Stewart1 , S. M. Huang3 , P. B. Watkins1 , 1University of North Carolina, Chapel Hill, NC, 2OPS, CDER, Rockville, MD, 3OCPB, CDER, Rockville, MD
Background. The higher systemic clearance of some CYP3A4 (whether also P-gp) drug substrates in women compared to men is attributed in part to a higher hepatic CYP3A4 content in women. This, combined with the general paucity of reported sex differences in the apparent oral clearance of CYP3A4 substrates suggested a sex-dependent expression of CYP3A4 in the intestine but in a pattern opposite to the liver.
Methods. Duodenal biopsies obtained from healthy men (n=46) and women (n=45) were analyzed, by Western blot, for relative CYP3A4, as well as for CYP3A5 and P-gp, expression levels.
Results. Among all subjects, CYP3A4 and P-gp varied 8- and 10-fold, respectively. CYP3A5, which was readily detected in 27% of these predominantly white individuals, varied 7-fold. For all 3 proteins, a sex difference was not detected (p>0.55). The lack of a difference remained for CYP3A4 and P-gp when the analysis was restricted to white subjects (n=74) or to individuals with undetectable CYP3A5. Comparing the 21 pre-menopausal women (all were aged <45 years) with the 43 men aged <45 years, again no sex differences were detected in CYP3A4 and P-gp content. Comparing the pre- with post-menopausal women, mean CYP3A4 content was 20% lower in the post-menopausal individuals (p=0.01).
Conclusions. The lack of a sex-dependent difference in proximal intestinal CYP3A4 could account in part for the lack of reported sex differences in the oral, relative to systemic, clearance of some CYP3A4 substrates. Ramifications of lower intestinal CYP3A4 content in post- vs. pre-menopausal women require further investigation.
Dose Dependent Inhibition of Midazolam Elimination by Ketoconazole: Effect of CYP3A5 Genotype
A. Lucksiri1 , R. Vuppalanchi2 , J. K. Hilligoss2 , M. A. Hamman2 , L. Li2 , J. Y. Chien3 , S. M. Huang4 , S. Hall2 , 1Purdue University, 2Indiana University, 3Eli Lilly &Co, 4OCPB, CDER, FDA
AIMS: Ketoconazole (K), a potent inhibitor and substrate of CYP3A enzymes, is commonly used to determine the worst-case drug interaction for CYP3A substrates. The effect of K dose and CYP3A5 genotype on in vivo CYP3A activity was assessed using the probe drug, midazolam (M).
METHODS: 15 healthy volunteers (12 male), weighing (mean+SD) 76+12kg, completed a 3-phase, randomized, crossover study. Intravenous (IV, 0.05mg/kg over 30 minutes) and oral (4mg) M were administered on separate days before and during 7 days of dosing with K 200 or 400mg (K 1hr prior to IV M on day 6 and oral M on day 7). Serum samples were assayed for M and K by HPLC-MS. CYP3A5 genotype was obtained by allele-specific real-time PCR.
RESULTS: The baseline AUCs after IV and oral M were 149+29 and 45+16 ug/L/hr. The baseline oral bioavailability, systemic and oral clearance of M were 0.27+0.06, 26+6L/hr and 99+32L/hr, respectively. The average steady state concentration of K after 200mg and 400mg dosing was 1.3+0.5 and 3.9+1.4 mg/L, respectively. The fold increase in IV & oral M AUC was significantly greater (P<0.02) after 400mg K (4.2+0.6 & 15+4, respectively) compared to 200mg K (3.4+0.7 & 11+4, respectively) daily. CYP3A5 genotype (*1/*1, *1/*3; n=7 vs. *3/*3; n=8) did not significantly alter the extent of inhibition observed after K dosing (P>0.05).
CONCLUSIONS: The extent of CYP3A inhibition by K is dose dependent with significantly greater effect by 400mg compared to 200mg K dosing. Grant support FDT-001756 from FDA and M01-RR00750 from NIH.
SB-497115, a Novel, Oral, Small Molecule Thrombopoietin Receptor Agonist, Increases Platelet Counts in Healthy Subjects
J. M. Jenkins1 , D. Williams2 , D. A. Collins2 , V. Kitchen1 , Y. Deng3 , C. L. Erickson-Miller1 , 1GSK, Collegeville, PA, 2GSK, Research Triangle Park, NC, 3GSK, King of Prussia, PA
SB497115, is a novel, orally bioavailable, small molecule thrombopoietin receptor agonist that induces differentiation and proliferation of megakaryocytes, the first such agent to be tested in human subjects. In a randomized, single blind, placebo-controlled, parallel group, phase I study in 72 healthy male subjects, SB497115 was administered as oral capsules once daily for 1 day and, after a 1 week washout, for 10 days at doses of 5 to 75 mg.
SB497115 was shown to be orally bioavailable in humans with a linear pharmacokinetic profile suitable for a once daily oral medication. When administered at oral doses of 30mg to 75mg for 10 days a dose dependent increase in the platelet count was observed, maximum platelet count (1.5 fold) was observed on days 14 to 16 following initiation of dosing. This effect was predicted using preclinical and clinical PK and PD data.
SB497115 was well tolerated in the study, there were no serious adverse events, no significant changes in laboratory or cardiovascular safety parameters and there was no observed relationship between the incidence or severity of adverse events and dose. Most adverse events were mild in intensity and self-limiting. Platelet function was not affected by SB497115 when administered at up to 75mg for 10 days, as measured by platelet activation and aggregation.
On the basis of this safety, pharmacokinetic and pharmacodynamic data the oral thrombopoietin receptor agonist, SB497115, is being tested in phase II studies involving patients with ITP, hepatitis C virus associated thrombocytopenia and cancer patients receiving thrombocytopenic chemotherapy.
PREDICTION OF DRUG CLEARANCE FROM IN VITRO DATA FOR 11 DRUGS IN NEONATES, INFANTS AND CHILDREN USING SIMCYP
T. N. Johnson1 , G. T. Tucker2 , A. Rostami-Hodjegan2 , 1Simcyp Limited, Blades Enterprize Centre, John Street, Sheffield UK., 2Simcyp Limited and Academic Unit of Clinical Pharmacology, University of Sheffield, Sheffield UK.
Purpose. Previously, the Simcyp algorithm has been applied successfully to predict drug clearance (CL) in adults1 and children >2 years2 from in vitro data. The purpose of this study was to incorporate information on early physiological development and the ontogeny of specific cytochromes P450 into the model, and to test its ability to predict CL from birth onwards.
Methods. In vitro (Vmax, Km) and in vivo CL values were obtained from the literature for 11 drugs commonly used in pediatrics - midazolam (oral and iv), caffeine, carbamazepine, phenytoin, omeprazole, diclofenac, cisapride, S-warfarin, theophylline, gentamicin and vancomycin. Median predicted CL values and their variability (95% CI) derived by in vitro to in vivo extrapolation using Simcyp were compared with in vivo values across the age range.
Results. Nine of twelve predicted medium CL values (CLpred) in neonates were within 2-fold of observed values (CLobs). Corresponding results for infants, children and adolescents were 12/13, 21/25 and 16/19, respectively. CLpred/CLobs ratios varied between 0.34 - 3.2, 0.1 - 1.61, 0.1 - 1.4 and 0.16 - 2.41, respectively. Predicted variability was within 2-fold of the observed values in 7/12, 9/13, 17/25 and 11/19 cases respectively.
Conclusions. In silico prediction cannot replace clinical studies. However, it can provide a valuable aid to the selection of appropriate doses for such studies in neonates and young children, where allometric scaling fails to account for ontogeny.
THE EFFICIENCY OF MIXED EFFECT MODELLING TO DETECT METABOLISM-BASED DRUG-DRUG INTERACTIONS (mDDI)
T. N. Johnson1 , T. Kerbusch2 , P. A. Milligan2 , B. Jones3 , G. T. Tucker4 , A. Rostami-Hodjegan4 , 1Simcyp Limited, Blades Enterprize Centre, John Street, Sheffield, UK, 2Clinical Pharmacology, Pfizer Ltd, Sandwich, Kent, UK, 3Pharmacokinetics Dynamics and Metabolism, Pfizer Ltd, Sandwich, Kent, UK, 4Simcyp Limited and Academic Unit of Clinical Pharmacology, University of Sheffield, UK
Purpose. To assess, by simulation, factors that influence the detection of mDDIs in phase 2/3 clinical trials using population pharmacokinetics (POPPK).
Method. Steady state plasma concentrations of a candidate drug in the presence and absence of enzyme inhibitors, were generated from in vitro data using the Simcyp program. Population (Caucasian, 50% male, 20-50 y) size was varied from 80 - 2000. The compound was metabolized mainly by CYP3A4 with a contribution from CYP2D6. Concomitant medications (COMEDs) with different Iu/Ki ratios (Iu = population average unbound plasma concentration, Ki = inhibition constant)(0.006, 0.026, 0.38, 3.3, 11, 22 for CYP3A4; 0.06, 0.9, 6.75, 13.5 for CYP2D6) were evaluated. The frequency of COMED was varied from 1.25% - 10%. The extent of interaction was determined using NONMEM (p > 0.001 backward; p > 0.01 forward selection of covariate).
Results. No false negative (Iu/Ki ¡Ý 0.38) or false positive (Iu/Ki < 0.38) interactions were detected using a population size of 2000. However, at Iu/Ki = 0.38 (e,g. fluconazole 50 mg/day) and COMED level of 2.5%, a statistically significant interaction could only be detected using > 480 subjects.
Conclusion. Simulations are recommended to define the size of study populations necessary to detect mDDIs with confidence using the POPPK approach.
Laboratory of Cardiovascular and Interventional Therapeutics: Program Overview
J. W.. Karanian, O. A. Chiesa, A. D.. Shah, T. L.. Murray, S. L.. Hilbert, W. F.. Pritchard, Laboratory of Cardiovascular and Interventional Therapeutics, DB/OSEL/CDRH, MRC/MODII, FDA, Laurel, MD
The Laboratory is investigating the safety and effectiveness of a range of interventional therapeutics, including cardiovascular and minimally invasive devices and related adjunctive therapeutics and their interaction with the body in both normal and health-compromised swine models. As part of this effort, we identify, evaluate and develop more predictive in vivo and in vitro preclinical models of device use and related failure modes to be used as preclinical device development tools. The laboratory studies swine models of disease include those with vascular disease induced by diet, mechanical interventions, hormonal manipulation or hemodynamic alterations. New techniques for the study of both the pharmacokinetics and pharmacodynamics of local drug delivery are being developed for use with these models of disease. These techniques include new methods of drug delivery, drug detection and tissue sampling. These studies improve understanding of current preclinical models and their limitations and identify and address regulatory science issues associated with novel interventional and combination therapeutics and delivery technology. The development of new preclinical tools for evaluation of the safety and effectiveness of devices and combination products will assist in the progress of new technology from concept to clinical trial and marketing. In particular, improved pre-clinical modeling and data may reduce the size of preclinical animal trials and the scope of the pivotal clinical trials, accelerating progress to marketing approval with reduced costs.
Blood flow and artery type are key determinants of endothelial cell gene expression and the development of vascular disease
W. F.. Pritchard1 , A. G.. Passerini2 , C. Shi2 , N. M.. Francesco2 , P. Chuan2 , E. Manduchi2 , G. R.. Grant2 , C. J.. Stoeckert2 , D. Wray-Cahen1 , J. W.. Karanian1 , P. F.. Davies2 , 1Laboratory of Cardiovascular and Interventional Therapeutics, DB/OSEL/CDRH, MRC, MODII, Laurel MD 20708, 2University of Pennsylvania, Philadelphia, PA
Background: Atherosclerosis preferentially develops in susceptible areas of the arterial system that are defined by the regional vascular hemodynamics. Regions of disturbed flow (DF) are susceptible to atherogenesis whereas regions of undisturbed laminar flow (UF) appear protected. Previous work in adult castrate male pigs revealed the coexistence of pro- and anti-atherosclerotic gene expression profiles in susceptible regions. The vascular response to therapeutic interventions varies with the character of the target vascular bed.
Methods: In fifteen adult pigs, endothelial transcript profiles from arterial regions of disturbed flow (aortic arch) and undisturbed flow (carotid artery, descending thoracic aorta) were compared in males, females, and males fed a short-term (2 week) diet high in fat and cholesterol, using microarray analysis and false discovery rate statistical methods.
Results: Hierarchical clustering analysis and differential gene expression showed that regional determinants of endothelial phenotype dominated any influence of gender or diet. In particular, the arterial samples from the two UF regions, carotid artery and descending thoracic aorta were in separate clusters. This clustering based on arterial bed and that based on hemodynamics dominated the influence of gender or the short exposure to diet.
Conclusions: The study highlights the importance of in vivo regional endothelial heterogeneity to atherosusceptibility associated with hemodynamic characteristics and may contribute to our understanding of differences in therapeutic responses among vascular beds. Identification of the differences in gene expression in these areas may lead to targets for therapeutic intervention. Studies of longer exposure to a hyperlipidemic diet and manipulation of vascular hemodynamics are planned.
Pharmacokinetics of Local Drug Delivery Depends on Mode of Delivery and Hemodynamics in an In Vitro Vascular Flow Model
A. D.. Shah1 , L. E.. Olsen2 , W. F.. Pritchard1 , S. L.. Hilbert1 , W. K. Riemenschneider1 , J. W.. Karanian1 , 1Laboratory of Cardiovascular and Interventional Therapeutics, DB/OSEL/CDRH, MRC, MODII, Laurel MD 20708, 2Department of Biomedical Engineering, Marquette University, Milwaukee, WI 53201
Background: Drug eluting stents that locally deliver drugs such as paclitaxel and rapamycin reduce restenosis after balloon angioplasty and stenting. Little is known regarding the physiologic and physical properties that influence local drug kinetics. These factors, dosing and the specific mode of delivery influence the effectiveness and safety (e.g., toxicity) of a drug
Methods: An in-vitro vascular flow system has been developed to model arterial flow. Swine coronary arteries were mounted in the closed flow loop. Oregon Green-labeled paclitaxel was delivered utilizing two different local delivery catheters: needle injection catheter and weeping balloon. Longitudinal and circumferential paclitaxel distribution was determined with confocal fluorescent microscopy.
Results: Drug deposition and transport kinetics are influenced by a variety of factors such as hemodynamics, physicochemical properties of drug, drug potency, tissue architecture, availability of binding sites, thickness of the barrier, drug solubility and local drug concentration. Paclitaxel is highly hydrophobic resulting in high tissue retention and slow diffusion rates. This property, along with its binding to the microtubule structure of vascular smooth muscle, is a determinant of paclitaxel kinetics in arterial tissue. Tissue paclitaxel distribution will be presented
Conclusions: These in-vitro blood vessel perfusion studies will help (i) define the effects of physiologic forces and tissue architecture on the pharmacokinetics of locally delivered drugs such as paclitaxel, and (ii) determine the effects of physical parameters such as transvascular pressure gradient, flow rate and pressure on the pharmacokinetics of different modes of delivery. The data obtained from these experiments will be used to develop a predictive model for pharmacokinetic evaluation of locally delivered therapeutics. The model will help refine the preclinical and clinical safety testing requirements for new therapies.
Minimally Invasive Endoscopic Techniques Allow Serial Tissue Visualization and Sampling for Pharmacokinetics and Safety Studies in Large Animal Models of Vascular Disease and Intervention
O. A. Chiesa, W. F.. Pritchard, T. L.. Murray, A. D.. Shah, J. W.. Karanian, Laboratory of Cardiovascular and Interventional Therapeutics, DB/OSEL/CDRH, MRC, MODII, Laurel MD 20708
Background: Standard pre-clinical pharmacokinetics studies (temporal and spatial drug distribution) generally require use of multiple animals in which the animals are dosed then sacrificed at different time points for tissue collection. Endoscopic surgical techniques allow access to the chest and major organs as a minimally invasive, survival procedure. Since major surgical exposure of the target tissue is avoided, animals may be studied repeatedly, reducing the number of animals required for collection of critical drug safety data.
Methods: Mature domestic swine underwent diagnostic coronary angiography. Using a novel microsyringe catheter, fluorescently labeled paclitaxel was delivered to the perivascular space by injection from within the coronary artery. The animals were allowed to recover. From 1 to 28 days later, each animal again underwent diagnostic angiography. The abdomen and chest were accessed using endoscopic techniques.
Results: Coronary angiography and local drug delivery was successfully performed in all animals. At endoscopy, the coronary vessels could be directly observed. In the 1 day animals, local hemorrhage at the drug delivery site could be identified through the pericardium. In the 30 day animals no hemorrhage was noted utilizing this approach. Catheter placement into the pericardial space allowed sampling of pericardial fluid. Entry into the pericardial space allowed direct inspection of the epicardium and coronary vessels.
Conclusions: Minimally invasive thoracoscopic access allows multiple observations of induced lesions and serial tissue sampling, providing more efficient study of the pharmacokinetics of locally delivered drugs. This may serve as a preclinical development tool for the evaluation of therapeutic devices and combination products, enhancing the data collected while reducing the number of animals required for preclinical safety studies.
Moderate Hyperlipidemia in Swine Results in Human-Like Restenosis Following Coronary Stenting
J. W.. Karanian, A. D.. Shah, T. L.. Murray, O. A. Chiesa, S. L.. Hilbert, W. F.. Pritchard, Laboratory of Cardiovascular and Interventional Therapeutics, DB/OSEL/CDRH, MRC, MODII, Laurel MD 20708
Background: The predictive value of existing animal models for establishing safety and effectiveness of interventional cardiovascular devices is limited. Experimental studies are typically performed in normal vessels of juvenile female and castrate male swine as models for diseased vessels in humans. The time-course of vascular healing differs between swine and humans with accelerated healing in swine relative to humans. This study was designed to define the effects of a high fat-high cholesterol diet on coronary restenosis and healing following balloon angioplasty and stenting in male and female swine.
Methods: After 16-20 weeks on a normal diet (ND) or hyperlipidemic diet (HD), the coronary arteries of domestic swine underwent balloon angioplasty, stenting or no therapy (control). Each animal was continued on its diet for an additional 8 weeks before pathologic analysis of the coronary arteries. Histomorphometry and characterization of the stenosis were assessed using IP Lab for Mac (Scanalytics).
Results: Restenosis rates were established following morphometric evaluation. Coronary arteries obtained from stented HD animals showed a proteoglycan-rich/fibromuscular-poor hyperplasia and inflammation around the stent struts as compared to the ND animals. A developing necrotic lipid core and fibrous cap were also noted in the HD animals with significant restensosis in comparison to comparable ND animals. The impact of gender and hyperlipidemia on lesion morphology and restenosis rates after coronary interventions will be defined
Conclusions: Vascular healing following stent placement was delayed in swine on HD diet with in-stent lesion development comparable to human restenosis. The hyperlipidemic animal model of vascular disease may represent a more predictive model of human disease and therapeutic intervention than the current normal juvenile models.
Safety and Effectiveness of Drugs to Treat Vascular Disease Depend on Mode of Delivery: Peri-Vascular Effects of Paclitaxel in Swine
J. W.. Karanian, S. L.. Hilbert, W. K. Riemenschneider, O. A. Chiesa, T. L.. Murray, W. F.. Pritchard, Laboratory of Cardiovascular and Interventional Therapeutics, DB/OSEL/CDRH, MRC, MODII, Laurel MD 20708
Background: Local drug delivery by coronary stents is a clinically effective method of preventing restenosis, a failure mode associated with balloon angioplasty and stenting, but the pharmacokinetics are not well defined. Local and regional tissue toxicity is a potential safety issue with locally delivered drug. We have previously shown that coronary arteries in swine develop stenosis .by 30 days after balloon angioplasty. Local delivery of 17β-estradiol with a needle injection catheter inhibited balloon angioplasty-induced stenosis.
Methods: Domestic swine underwent balloon angioplasty and transmural adventitial injection of 10ug fluorescently labeled paclitaxel with a needle injection catheter. Tissue healing response (morphometry and morphology) and drug distribution were determined at 1, 14 and 28 days after angioplasty and drug delivery.
Results: Angiographic evaluation demonstrated perivascular distribution of the drug at the intervention site without complication. Minor hemorrhage was noted acutely but was resolved by thirty days. Fluorescently labeled paclitaxel was distributed circumferentially and longitudinally following injection into a coronary artery in a time-dependent manner. The pharmacodynamic results combined with the drug distribution will be presented.
Conclusions: Following coronary intervention, the needle injection catheter delivered drugs reliably and safely to the peri-vascular space with no vascular complications. Minor hematomas were evident acutely, but not at 28 days. Fluorescent paclitaxel was present circumferentially at the injection site with longitudinal extension. These data are consistent with the proposition that local drug delivery, via a needle injection catheter, may provide a reliable and safe therapy for treatment of vascular disease and restenosis. Both safety and effectiveness may be dependent on the mode of delivery.
Single and Multiple Dose Sparse Sampling PK/PD Modeling of Pregabalin Effect in Rat Capsaicin Induced Allodynia with Translation to Human Response
J. Koup, D. Beidler, C. Lepsy, K. Kolbasa, Pfizer Global R&D, Ann Arbor, MI
Purpose: Pregabalin (Lyrica®, PGB) is clinically effective in the treatment of neuropathic pain. A sparse sampling PK/PD study was conducted to assess the PK/PD relationships of PGB plasma concentrations to inhibition of capsaicin induced allodynia in rat, following single and multiple dose drug administration.
Methods. Following a protocol approved by Pfizer IACUC, PGB was administered to rats, following induction of stable, bilateral allodynia. Inhibition of allodynia was measured at various times after drug administration. Plasma samples were collected following assessment of allodynia response. PK/PD modeling was performed using NONMEM. PD parameters obtained were utilized to predict the time-course of human response following administration of therapeutic doses of PGB
Results: Improvement in allodynia in relationship to plasma PGB concentrations was well described by a "link" PK/PD model, with peak response occurring later than peak plasma PGB concentrations. The relationship between effect site PGB concentration and response was described by an Emax function. EC50 was 2.74 µg/mL and a Ke0 of 0.2 hr-1. The response to PGB was similar following single and multiple dose administration. PD parameters predicted a 50% improvement in patients given a therapeutic dose of PGB. Improvement was maintained during Q12 hour dosing.
Conclusion: The capsaicin induced allodynia model in the rat provides PK/PD estimates that translate well to patients.
Simulation of Absorption of Mesalamine from Delayed Release Tablets
H. Kwon1 , R. Lionberger1 , L. X. Yu1 , T. Moore2 , Z. Gao2 , B. J. Westenberger2 , L. F. Buhse2 , 1CDER, FDA, ROCKVILLE, MD, 2CDER, FDA, St.Louis, MO
Background: Mesalamine provides topical anti-inflammatory action in the colon [k1]. Mesalamine is highly and rapidly absorbed in the intestine and thus, various delayed release mechanisms are used to prevent mesalamine from being absorbed until it reaches the colon. One example, Asacol® delayed release (DR) tablets, is formulated by coating mesalamine with an acrylic-based resin that dissolves at pH 7 or greater. The variability in Asacol® pharmacokinetics is high. Recently DPA has evaluated the variability of Asacol® in dissolution studies at pH conditions found in the gastrointestinal tract.
Method: Using a compartmental absorption and transit model for the intestine and the colon, we carried out absorption simulations of mesalamine. Data from IV, oral solution, and other mesalamine DR formulations determined the model parameters and their variabilities.
Results: The distribution of AUC, Cmax, and Tmax were calculated using the observed variability of Asacol® dissolution. Mean values of AUC, Cmax, and Tmax agree with the published results. For each Asacol® tablet, the location of drug absorption was identified and correlated with the observed pharmacokinetic parameters.
Conclusions: Asacol® is highly variable because of tablet to tablet variability in where the drug is released. When a tablet encounters pH 7 in the intestine, it dissolves rapidly, mesalamine is efficiently absorbed, and considerable plasma concentrations result. A tablet that does not encounter pH 7 until the colon results in less absorption and negligible plasma concentrations. This interaction between formulation performance and in vivo environment results in higher variability.
Development Of A Flow Through Dissolution Method For The Determination Of Metformin Hydrochloride And Comparison Of An In-house Versus Commercial Controlled Release Formulation Using GastroPLUS
C. L. Li, L. G. Martini, J. Taylor, GlaxoSmithKline
Introduction: The aim was to develop and validate a flow through dissolution method using a 100 micron path length flow cell for determination of metformin hydrochloride. Followed by establishment of an in-vitro in-vivo correlation using GastroPlus for comparison of three metformin controlled release formulations.
Methods: This method utilised an automated on-line flow through USP 2 paddle dissolution system, at 50rpm using 900ml of degassed 0.05M 0.68% potassium dihydrogen orthophosphate buffer pH6.8 at 37µC ± 0.5µC. Samples were filtered through in line Gelman, 25mm 1um glassfibre acrodisc filters fitted prior to the 100 micron path length quartz flow cells.
Gastroplus was used to interpret data and predict in-vitro in-vivo correlations followed by f2 similarity factor analysis.
Results: Across a range of 18% to 154% working concentration, method developed was linear with correlation coefficient of 0.9996, recoveries from media were 96.9 % to 100.9%, stability was 99% to 101% after 48 hours at 37µC, correlation coefficients for precision were all <5%, filter validation confirmed no adsorption of drug to the filter.
In-vitro in-vivo correlation was achieved and f2 similarity factor of 50 was achieved with the in-house formulation when compared with the commercial control.
Conclusion: A sensitive, precise and accurate flow through dissolution method was developed which alleviated the requirement for dilution of samples prior to analysis due to the strong UV chromophore of metformin. Data was used to achieve in-vitro in-vivo correlation. The three controlled release formulations were compared and the in house formulation successfully confirmed as having statistically similar profile.
PK/PD MODELING OF THE INTERACTION BETWEEN IV SCOPOLAMINE (SCP) AND PHYSOSTIGMINE (PHY) IN HEALTHY ELDERLY VOLUNTEERS
A. Men1 , K. Wesnes2 , J. Venitz3 , 1CDER, FDA, Rockville, MD, 2Cognitive Drug Research Ltd.,UK, 3Dept. of Pharmaceutics, VCU, Richmond, VA
Aims: 1. To assess the PK/PD interaction between IV SCP and PHY in elderly subjects; 2. To estimate in vivo muscarinic receptor affinities for SCP.
Methods: In a randomized, placebo-controlled, four-way crossover clinical trial, sixteen volunteers (> 65 years) received 6.7 µg/kg IV SCP/placebo, followed after 60 minutes by 6.7 µg/kg IV PHY/placebo. An integrated SCP PK/PD model was used to simultaneously fit the plasma concentration and PD endpoints (heart rate, BPCHR, saliva flow, SF, simple reaction time, SRT, numerical working memory, NWM). A competitive PD model described the PD interaction between SCP and PHY.
Results: SCP and PHY PK profiles fit two- and one compartment models, respectively. Median PK and PD parameter estimates (n= 16) are as follows:
| PK Parameter: | SCP | PHY | PD Parameter: | BPCHR [bpm] | SF [g/2 min] | SRT [ms] | NWM [ms] |
| V1 [l] | 55 | 77 | E0 | 0 | 0 | 372 | 880 |
| k12 [1/hr] | 14.1 | keo [1/hr] | 10 | 0.4 | 0.5 | ||
| k21 [1/hr] | 4.9 | Emax | 23 (Tachycardia) -5 (Bradycardia) | -3.3 | 2314 | 6099 | |
| k10 [1/hr) | 2.9 | 2.6 | EC50 [ng/ml] | 1.5(Tachycardia) | 0.1 | 1.2 | 2.0 |
| n | 4.1 | 1.3 | 2.3 | 1.5 | |||
| IC50 [ng/ml] | 4.9 | 10 | 1.0 | 0.7 |
Conclusions: PHY IC50s were estimated successfully: IC50CNS<IC50HR<IC50SF, which is helpful in predicting the clinical interaction between SCP and PHY in future studies. PK/PD modeling of SCP also succeeded in estimating EC50s for CNS and PNS endpoints: EC50brady(M1)<EC50SF(M3)<EC50Tachy(M2). CNS effects may be attributed to central-M2 receptors.
Exposure-response analyses lead to more informed regulatory decisions: The Oncology Drug Experience.
R. P. Ramchandani, B. P. Booth, J. Z. Duan, G. Williams, A. Men, A. Rahman, M. U. Mehta, J. V.S. Gobburu, DPE-1/OCPB/CDER/FDA
Background:
Development of quantitative relationships between drug exposure and response allows for a better understanding of the sources of variability in effectiveness and toxicity of drugs, and also guides regulatory decisions regarding the safe and effective use of these drugs. This approach has recently been applied to the review of several oncology drugs, including busulfan, zoledronic acid, gemtuzumab, imatinib, irinotecan and erlotinib.
Methods:
Reviews of new drug applications (NDAs) for several oncology drugs were examined to determine the type of exposure-response analyses conducted and their impact on regulatory decisions regarding these drugs.
Results:
Pharmacokinetic and pharmacodynamic models were developed by reviewers based on data included in the NDAs for the above-listed oncology drugs. Exposure-response models for measures of toxicity were developed for irinotecan (diarrhea and neutropenia), gemtuzumab (venous occlusive disease, hepatomegaly), imatinib (edema), zoledronic acid (renal deterioration) and erlotinib (rash, diarrhea). Models for measures of effectiveness and clinical outcomes were developed for zoledronic acid (bone markers and skeletal related events), imatinib (hematological response, survival) and erlotinib (survival). In most cases, the analyses led to labeling changes to highlight safety concerns about the drug. In some cases, specific recommendations for dosing adjustments were made, e.g., in pediatric patients receiving busulfan, and renally-impaired patients receiving zoledronic acid.
Conclusions:
Quantitative exposure-response relationships led to an improved understanding of the effectiveness and safety of several oncology drugs. This approach also provided a basis for dosing recommendations in special populations and aided in more informed regulatory decisions.
The pharmacokinetics of TMC125 in different mouse strains: impact on carcinogenicity testing strategy
A. Raoof1 , S. Lachau-Durand1 , B. Willems1 , L. De Zwart1 , M. Bouche1 , K. Steemans1 , J. Verbeeck1 , H. Van Cauteren2 , 1Johnson & Johnson Pharmaceutical Research and Development, Turnhoutseweg 30, B- 2340 Beerse, Belgium, 2Tibotec BVBA, Generaal de Wittelaan L11B3, 2800 Mechelen, Belgium.
Purpose: Carcinogenic potential of pharmaceuticals can be evaluated using one long term conventional study in a rodent species plus another short or medium term study using transgenic animal models. The present study aimed to investigate the pharmacokinetics of TMC125, a non-nucleoside reverse transcriptase inhibitor in development for treatment of human immunodeficiency virus (HIV), in Swiss CD1 mice and non-transgene rasH2 littermates produced by crossing C57BL/6J transgenic males with BALB/cByJ non-transgenic females.
Methods: Animals were dosed daily via gavage with TMC125 HBr at 10, 50, 200, and 800 mg/kg to CD1 Swiss mice for 3-months and at 50, 200, 400, and 800 mg/kg to non-transgenic littermates for 1-month. Vehicle groups were dosed in both studies. Toxicokinetic evaluations, at the first and last days of dosing, were performed on satellite animals.
Results: TMC125 systemic exposure (Cmax and AUC) was significantly lower in the rasH2 littermates relative to CD1 mice at comparative tested doses. This reduction was observed on both first and last days of dosing. No change in toxicity profile was seen between the different mice strains.
Conclusions: The study demonstrates differences in pharmacokinetics between various mouse strains. This may lead to discrepancies in the selection of the highest tolerated doses that can be employed in carcinogenicity studies. The guidelines are not clear about the suitability of a particular strain for dose range finding studies when using transgenic animals for carcinogenicity testing. Based on our data, the same strain of mouse should be used in dose range finding studies that precede carcinogenicity testing.
Pharmacokinetic-Probabilistic Simulations of Risk of Blood Pressure Changes Associated with the Cyclamate Metabolite Cyclohexylamine.
W. L. Roth, S. J. Chirtel, Center for Food Safety
Background: Cyclohexylamine (CA) is the primary metabolite of cyclamate (CM) detected after oral administration of CM. CA is formed from CM by bacterial metabolism in the intestines, at rates which depend on the flora and activity of the individual exposed (Drasar et al., 1972). CA has been shown to increase blood pressure (BP) in humans given oral CA, and was thought to be produced in sufficient quantity by some individuals consuming CM to result in adverse effects.
Methods: A pharmacokinetic model (Roth and Ekelman, 2001) was used to predict plasma levels of CA from CM. Data on BP changes in volunteers treated with CA (Eichelbaum et al., 1974) was used topredict BP changes from plasma CA levels. Data on the daily conversion of CM to CA in "high converters" over a period of 13 weeks was analyzed to estimate the frequency (F) of episodes of high conversion. Conversion rates for "non-converters" were compared to F for "converters" to develop probabilistic models of BP. The F of BP changes predicted for high converters was compared with the normal range of BP in studies of normal and hypertensive subjects reported by Littler et al. (1978).
Results: The conditional probability of greater than 50 % conversion of CM to CA in "converters" , P(>50%) was 0.05 (Confidence interval = 0.012-0.131). The probability of any one subject being a "converter" , P(>2%) was found to be 0.0722 (Confidence interval = 0.039-0.118). The overall probability for such events in the general population was computed as the product of these conditional probabilities:
P(>10 mm | CM) = P(>2%)*P(>50%), giving an average of 3.61 x 10-3 and range of 0.474 - 15.44 x 10-3/day.
Conclusion: When model-predicted plasma CA levels are compared with observations, it appears that even model-predicted BP changes are small in comparison to the normal range of variation. Since daily CM to CA converting activity is almost randomly distributed even for high converters, BP changes associated with daily CM consumption would be rare (3.61 x 10-3 /day).
Simulation of the Influence of Formulation Factors and Dissolution Rates on the Absorption of Phenytoin Sodium In Vivo.
P. M. Sathe1 , L. X. Yu1 , A. S. Hussain1 , J. Chittenden2 , W. S. Woltosz2 , M. B. Bolger2 , 1CDER, FDA, Rockville, MD, 2Simulations Plus, Inc., Lancaster, CA
Objective: To validate the application of advanced compartmental absorption and transit (ACAT) model simulations and Weibull function release for phenytoin sodium formulations.
Methods: GastroPlusTM (Simulations Plus, Inc., Lancaster, CA) was used to simulate human plasma concentration-time profiles expected for phenytoin sodium capsules when administered at doses of 100 mg and 300 mg. The resulting simulated plasma concentration vs. time profiles were compared to corresponding literature data and actual data submitted to the FDA. A Weibull function was fitted to the in vitro dissolution profile for the sodium salt based on a basket dissolution apparatus. We used GastroPlus simulations to compare slow and fast dissolution of the sodium salt by changing the time scale parameter of a Weibull function and we compared absorption under fasted and fed conditions.
Results: The simulations predict that the sodium salt of phenytoin dissolves rapidly in the stomach and then precipitates due to low solubility of the acid drug at physiological pH values. Factors such as precipitation time, particle size and Weibull time scale factor for initial dissolution of the sodium salt were shown to significantly affect the total amount of undissolved drug (1.1% at 100 mg and 6.5% at 300 mg) remaining in vivo. This resulted in fraction absorbed of 99% and 93% for the 100 mg and 300 mg doses, respectively.
Conclusions: In vivo precipitation time, particle size and rate of release appear to be important contributors towards the dissolution, absorption, and bioavailability of phenytoin sodium capsules.
Improving Pediatric Labels Through the Pediatric Initiatives, What We Have Learned
A. Selen, W. J. Rodriguez, C. S. Chaurasia, D. M. Chilukuri, T. L. Crescenzi, J. L. DiGiacinto, G. Gieser, S. M. Huang, M. J. Kim, P. I. Lee, L. J. Lesko, P. J. Marroum, L. L. Mathis, M. D. Murphy, S. J. Murphy, R. Roberts, H. C. Sachs, D. R. Stanski, S. Suarez-Sharp, V. Tandon, R. S. Uppoor, J. J. Zheng, FDA, Rockville, MD
Background: Pediatric initiatives for labeling of drugs and biologics for their safe and effective use in pediatric patients have led to development of policy and regulations, which in turn continues to generate research for greater understanding and knowledge for safe and effective use of drugs and biologics in pediatric patients.
Methods: The methodology, data/results and the respective labeling changes of the pediatric studies conducted to date are reviewed and summarized to determine trends and differences across studies as a means to illustrate the impact of improved dose and dosing recommendations for pediatric patients.
Results: Since the initiation of pediatric exclusivity efforts, approximately 87 drugs have new or revised pediatric labeling that include new doses or dosing changes, new and/or enhanced safety information, dosing information for younger patients and information on new pediatric formulations. Specific examples illustrating the above changes, and the lessons learned as derived from the differences and similarities in drug pharmacokinetics, and pharmacodynamics, and adverse drug reactions between pediatric and adult patients will be presented.
Conclusion: The Agency efforts and legislation have resulted in a significant increase in the number of pediatric studies conducted for safe and effective use of drugs and biologics. These efforts have resulted in critical changes in drug labels for pediatric patients and have shown that unique pediatric dosing may be necessary.
The continuation of efforts emphasizing creative and harmonized methodology and commitment for optimizing knowledge gained from pediatric studies are of utmost importance for developing therapies for pediatric patients.
Physicochemical profiling of food-effect for immediate-release formulations
B.N. Singh, College of Pharmacy and Allied Health Professions, St. John's University, Jamaica, New York (Present address: Pharmaceutics R & D, Emisphere Technologies, Inc., Tarrytown, New York)
Background: There is a general consensus that co-administration of standardized food results into increased absorption of lipophilic, poorly-soluble (aqueous solubility < 100 µg/ml; dose/solubility ratio > 250 ml) drugs when administered as immediate-release formulations, although there are sporadic examples of drugs which exhibit reduced or unaffected absorption in the fed state, despite the fact that they are poorly-soluble and/or have log P values in the range typically required for optimal passive transcellular transport (i.e., log P = 2-3).
Methods: Mean AUC data obtained under fasted and fed states of over 100 structurally diverse orally active drugs with different physicochemical properties were obtained from the primary source of literature. Correlations of AUC ratio (Fed/Fasted) with aqueous solubility, dose/solubility ratio and log P were derived and statistically evaluated by Pearson's correlation test (two-tailed).
Results: A negative correlation was obtained between the logarithm of the aqueous solubility and the AUC ratio (r = -0.5982, N = 93), whereas a positive correlation existed between AUC ratio and log P (r = 0.5147, N = 110) and between AUC ratio and dose/solubility ratio (r = 0.5511, N = 87). All these correlations were statistically significant (p < 0.0001). Based on this study, the estimated range within which a drug is not expected to be significantly affected by food falls between 0.148-89.39 mg/ml for aqueous solubility and between 0.23-624 ml for dose: solubility ratio. The corresponding range of log P for expecting a lack of food-effect lies between -1.13 and 2.98.
Conclusions: It is concluded that aqueous solubility, dose/solubility ratio and partition coefficient can be used as useful parameters to probe the dependence and correlation of food-effect with these physicochemical parameters for immediate-release formulations. Quantitatively, the effect of food is most pronounced for lipophilic, poorly water-soluble drugs (with only a few exceptions), irrespective of whether the drug is acidic, basic or neutral.
Exposure-response analysis of the novel oral taxane BMS-275183 in patients with advanced solid tumors.
S. Song1 , M. H. Woo1 , M. B. Cohen1 , L. E. Broker2 , G. Giuseppe2 , M. Voi3 , T. W. Griffin1 , 1Bristol Myers Squibb, Princeton, NJ, 2Free University Medical Center, Amsterdam,Netherland, 3Bristol Myers Squibb, Wallingford,CT
Background: BMS-275183 is a novel orally bioavailable taxane with clinical activity against non-small cell cancer and other tumor types. Phase I dose-ranging studies have assessed the safety, tolerability, pharmacokinetics (PK), and antitumor activity of BMS-275183. This analysis evaluated the relationship between BMS-275183 exposure and response or toxicity.
Methods: BMS-275183 was given on a continuous weekly schedule to patients with advanced solid tumors. Blood samples for PK analysis were collected over 48 hrs on Days 1 and 15. Plasma concentrations of BMS-275183 were evaluated using LC/MS/MS. BMS-275183 PK parameters were derived by non-compartmental analysis. Because of the low intra-patient variability, Day 1 PK parameters were used for the exposure-response analysis.
Results: A total of 48 patients were treated at various dose levels ranging from 2.5 to 320 mg/m2. Across the dose range studied, systemic exposure (AUC) increased linearly with dose level. Parameters of exposure were predictive of response or toxicity. Patients who responded to treatment or who experienced toxicity had a higher AUC than those who did not. The median AUC in patients who had a PR (n=10), SD (n=15), or PD (n=17) was 3620, 3680, and 1000 ng/mL*h, respectively. Neurotoxicity was the dose limiting toxicity and a major cause of toxicity-related study withdrawal and early dose reduction. The median AUC of patients who had Grade 0 or 1 neurotoxicity (n=23) was 1570 ng/mL*h and those of patients who developed Grade 2 (n=19) or 3 (n=6) neurotoxicity were 5570 and 5800 ng/ml*h, respectively. Patients withdrawn from the trial due to drug related adverse effect on cycle 1 (n=3)or who required a dose reduction or delay on cycle 1 (n=7) due to neurotoxicity had an median AUC of 6620 and 6530 ng/mL*h, respectively. The median AUC of patients who developed Grade 3 or 4 myelosuppression (n=8) was 5727 ng/mL*h. The relationship between Cmax and response or toxicity was similar to that with AUC except for greater variability.
Conclusions: Development of response, toxicity, and severity of toxicity appear to be related to higher exposures of BMS-275183.Future evaluations will identify exposures that optimize activity and reduce toxicity.
Virtual Bioequivalence Trials Can Help Reduce Risk of Failure
W. S. Woltosz, J. Chittenden, J. DiBella, Simulations Plus, Inc.
Purpose: To demonstrate the application of virtual bioequivalence trials in assessing the likelihood that a test formulation will demonstrate bioequivalence in human trials at different study powers (numbers of subjects).
Methods: Two bioequivalence studies failed to demonstrate bioequivalence for a generic version of a reference product. A third generic formulation was developed for which the in vitro release rate was closer to the reference product. Data from the two failed bioequivalence studies were used to fit pharmacokinetic parameters and to determine variabilities and covariances for pharmacokinetic parameters. Genetic polymorphism was an issue, so PK parameters were fitted independently to three different groups representing poor, average, and extensive metabolizers. Virtual trials were simulated with different numbers of subjects ranging from 25 to 240 to assess the probability that bioequivalence would be demonstrated at each level.
Results: Virtual bioequivalence trials showed that the newest formulation was less likely to demonstrate bioequivalence than the first two. Release failures (very late release - sometimes more than 24 hours after dosing) of the reference product resulted in depressed values for Cmax and AUC over those that would have been obtained if all reference doses had released properly. The generic dosage forms did not experience the same rate or extent of late releases, so they produced higher mean Cmax and AUC values.
Conclusions: (1) Virtual bioequivalence trials can provide understanding of the complex interactions that exist among physicochemical properties, physiological variables, pharmacokinetics, and formulation variables, and with that understanding, contribute to reducing the risk of failure. (2) The FDA should consider whether generic dosage forms should be designed to match a reference product's Cmax and AUC determined when obvious release failures have occurred, or whether those subjects with failed release should be excluded, enabling the generic product to demonstrate bioequivalence only against reference product that releases properly.
Incorporation of inter-individual variability into the prediction of in vivo drug clearance from in vitro data.
E. M. Howgate1 , K. Rowland Yeo1 , N. J. Proctor1 , G. T. Tucker2 , A. Rostami-Hodjegan2 , 1Simcyp Ltd, 2University of Sheffield & Simcyp Ltd
A number of scaling methods can be applied to extrapolate pre-clinical data to estimate average values of in vivo drug clearance. In contrast, Simcyp® software combines genetic, physiological, demographic and ethnic variabilities with in vitro metabolism data to allow prediction of population distributions of clearance values.
Data on in vitro metabolism (Vmax, Km) and observed in vivo clearance values for 21 drugs given orally and 14 drugs given intravenously were collated from the literature. Version 5 of the software was used to predict oral (CLpo) and systemic (CLiv) clearances.
For median population clearance, 86% of CLpo and 100% of CLiv predictions were within 2-fold of the observed values. For fold variability (90% confidence interval/median) 71% of both CLpo and CLiv predictions were within 2-fold.
The software can predict median clearance and population variability with reasonable confidence.
Implications of the disparity in relative holo:apo-protein contents of different standards used for immuno-quantification of hepatic cytochrome P450 3A4 (CYP3A4)
Z. E. Wilson1 , S. W. Ellis1 , R. J. Edwards2 , K. Rowland Yeo3 , G. T. Tucker4 , A. Rostami-Hodjegan4 , 1University of Sheffield, 2Imperial College School of Medicine, 3Simcyp Ltd, 4University of Sheffield & Simcyp Ltd
Reported values of the abundance of hepatic CYP3A4 are based on immuno-quantification using different protein standards (purified enzyme, human liver microsomes (HLM) and recombinantly expressed enzyme (rCYP)). The immuno-detectable (IMD) CYP3A4 protein content of 3 rCYP systems and a sample of HLM quantified for CYP3A4 apoprotein (HLMSTD) were determined by ELISA in this study. Equal amounts of CYP3A4 (as stated in source information) produced relative values of 2.0, 2.4 and 3.2 for IMD CYP3A4 in human lymphoblastoid (LYMPH) (Gentest®), baculovirus-insect cell (SUP) (Supersomes - Gentest®) and E.Coli (Bactosomes - Cypex®) systems compared to HLMSTD. Application of these ratios to reported HLM CYP3A4 abundance in the literature [1,2], where authors used rCYP as their standard for IMD, raised mean values from 73 to 176 pmol/mg protein (SUP) and from 70 to 139 pmol/mg protein (LYMPH), respectively. These are more consistent with the values reported using HLMSTD as standard (175 pmol/mg protein [3]). Different ratios of holo:apoprotein in standards used in the immuno-quantification of HLM CYP3A4 may explain the wide variability observed in different literature reports.
Inter-individual variability in the catalytic activity of CYP3A4 per unit of enzyme (kcat)
Y. Lei1 , Z. E. Wilson1 , K. H. Crewe1 , K. Rowland Yeo2 , G. T. Tucker3 , A. Rostami-Hodjegan3 , 1University of Sheffield, 2Simcyp Ltd, 3University of Sheffield & Simcyp Ltd
Inter-individual variation in in vitro CYP3A catalytic activity (metabolite formation per minute per mg microsomal protein; /mmp) is considerable. Differences in individual levels of hepatic CYP3A4 protein are considered to be the main source of this variation, and significant correlations between hepatic CYP3A4 abundance and in vitro catalytic activity (/mmp) have been reported. Our aim was to re-evaluate the variability based on the intrinsic activity of the enzyme (kcat). Microsomal CYP3A4 abundance was measured using a validated ELISA in duplicate samples from 53 livers. Testosterone [6β] hydroxylase activity (nmol/min/pmol CYP3A4 at 200 µM testosterone) was available for 35 of these livers.
Inter-individual variation in kcat was lower than that of catalytic activity (/mmp): 22 vs 42 fold, respectively. The apparent variability in CYP3A4 related kcat may be due to additional variable contribution from CYP3A5 in different livers and differences in the levels of accessory proteins such as cytochrome b5. However, CYP3A5 contribution to CYP3A mediated liver metabolism is expected to be small in Caucasians, and correlation of cytochrome b5 levels with catalytic activity (/mmp) as part of a multiple regression analysis in 26 of our livers did not reach statistical significance.
Inter- and intra-individual variability in gastro-intestinal physiology has significant effects on the prediction of fraction of dose absorbed (fa)
M. Jamei1 , J. Yang1 , K. Rowland Yeo1 , A. Rostami-Hodjegan2 , G. T. Tucker2 , 1Simcyp Ltd, 2University of Sheffield & Simcyp Limited
Predictive physiologically-based PK models are being applied increasingly in drug development. However, the application of such models without consideration of inter- and intra-individual variability of the physiological parameters in the target population may lead to flawed conclusions.
The Compartmental Transit and Absorption (CAT) model (1) and literature values defining the variability of relevant aspects of gastrointestinal physiology were implemented in Simcyp®. The parameters were: gastric emptying time, Ts, small intestinal transit time, Tsi, and the radius and length of the small intestine, R and L respectively. Model drugs with a wide range of permeability characteristics were studied, including enalaprilat (a poorly permeable compound; Peff= 0.079 cm/h) and antipyrine (a highly permeable compound; Peff= 2.02 cm/h) (2).
Simulations were done for a population of 100 individuals repeated for 10 separate trials. For example, the predicted fa values of enalaprilat and terbutaline varied from 0.12 (5th percentile) to 0.42 (95th percentile) and 0.34 (5th percentile) to 0.71 (95th percentile), respectively. The variability was greater for less permeable compounds. Inconsistency between point estimates of fa and observed values from small clinical studies may be expected if inter- and intra-individual variability is not considered.
A critical evaluation of the experimental design in reports of mechanism based inhibition (MBI) studies and the assessment of their implication for in vitro-in vivo extrapolation
F. Ghanbari1 , K. Rowland Yeo2 , M. S. Lennard1 , G. T. Tucker3 , A. Rostami-Hodjegan3 , 1University of Sheffield, 2Simcyp Ltd, 3University of Sheffield & Simcyp Ltd
An increasing number of drugs are reported to cause MBI of cytochrome P450 enzymes (CYPs). Determination of the kinetics of MBI involves a pre-incubation followed by an incubation stage (Silverman, 1995), although experimental design differs considerably between laboratories. We have evaluated the experimental protocols reported in the literature for >100 mechanism based inhibitors of human CYPs in vitro. Wide variability in the timing of the first sample during the "pre-incubation" stage (range: 0.5 - 6 min; mode: 2 min), the dilution factor (range: no dilution - 100 fold; mode: 20 fold), and the incubation time for measurement of the remaining enzyme activity (Range: 2 - 20 min; mode: 10 min) was apparent. No correction was made for the natural decline in enzyme activity in 9% of the studies, and 14% of the reports used the slope of the log to base 10 instead of the natural log for calculating inactivation constants. The use of non-linear fitting was popular, although 55% of the data analyses were carried out using Kitz-Wilson plots. Because study design can have a substantial effect on the accuracy of the resulting MBI kinetic parameters, literature values should only be used for in vitro-in vivo extrapolation after due attention to experimental design issues.
Silverman RB (1995) Mechanism-based enzyme inactivation. Methods in Enzymology 249: 240-283.
EFFECTS OF DELAY IN TISSUE PROCESSING/FIXATION ON PROTEIN EXPRESSION PROFILES OF NORMAL AND MALIGNANT COLON TISSUE.
J. A. Aquino1 , E. Castellano Sanchez2 , J. D. Wulfkuhle1 , C. Zornig3 , K. David4 , H. Juhl5 , L. A. Liotta1 , E. F. Petricoin III6 , 1FDA-NCI Clinical Proteomics Program, National Cancer Institute, Bethesda, MD, 2Centro de Investigcion del Cancer, Salamanca, Spain, 3Dept of Surgery, Israelitic Hospital, Hamburg, Germany, 4.Indivumed, Center for Cancer Research at the Israelitic Hospital, Hamburg, Germany, 5Indivumed, Center for Cancer Research at the Israelitic Hospital, Hamburg, Germany, 6FDA-NCI Clinical Proteomics Program, Food and Drug Administration, Bethesda, MD
Knowledge and control of collection procedures of biospecimens are necessary for correct interpretation of experimental results. In this study, we analyzed and compared the impact of increased time from surgical removal to fixation/freezing of tissue. We used reverse-phase protein microarrays to examine the expression levels and phosphorylation state of several key signal transduction proteins that might be affected by collection parameters.
Normal and tumor colon tissue samples were collected from 5 cancer patients and frozen in liquid nitrogen at defined time points (2 to 45 minutes), immediately following surgical resection. Whole tissue and laser-capture microdissected (LCM) epithelial cell lysates from each time point were printed on nitrocellulose-coated glass slides. These arrays were analyzed for changes in the expression and activation status of key signaling proteins.
We observed significant variability in the levels of various phosphoproteins in both normal and tumor samples at each time point, and differences in expression trends between normal and tumor samples both within a single patient and between patients. Differences in expression levels of various phosphoproteins were observed between whole cryostats and LCM-derived samples from the same time point, and also in the expression change over time within each patient. These results suggest that cellular response to tissue processing is variable, and there were no generalized effects on the phosphoproteome in tissues that could be directly correlated to collection time. These results also reveal the potential caveats of extrapolating experimental data obtained from whole tissue specimens to individual cell populations within tissues
Evaluation of a Genetically Modified Leishmania Parasite towards the Development of a Safe and Effective Vaccine
R. C. Duncan, A. Selvapandiyan, H. Hairston, K. Tomioka, H. L. Nakhasi, CBER, FDA, Bethesda, MD
Purpose: Leishmania donovani is a blood borne protozoan parasite causing a disease that is fatal if not treated in 500,000 new cases each year. The health risk is emphasized by US soldiers acquiring infections in Iraq. A live attenuated parasite vaccine has potential for the control of this disease.
Method: We have developed a centrin gene deleted cell line of this parasite that can be grown in culture, but survives only a short time in the human infecting, amastigote, form (Selvapandiyan et al. JBC, 2004). To further examine this cell line's potential as a vaccine candidate, we identified additional genes that are differentially expressed using a L. donovani genomic microarray. The changes in expression of these same genes were also examined in wild type cells as they undergo stage differentiation, changes in growth phase, or programmed cell death, because these phenomena are altered in the centrin gene deleted parasite relative to wild type.
Results: The observed expression patterns suggested what roles these genes are playing in the altered physiology of the centrin deleted parasite.
Conclusion: This study is part of monitoring the genetic stability of the potenital live attenuated vaccine to avoid reversion to wild type and further characterization of these genes could contribute towards development of a safe and effective vaccine against leishmaniasis. This research contributes to the regulatory need to maintain expertise in evaluating new vaccine applications. A better understanding of the biology of the parasite serves our mission to protect the blood supply from this blood borne infectious agent.
Changes in Protein Expression of Saccharomyces cerevisiae Cultured on Different Carbon Sources Determined With LCMS and Informatics Tools
B. Kenney, C. Dorschel, J. Silva, S. Geromanos, Waters Corporation
Background: Differential qualitative and quantitative proteomics of yeast grown on different
media were determined with novel liquid chromatography mass spectrometry (LCMS) data
acquisition and informatics methods.
Methods: Yeast (Saccharomyces cerevisiae) was cultured in media containing different carbon sources. The lysates obtained from the harvested cells were concentrated to approximately 12 ug of protein per uL by means of centrifugal ultrafiltration devices (5000 MWCO). The concentrated lysates were reduced, alkylated, and digested with trypsin. For LCMS, triplicate injections of ca. 6 ug of digest were made onto a reversed phase capillary column (300 micron ID) and eluted with an acetonitrile/water/0.1% formic acid gradient. Column effluent was directed to the electrospray source of a hybrid quadrupole-time of flight mass spectrometer. Data were collected in alternating elevated and low collision energy scans interspersed with scans of a reference mass solution.
Results: The data from the replicate injections were clustered, and the results from the various samples were normalized and analyzed for quantitative differences. The determined exact masses of the tryptic peptides and the sequence information obtained from the elevated collision energy scans permitted qualitative identification of the proteins. Yeast grown on glucose was chosen as the baseline sample for comparison. Samples grown on lactose and glycerol were observed to grow at a slower rate than the glucose sample. This is reflected in an observed notable down regulation of a substantial number of ribosomal proteins in the lactose/glycerol samples, which in turn reflects a reduced rate of protein synthesis in these samples. Significant up regulations of proteins involved in carbon source metabolism include phosphofructokinase, fructose biphosphate aldolase, glucose-6-phosphate isomerase and galactokinase in the lactose sample compared with the glucose sample, and glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, fructose biphosphate aldolase, and DL-glycerol-3-phosphatase in the glycerol sample compared with the glucose sample. More detailed lists of differentially expressed proteins and numerical fold-change values will be provided in the poster.
Conclusions: These results clearly demonstrate that this new method is capable of providing a great deal of information regarding the response of the yeast proteome to the growth environment of the organism while requiring only a small amount of experimental time and effort to acquire and analyze the data. This same method has been applied to differential proteomic studies of tissue, plasma, and other microorganisms
Reverse Protein Microarray Analysis of Lymphangiogenesis
L. V.. Leak1 , L. A. Liotta2 , V. S. Calvert1 , J. Wulfkuhle1 , E. F. Petricoin1 , 1CBER,FDA, Bethesda, MD, 2NCI,NIH, Bethesda, MD
Results from our laboratory and others have shown that VEGF-C is a potent mitogen for the lymphatic endothelium and a key lymphangiogenic factor. However, the biochemical signaling pathways activated via VEGFR-3 upon stimulation by VEGF-C in the lymphatic endothelium are not well characterized. Likewise, the molecular mechanisms that regulate the discrete phenotypic modulations of: (1) proliferation, (2) sprouting and branching, (3) cell elongation and migration, (4) cell folding into tubes and (5) vascular networks, during the process of lymphangiogenesis have not been defined. To profile and map multiplexed protein phosphorylation and determine the linkage of signal transduction events during these various stages, we have employed an in vitro cell culture model of lymphangiogenesis coupled with reverse-phase protein microarrays and phosphor-specific antibodies to examine the activation status of key molecular steps in LEC survival, proliferation signaling leading to differentiation. With VEGF-C stimulation, the levels of activated ERK ½ showed variations between proliferating and tube forming. In addition p38 showed variations in LEC stimulated with VEGF-C and marked changes were noted between the various stages of lymphangiogenesis for phosphoro and total p38, of the MAPKK pathway. Activated AKT, an indicator of the state of PI3kinase/AKT pro-survival pathway also showed intense and varied activation within the different stages of lymphangiogenesis. By employing this high throughput technology for the analysis of signal pathway profiling in LEC during lymphangiogenesis it will be possible to gain further insight into the molecular modification of proteins in the lymphatic endothelium during various physiological and pathological conditions.
CHOLESTERYL ESTER TRANSFER PROTEIN VARIANTS HAVE DIFFERENTIAL STABILITY BUT UNIFORM INHIBITION BY TORCETRAPIB
D. B. Lloyd1 , M. E. Lira1 , L. S. Wood1 , L. K. Durham2 , T. B. Freeman3 , G. M. Preston3 , X. Qiu4 , E. D. Sugarman5 , P. C. Bonnette5 , A. J. Lanzetti4 , P. M. Milos1 , J. F. Thompson1 , 1Discovery Pharmacogenetics, Pfizer Global Research and Development, Groton, CT, 2Clinical Biostatistics, Pfizer Global Research and Development, Groton, CT, 3Translational Biomarkers, Pfizer Global Research and Development, Groton, CT, 4Protein Expression and Structure, Pfizer Global Research and Development, Groton, CT, 5Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT
Cholesteryl ester transfer protein (CETP) is an important modulator of HDL-C in humans and thus considered to be a therapeutic target for preventing cardiovascular disease. The gene encoding CETP has been shown to be highly variable with multiple SNPs responsible for altering both its transcription and sequence. Examining nine missense variants of CETP, we have found some with significant associations with CETP mass and HDL-C levels. Two variants, P373 and Q451, appear to be more stable in vivo. This observation is mirrored by partial proteolysis studies performed in vitro. Since these naturally occurring variant proteins are potentially present in clinical populations that will be treated with CETP inhibitors, all commonly occurring haplotypes were tested to determine whether the proteins they encode could be inhibited by torcetrapib, a compound currently in clinical trials in combination with atorvastatin. Torcetrapib behaved similarly with all variants, with no significant differences in inhibition.
Mapping of Genes That Affect the Antibody Response to Human Factor VIII and IX in Mice.
J. N. Lozier, N. Tayebi, P. Zhang, CBER, FDA, Rockville, MD
Factor VIII or IX antibodies (inhibitors) are the most important serious adverse events associated with the use of therapeutic coagulation factor products. Manufacturing changes can cause inhibitors to appear in patients who previously had none despite extensive factor VIII exposure. Many new recombinant products have been introduced recently with manufacturing changes designed to eliminate animal-derived intermediates (e.g., serum, monoclonal antibodies, detergents) in response to the demands of the hemophilia community, and each new product needs to be assessed for its immunogenicity. The immune response is affected by product attributes, environmental factors, and the patients' genetic makeup. To detect genes that influence inhibitor development we assessed AXB and BXA mice derived from high and low responder strains and established linkage with genetic markers near the H2, IL-10, and interferon-γ genes (LOD scores 2.3-4.8). We measured the antibody response of B10.A mice (which have the H2 genes of the high-responder A/J mice in the background of low-responder C57 mice) and confirmed the importance of H2 genes for the immune response to factor IX. In related work, we have shown C57BL/6ByJ mice to be low responders and Balb/cByJ mice to be high responders to human factor VIII. Measurement of antibody levels in CXB recombinant inbred mice derived from these strains show linkage between markers near H2 genes and zinc alpha-2-glycoprotein I (LOD scores 2.8). The latter is a plasma glycoprotein that has significant homology to class I proteins and Ig constant regions. We are assessing the importance of this protein in the factor VIII antibody response in mice, and hope to extend our research to other animals and humans with hemophilia. Identification of genetic factors that influence inhibitor development will facilitate review of clinical trials whereby immunogenicity of clotting factors is evaluated.
Analysis of single nucleotide polymorphisms (SNPs) using microarrays
R. C.K.. Panguluri, R. Elespuru, Food and Drug Administration, Center for Devices and Radiological Health, Office of Science and Engineering Laboratory, Division of Biology, 10903 New Hampshire Ave, Silver spring, Maryland 20903.
Background: Resolving single base changes in human target DNA requires a high degree of specificity and sensitivity. Here, we address the technical issues pertaining to genotypic microarrays in the context of probe purity, chemical labeling vs. dye incorporation during polymerase chain reaction, choice of dyes (Cy3 vs. Cy5), fragmentation of target DNA, and hybridization conditions.
Methods: Ten oligonucleotide probes specific to p53 hotspot codon SNPs were analyzed using microarrays.
Results: All ten p53 hotspot codon SNPs were resolved simultaneously on a single array. As conditions were optimized, multiple technical issues were addressed, in order to achieve high specificity and sensitivity. (1) Oligonucleotide probes for detecting SNPs require purification in order to minimize n-1 sequences typically formed during oligo probe synthesis. (2) Chemical labeling of target DNA produced more uniform signal intensities than dye incorporation. (3) The Cy5 dye exhibited higher signal intensity than Cy3 when both were used as chemical labels. (4) Re-hydrating the spots after printing reduced donut shaped spots and increased the availability of probe on the spot. (5) Fragmentation of the target amplicons with DNAseI increased specificity and sensitivity. (6) During hybridization of target DNA to immobilized probes, agitating the slides resulted in improved hybridization signal intensities and reduced variability across the slide.
Conclusions: Microarray based detection of SNPs in target DNA can be efficient and robust. However, careful attention to several technical issues would be appropriate if used for standardized DNA diagnostics. Factors studied here will facilitate regulatory assessment of genotypic (DNA sequence-based) diagnostic microarray devices.
Pharmacogenomic Data Submissions: Impact on FDA's Submissions and Review Process
A. Rudman, F. W. Frueh, F. M. Goodsaid, L. J. Lesko, CDER, FDA, Rockville, MD
Pharmacogenomic Data Submissions: Impact on FDA's Submissions and Review Process
Background:
The use of pharmacogenomics has increased rapidly over the last several years as indicated in the increasing number of regulatory submissions to the FDA containing pharmacogenomic information. Initiatives such as FDA's Critical Path, the recently issued Guidance to Industry: Pharmacogenomic Data Submissions, the creation of a new voluntary genomic data submission (VGDS) process and the formation of an Agency-wide Interdisciplinary Pharmacogenomics Review Group (IPRG) not only encourage industry to use pharmacogenomics in drug development, but also provide the support and resources needed to review genomic data at the FDA.
In addition to review VGDS data, the IPRG also works on policy development, and, upon request, advises review divisions on interpretation and evaluation of pharmacogenomic data that is part of regular submissions.
Process:
Sponsors can submit a VGDS to the IPRG by following the procedures and decision trees outlined in the Guidance and the associated operating policies (MaPPs) that have been published.
Conclusions:
Early experience with VGDSs have been very positive. Among the benefits to sponsors are the possibility to: meet informally with FDA and receive peer review assessments of scientific data from pharmacogenomic experts at the Agency, obtain insight into the evolving regulatory decision making process as it relates to genetic and genomic information, familiarize FDA scientists with novel pharmacogenomic experiments, data analysis, and interpretation approaches at an early stage.
Cross-platform Comparability of Microarray Technology: Intra-platform Consistency and Appropriate Data Analysis Procedures Are Essential
L. Shi1 , W. Tong1 , H. Fang1 , U. Scherf2 , J. Han3 , R. K. Puri3 , F. W. Frueh4 , F. M. Goodsaid4 , L. Guo1 , Z. Su1 , T. Han1 , J. C. Fuscoe1 , Z. A. Xu1 , T. A. Patterson1 , H. Hong1 , Q. Xie1 , R. G. Perkins1 , J. J. Chen1 , D. A. Casciano1 , 1NCTR, FDA, Jefferson, AR, 2CDRH, FDA, Rockville, MD, 3CBER, FDA, Bethesda, MD, 4CDER, FDA, Rockville, MD
The FDA is actively assessing the applicability of microarrays as a tool in pharmacogenomic and toxicogenomic studies. A recent report (E. Marshall, Science, 306, 630-631, 2004), by extensively citing the study of Tan et al. (Nucleic Acids Res., 31, 5676-5684, 2003), portrays a disturbingly negative picture of the cross-platform comparability, and, hence, the reliability of microarray technology. We reanalyzed Tan's dataset and found that the intra-platform consistency was low, indicating a problem in experimental procedures from which the dataset was generated. Furthermore, by using three gene selection methods (i.e., p-value ranking, fold-change ranking, and Significance Analysis of Microarrays (SAM)) on the same dataset we found that p-value ranking (the method emphasized by Tan et al.) results in much lower cross-platform concordance compared to fold-change ranking or SAM. Therefore, the low cross-platform concordance reported in Tan's study appears to be mainly due to a combination of low intra-platform consistency and a poor choice of data analysis procedures, instead of inherent technical differences among different platforms, as suggested by Tan et al. Our results illustrate the importance of establishing calibrated RNA samples and reference datasets to objectively assess the performance of different microarray platforms and the proficiency of individual laboratories as well as the merits of various data analysis procedures.
The MAQC (Microarray Quality Control) Project: Calibrated RNA Samples, Reference Datasets, and QC Metrics and Thresholds
L. Shi1 , F. W. Frueh2 , U. Scherf3 , R. K. Puri4 , S. A. Jackson5 , H. C. Harbottle6 , J. A. Warrington7 , J. Collins8 , D. Dorris9 , G. P. Schroth10 , L. Lamarcq11 , Y. Wang12 , T. J. Sendera13 , S. C. Baker14 , G. M. Fischer15 , B. Bromley16 , D. J. Dix17 , M. Salit18 , Z. Szallasi19 , E. S. Kawasaki20 , C. Wang21 , R. V. Jensen22 , J. Han4 , F. M. Goodsaid2 , G. A. Pennello3 , T. A. Cebula5 , J. E. LeClerc5 , W. Tong1 , J. C. Fuscoe1 , T. A. Patterson1 , T. Han1 , L. Guo1 , H. Fang1 , J. J. Chen1 , Y. P. Dragan1 , W. Slikker, Jr.1 , D. A. Casciano1 , 1NCTR, FDA, Jefferson, AR, 2CDER, FDA, Rockville, MD, 3CDRH, FDA, Rockville, MD, 4CBER, FDA, Bethesda, MD, 5CFSAN, FDA, Laurel, MD, 6CVM, FDA, Laurel, MD, 7Affymetrix, Santa Clara, CA, 8Agilent, Palo Alto, CA, 9Ambion, Austin, TX, 10Applied Biosystems, Foster City, CA, 11BD Clontech, Palo Alto, CA, 12Full Moon BioSystems, Sunnyvale, CA, 13GE Healthcare, Chandler, AZ, 14Illumina, San Diego, CA, 15Stratagene, La Jolla, CA, 16ViaLogy, Altadena, CA, 17NHEERL, EPA, Research Triangle Park, NC, 18NIST, Gaithersburg, MD, 19Harvard, Boston, MA, 20NCI, Bethesda, MD, 21UCLA, Los Angeles, CA, 22UMass, Boston, MA
FDA's Critical Path Initiative identifies pharmacogenomics and toxicogenomics as key opportunities in advancing medical product development and personalized medicine, and the ¡°Guidance for Industry: Pharmacogenomic Data Submissions¡± has been released. Microarrays represent a core technology in pharmacogenomics and toxicogenomics; however, before this technology can successfully and reliably be used in clinical practice and regulatory decision-making, standards and quality measures need to be developed.
The Microarray Quality Control (MAQC) project involves six FDA Centers, major providers of microarray platforms and RNA samples, EPA, NIST, academic laboratories, and other stakeholders. The MAQC project aims to establish QC metrics and thresholds for objectively assessing the performance achievable by various microarray platforms and evaluating the advantages and disadvantages of various data analysis methods. Two RNA samples will be selected for three species, human, rat, and mouse, and differential gene expression levels between the two samples will be calibrated with both microarrays and QRT-PCR. The resulting microarray datasets will be used for assessing the precision and cross-platform/laboratory comparability of microarrays, and the QRT-PCR datasets will enable evaluation of the nature and magnitude of any systematic biases that may exist between microarrays and QRT-PCR. The availability of the calibrated RNA samples combined with the resulting microarray and QRT-PCR datasets, which will be made readily accessible to the microarray community, will allow individual laboratories to more easily identify and correct procedural failures. The MAQC project will help improve the microarray technology and foster its proper applications in discovery, development and review of FDA regulated products.
Microarray Scanner Calibration Curves: Characteristics and Implications
L. Shi1 , W. Tong1 , Z. Su1 , T. Han1 , J. Han1 , R. K. Puri1 , H. Fang1 , F. W. Frueh2 , F. M. Goodsaid2 , L. Guo1 , W. S. Branham1 , J. J. Chen1 , Z. A. Xu1 , S. C. Harris1 , H. Hong1 , Q. Xie1 , R. G. Perkins1 , J. C. Fuscoe1 , 1NCTR, FDA, Jefferson, AR, 2CDER, FDA, Rockville, MD
Microarray-based measurement of mRNA abundance is based on the assumption of a linear relationship between fluorescence intensity and dye concentration. By scanning a microarray scanner calibration slide containing known concentrations of fluorescent dyes under 18 PMT gains, we were able to evaluate the differences in calibration behavior of Cy5 and Cy3. First, the calibration curve for the same dye under the same PMT gain is nonlinear at both the high and low intensity ends. Second, the degree of nonlinearity of the calibration curve depends on the PMT gain, and there is an optimal range of gain setting for each dye. Third, the two PMTs (for Cy5 and Cy3) behave differently even under the same gain. Fourth, the background intensity for the Cy3 channel is higher than that for the Cy5 channel. The impact of such characteristics on the accuracy and reproducibility of measured mRNA abundance and the calculated ratios is demonstrated. Combined with simulation results, we provide possible explanations to the existence of ratio underestimation, intensity-dependence of ratio bias, and anti-correlation of ratios in dye-swap replicates. By using simulated data based on the different nature of the observed calibration curves for Cy5 and Cy3, we found that although Lowess normalization effectively eliminates the intensity-dependence of ratio bias and makes dye-swap replicates more comparable, the systematic deviation from true ratios largely remains after normalization. A method of calculating ratios based on concentrations estimated from the calibration curves is proposed for correcting ratio bias.
Molecular Characterization of a Hemophilia A Dog Model
N. Tayebi, L. Wood, J. N. Lozier, CBER, FDA, Rockville, MD
Background: Hemophilia A (HA) is cause by FVIII gene mutations. The most prevalent mutations are in intron 22 with 40 to 50% of severe HA arising from intron 22 gene inversions. The best characterized animal model of HA is the Chapel Hill HA dog, which has an inversion similar to the common human mutation.
Method: Overlapping canine genomic DNA BAC clones 292 C4 and 314 O16 were shown to contain FVIII gene sequences. BAC clone 292 C4 contains exon 1-22 and BAC clone 314 O16 contains exons 23-26 of the FVIII gene. High throughput sequencing was used to establish 14 fragments of sequence for BAC clone 292 C4. We have established overlaps between 8 of the 14 known sequence fragments by PCR, subcloning and sequencing of overlap fragments from the 292 C4 BAC and dog genomic DNA. The sequence of intron 22 remains incomplete and we are in the process of sequencing this region from BAC clone 292 C4 and genomic DNA.
Results: Sequence analysis of FVIII intron 22 will facilitate characterization/cloning of the breakpoints of the abnormal HA inversion site in dog. Complete FVIII genomic sequencing will permit development of a rapid PCR diagnostic test for the common FVIII inversion in dogs and facilitate detection other HA mutations and normal polymorphic variants of dog FVIII among breeds.
Conclusions: A complete characterization of the dog FVIII gene will also facilitate our study of genetic factors that contribute to the inhibitor antibody response to therapeutic FVIII products in humans.
APPLICATION OF A CROSS-PLATFORM RNA STANDARD FOR ASSESSING MICROARRAY DATA COMPARABILITY
P. S. Pine1 , B. A. Rosenzweig1 , J. C. Fuscoe2 , C. A. Afshari3 , H. K. Hamadeh3 , J. D. Retief4 , Y. Turpaz4 , E. Blomme5 , R. Ciurlionis5 , J. Waring5 , R. S. Paules6 , C. J. Tucker6 , T. L. Fare7 , E. M. Coffey7 , Y. He7 , J. Collins8 , K. Jarnagin9 , S. Fujimoto9 , G. L. Kiser10 , T. Kaysser-Kranich10 , F. D. Sistare11 , J. Sina11 , K. L. Thompson1 , 1CDER, FDA, Silver Spring, MD, 2NCTR, FDA, Jefferson, AR, 3Amgen Inc., Thousand Oaks, CA, 4Affymetrix Inc., Santa Clara, CA, 5Abbott Laboratories, Abbott Park, IL, 6NIEHS, Research Triangle Park, NC, 7Rosetta Inpharmatics LLC, Seattle, WA, 8Agilent Technologies, Santa Clara, CA, 9Iconix Pharmaceuticals Inc., Mountain View, CA, 10GE Healthcare, Chandler, AZ, 11Merck & Co. Inc., West Point, PA
Microarray data comparability remains a challenge for the integrated use of genomic data generated across sites and platform formats. Tools needed to address this issue include an RNA standard that performs similarly on multiple microarray formats and serves a function analogous to a standard reference material in single analyte assays. We have designed and tested a reagent that can provide a basis for performance assessments on Affymetrix RAE230A, CodeLink UniSet Rat I, and Agilent G4130A rat oligonucleotide arrays. The reagent is a set of two mixed tissue RNA samples formulated to contain different ratios of RNA for each of four rat tissues (brain, liver, kidney, and testis). The analytes in this assay bind to a subset of gene probes that were matched across platforms to the same UniGene cluster or GenBank accession number, were determined to be tissue-selective on each of the 3 platforms using average baseline expression levels in single tissue samples from control animals, and have signal intensities that span the dynamic range of each platform. This reagent forms the basis of a ratiometric assay in a complex biological background that can be used to objectively assess the effect of different array processing methods and conditions on microarray data comparability within and across platforms, and could be a paradigm for similar performance standards for mouse and human arrays.
Influence of the UGT1A1*28 and UGT2B7*2 genetic polymorphisms on enzyme activities: How can we be confident that we are not missing functionally relevant polymorphisms in other enzymes and transporters?
V. Peterkin, S. Myrand, J. Paulauskis, K. Johnson, M. Man, J. Cook, J. A. Williams, Pfizer Global R&D
Purpose: To evaluate two components of an approach to 'unequivocally' determine if gene polymorphisms have functional relevance on drug disposition.
Methods: Component 1: Genotype influence on enzyme activities and immunoreactive protein levels (n=59 livers) for UGT1A1 and UGT2B7 was investigated. Component 2: Polymorphism-associated differences and the intrinsic pharmacokinetic variability was simulated to define subject numbers per study group required to detect two to 10-fold differences in drug exposure ratios (AUCvariant/AUCwild-type) between predicted extensive metabolizers/transporters (homozygous wild-types) versus predicted poor metabolizers/transporters (homozygous variants).
Results: Component 1: For the UGT1A1*28 polymorphism, livers from homozygous wild-type individuals had 4-fold higher mean UGT1A1 activity than homozygous variants. Heterozygotes had intermediate activities. The UGT2B7*2 polymorphism had no influence on UGT2B7 enzyme activity or immunoreactive protein. Component 2: To be confident (80% power) of detecting a 2-, 4- or 10-fold difference in drug exposure between predicted extensive and poor metabolizers/transporters, 11, 4 or 2 subjects would be required per study group, for a drug with a coefficient of variation in exposure (AUC) of 70%.
Conclusions: The in vitro genotype/phenotype correlation for UGT1A1 provides a quantitative assessment of the influence of UGT1A1*28 on enzyme activity, and thus a 'positive control' to investigate genetic influence on enzyme activity for UGT2B7. The expected exposure difference, and variability in substrate pharmacokinetics are important factors to be considered when designing clinical trials so that they are adequately powered to detect effects on drug disposition that truly reflect polymorphism effects and not be equivocal due to pharmacokinetic variability of the probe substrates.
Assessing Treatment Differences Using a Weighted Responder Analysis
L. Chen, Q. H. Li, QMRS/OB CDER FDA Rockville, MD
When differences in means between treatments are not considered valid to quantify treatment differences for endpoints measured on continuous or ordinal scales in clinical trials because small treatment gains (losses) are not clinically relevant, data are often dichotomized into responders versus non-responders. Therefore, differences in responder rates between treatments are used to quantify the treatment differences, without incorporating the sizes of treatment gains (losses) in responders. Since sizes of treatment gains (losses) provide information on how good the treatment is in responders, while the sizes in non-responders are clinically irrelevant, it is important to incorporate information on treatment gains (losses) in responders when assessing the treatment difference. In this article, we propose a new way of quantifying treatment effect, referred to as weighted responder rates, by making more complete use of the information available in responders. Tests for treatment differences using weighted responder rates are proposed. Examples of using the weighted responder analysis are illustrated.
FDA's critical path initiative for evaluation of devices for the correction of presbyopia
M. Eydelman, B. Drum, D. Calogero, G. Hilmantel, CDRH, FDA, Rockville, MD
Background: Presbyopia is the progressive, age-related loss of visual accommodation (the eye's ability to change focus from distance to near). Presbyopia correction is currently limited to static optical devices (e.g., bifocal spectacles and reading glasses). Due to high patient demand, the ophthalmic community is currently investigating numerous methodologies to correct presbyopia by restoring active accommodation. Accurate, reliable measures of accommodative amplitude are essential to evaluate the clinical outcomes of these trials. Subjective accommodation measures have been traditionally used clinically and often are preferred for clinical trials by device manufacturers, but are susceptible to measurement bias and unreliability. Potentially more accurate and reliable objective measures are now available, but are not yet widely accepted for clinical testing.
Methods: FDA has arranged to collaborate with investigators at the University of Houston in a 12-month cross-sectional study of subjective and objective measurements of accommodation. We will test a balanced cohort of 200 subjects of age 40 to 50 years, using two objective and two subjective methods to determine residual accommodative amplitude. The data will be analyzed by age to determine the rate of accommodative amplitude loss. The four methods also will be compared for accuracy, precision and measurement bias.
Goals: We hope to demonstrate that objective measures of accommodation are superior to subjective measures. Identification of objective measures appropriate for clinical trials will facilitate FDA's evaluation of the safety and effectiveness of devices for the correction of presbyopia by providing more reliable evaluations of effectiveness, reduced subject testing time and faster FDA review of premarket submissions.
Estimation of Treatment Effect in the Presence of Non-compliance: A Simulation Study
Yonghong. Gao, CDRH, FDA, Rockville, MD
The intent-to-treat (ITT) analysis and the per-protocol (PP) analysis are the two main analysis methods to estimate treatment effect in randomized clinical trials (RCT). For clinical trials with non-randomized controls the propensity score adjusted estimation of the treatment effect is one of the approaches to adjust for the imbalance of the baseline covariates between the treatment and the control. In the randomized two-arm clinical trial the presence of non-compliance could break the balance of two arms and treatment effect could be confounded with the subject characteristics. In this simulation study the propensity score methodology is adopted in two-arm RCT to estimate the treatment effect under different non-compliance scenarios. Two different approaches of utilizing propensity scores are applied: stratification and weighting. The estimation results of ITT-based direct comparison, PP-based direct comparison, propensity score stratified estimation and propensity score weighted estimation are presented.
Statistical Considerations for Non-inferiority Trial Methodology in Regulatory Applications
H. J. Hung, S. J. Wang, R. O'Neill, C. Anello, Y. C. Wang, K. Mahjoob, E. Nevius, A. Chakravarty, CDER, FDA, Rockville, MD
Statistical methodology for design and analysis of non-inferiority trials without a placebo arm has been controversial in evaluation of regulatory applications, though a variety of methods are proposed in literature. Statistical considerations rest heavily on two unverifiable assumptions: 1) constancy assumption that the treatment effect of the selected positive control does not attenuate in the non-inferiority trial as compared to the historical trials, 2) assay sensitivity assumption that the non-inferiority trial can detect a treatment difference if it exists. In this poster presentation, the topics to be discussed are the pros and cons of some frequently proposed statistical methods, non-inferiority margin selection, and scenarios where these methods may have utility from a regulatory perspective. Also to be discussed are the contrast between the non-inferiority study designs with a placebo arm and without a placebo arm
Novel confocal laser method for intraocular lens power testing: How a laboratory born invention goes through the path from bench to independent clinical sample testing
I. K.. Ilev, R. W.. Faaland, R. J. Landry, R. W. Waynant, D. Calogero, CDRH, FDA, Rockville, MD
Background: Intraocular lens (IOL) dioptric power is a fundamental parameter whose precise measurement is of critical importance for characterizing and evaluating the effectiveness and safety of IOLs. Conventional methods currently used for IOL power testing are limited in terms of high accuracy, spatial sample alignments and the dynamic range over which measurements can be performed (for both positive and negative dioptric powers).
Methods: We present a novel confocal fiber-optic laser method (CFOLM) for precise IOL power testing (Pending Patent, March 3, 2005). The CFOLM operating principle is based on a simple apertureless fiber-optic laser confocal design. The key element is a single-mode fiber coupler that serves simultaneously as a point light source (3-5 µm diameter) used for formation of a collimated Gaussian laser beam profile, and a point receiver that is highly sensitive to spatial displacements of the focused backreflectance laser emission.
Results: Using the proposed CFOLM design, we have tested various clinical IOL samples with both positive (from +5 to +30 diopters) and negative (from -5 to -20 diopters) dioptric powers. The CFOLM principle setup provides high accuracy (<1 µm) in spatially locating the focal point and in measuring the IOL power. We have obtained a high levels of testing repeatability estimated by a standard deviation in the interval of 0.004-0.06 diopters and a relative error in the interval of 0.015-0.3 %.
Conclusions: The CFOLM operating principle and designs provide a simple, accurate, completely objective, quick and inexpensive method for measuring the focal length of various focusing optical elements and systems including positive and negative lenses, objectives, IOL's, contact lenses, eyeglasses, and mirrors. FDA is proposing that this new test method be incorporated into national and international standards for the optical quality of IOLs.
Model-based longitudinal data analysis can lead to more efficient drug development: A Case Study
P. R. Jadhav1 , J. V.S. Gobburu2 , 1CDER, FDA, Rockville, MD and MCV/VCU, Richmond, VA, 2CDER, FDA, Rockville, MD
Purpose. Longitudinal data analysis has been proposed as an alternative to conventional single-point analysis of data generated in typical clinical trials. The aim of this work was to compare the efficiency of conventional and model-based analysis.
Methods. Model based analysis of pivotal trial data was carried out for a drug to treat Parkinson's disease. The key issue was to discern between protective (disease-modifying) and symptomatic benefit of drugs in the treatment of disease. Among all the available choices, a linear model was finally selected due to its simplicity and practical usefulness. Moreover, comparison was made between conventional and model based analysis to assess relative power and type-1 error rates. Also, the impact of drop-outs due to lack of effectiveness or toxicity was assessed.
Results. Model based analysis was found to be twice powerful in comparison with the conventional analysis. Model based analysis preserves pre-specified type-1 error rate and probably it is insensitive to the mechanism of patient drop-out within the limits of model assumptions. Conventional analysis could lead to higher false positive conclusions due to use of last observation carried forward (LOCF) if there are considerable number of drop-outs.
Conclusions. Model based analysis of clinical trial data is more powerful compared to conventional analysis. The saved resources can be effectively utilized to optimize the dose better.
Time to Perceptible and Meaningful Pain Relief
H. L. Lu, M. Katzper, Y. Kim, A. M. Rahman, CDER, FDA,Rockville, MD
Background: A concern in acute pain relief medication is how long it will take till relief occurs.
Methods: We reviewed CDER data from a number of NDA submissions that used identical dental pain models - oral surgery involving molar extraction. Onset of pain relief is measured by a 2-stopwatch method: a patient is asked to click the first stopwatch when he first perceives pain relief, and to click the second stopwatch when sure that satisfactory pain relief (meaningful relief) is achieved. If a patient only reports perceptible pain relief but not meaningful pain relief, the time to onset of perceptible relief is set to a large value, usually the end of the single dose period. Median time to perceptible relief is reported in the drug label as an indicator for drug onset time.
Results: Investigation shows that there are a number of problems with this piece of information. Time of onset is influenced by subjects that get no relief. The public is not informed of the proportion of subjects obtaining relief. Graphs and tables show the extent of this problem and a suggested solution in terms of the median time and proportion of subjects with meaningful relief.
Conclusions: Our study shows that more informative labeling provides an onset measure which both accounts for median relief time of those obtaining relief from the trial drug and the proportion of subjects obtaining relief.
HIV-1 infections during vaccine trials: Identifying new peptides for differential diagnosis of HIV-1 infections in the face of vaccine-generated antibodies
S. Khurana, J. Needham, H. Golding, CBER, FDA, Bethesda, MD
Most of the HIV-1 prophylactic vaccines currently under development are complex products containing multiple viral genes or proteins. As a result, many vaccine-recipients' sera were found to be reactive in currently licensed HIV-1 detection assays, and produce patterns indistinguishable form sera obtained from infected individuals. This will have negative impact on future phase III efficacy trials of prophylactic HIV vaccines that require early detection of breakthrough infections. It will also exclude all vaccinees from the pool of potential blood donors, and may contribute to other social harms. Therefore, it is important to design new testing strategies for vaccine participants that will clearly discriminate between vaccine-induced reactivity and HIV-1 infection. Our goals were to identify new HIV-1 epitopes that: 1) Do not contain important neutralizing or CTL epitopes, and can be omitted from future HIV vaccine constructs, 2) Recognized by antibodies from HIV infected individuals at early times post-infection. 3) Highly conserved among clades and subtypes. Using Phage Display libraries constructed from whole HIV-1 genome, combined with panning with antibodies from early seroconvertors, we identified two new immunodominant epitopes, one in gp41 intracytoplasmic tail and one in p6, that do not contain known protective epitopes and are expendable for HIV vaccines.
These peptides were used for the development of new differential HIV-1 ELISA. To date, we tested more than 1000 seronegative samples as well as several well-characterized panels of early seroconvertors from Boston Biomedica. So far our assay performed well compared with results obtained with other FDA licensed HIV diagnostic kits. Analysis of five different early seroconversion panels showed that HIV-1 infection can be detected using these peptides within 2-3 weeks following HIV-1 RNA detection by PCR.
We have also tested mixed HIV-1 clade panels from around the world (including 100 specimens from Cameroon), and obtained data supporting the utilization of our assay in detecting antibodies in clades A, B, C, D, E, F infections. Currently, our assay performs at 99.3 % specificity and 99.1 % combined sensitivity.
Importantly we already screened samples from a Phase II trial of prophylactic HIV vaccines conducted by DOD. The vaccine regiment used in that trial was a prime-boost ALVAC-HIV (vCP205; gag-LAI + pro-LAI + gp120MN/gp41TM-LAI) with HIV-1 gp160 protein boost (gp120 MN, gp41 LAI-2) (with Gag-p6). So essentially this vaccine regimen is the worst case scenario, wherein both the peptide sequences that are being used in our CBER EIA are present as an antigen in the used vaccine candidate. In this trial, 80% of vaccine recipients gave false positive results in a commercial HIV-1 detection kits eventhough none of the samples were HIV infected. In contrast, although the vaccinees were boosted with complete HIV-1 Env gp160 protein, all samples scored negative in our new gp41-based EIA assay. Moreover, even though the vaccine included p6, very low number (2.5 %) of vaccinees scored positive in our p6-based EIA. We are currently testing specimens from other completed and ongoing vaccine trials, and are seeing very promising results.
In another 100 samples obtained from three vaccine trials from Vaccine Research Center (VRC), the results were even more promising. While 46 % samples tested positive by licensed EIA kit and western blots, none of the vaccine samples reacted with gp41 peptide and two samples reacted positive to the Gag-p6 peptide in our EIA (Fig. IX). To our surprise one of these p6 positive sample was actually obtained from an HIV infected individual that was part of the placebo group on these VRC trials. The other p6 positive samples had one of the highest titre of the antibodies to HIV antigens in the vaccine regimen as judged by licensed HIV serodiagnosis kits.
Issues of concern when applying the most popular statistical methods of survival analysis to medical device studies
Barbara. Krasnicka, CDRH, FDA, Rockville, MD
Statistical issues like non-randomized studies, clustered survival data (e.g., sites, surgeons, patient with two implanted devices), and repeated events (e.g., patients experiencing some adverse events) are often encountered in the time-to-event data in cardiovascular medical device clinical studies. Therefore, in many applications, one cannot assume that the study population is homogenous and the population is to be considered as a heterogeneous sample. However, in practice, evaluations of medical device effectiveness are based mostly on the Kaplan-Meier product limit estimator, log-rank test and/or Cox proportional hazard regression model (frequently only one treatment factor is taken into account), despite the fact that, in the case of heterogeneity among patients in a given population, these methods do not function well for many clinical studies. Problems such as misspecification of regression models, biases in treatment effect comparisons and losses of power are encountered. Additionally, sometimes highly significant and relatively strong prognostic factors have very low absolute predictive accuracy; i.e., a significant treatment may have small influence on the outcome for an individual patient.
In our presentation, when discussing use of survival analysis to cardiovascular medical device studies, we will concentrate on modeling the heterogeneity, and evaluation of explained variation and predictive accuracy of survival. Some examples of proper handlings of these problems will be presented.
Statistical Consideration of Group Sequential Method for Bioequivalence Assessment
H. Li, Y. Tsong, CDER, FDA, Rockville, MD
The two-stage group sequential design with one interim look at half information time is often used in bioequivalence trial of generic drug product. Often the experimenter adopted a Pocock boundary rather than an O'Brien-Fleming boundary with an optimistic expectation of establishing bioequivalence with half sample size. A simulation study is conducted to illustrate the advantage and disadvantage of using group sequential designs (Pocock and O'Brien-Fleming) and fixed sample design under various scenarios of the deviation from assumptions on cure rate of the reference product and the difference in cure rate between test and reference drugs.
A Retrospective Approach for Analyzing the Data Collected from the Adverse Events Reporting System
Peng.T. Liu, CFSAN, FDA, College Park, MD
The Adverse Events Reporting System (AERS) provides only the "numerator" information in risk estimation. The "denominator" is not available. Therefore, the usual estimations of the risk, such as the probability of an adverse event given an exposed case and the probability of an adverde event given a non-exposed case, are liable to be ineffective. The current signals or data mining techniques are based solely on the "numerator" information without associated denominator. Regardless of how you manipulate the data, the results are difficult to link to the true risk of compounds. To achieve the signal-risk linkage, we purpose a retrospective approach. We express the "numerator" and "denominator" in risk estimation in a reverse manner. Two inverse conditional probabilities--1) the probability of exposure given a case and 2) the probability of exposure given a control, are estimable from the AERS data. Therefore, the ratio of two inverse conditional probabilities could be one of the best signals for flagging high-risk compounds.
Patient Reported Outcomes: Comparison of Two Simulated Treatment Groups in which Extreme Values of Ordinal Responses are Under-Reported
T.J. Massie, T.S. Massie, CDER, FDA, Rockville, MD
Background: The use of Patient Reported Outcomes (PRO's) has been widely used in clinical trials. These outcomes can be continuous, categorical, ordinal and/or written text typically transcribed within a patient's diary. The data consisting of continuous, categorical and ordinal responses can be utilized as the primary, secondary or tertiary endpoints within clinical trials. This poster will focus on ordered categorical (ordinal) responses. There are many issues that arise with use and analysis of ordinal response PRO's. One particular issue of interest is the reluctance of patients to use extreme values within PRO's. This poster will examine comparisons of simulated treatment effects from ordinal responses in which the reporting rate of extreme values varies.
Methods: The authors simulated data to represent both pre-treatment and post-treatment responses for two treatment groups. The authors then manipulated these simulated datasets such that a predetermined percentage of simulated "patients", regardless of treatment group, would not utilize extreme values on a preset categorical scale, for both pre- and post-treatment responses. To do this, the original simulated data was modified so that a certain percentage of the extreme values were replaced with a one level reduction of the extreme value, which represented a moderating of extreme values. The authors will refer to this percentage as the "under-reporting percentage".
Results: First, statistical tests were performed to compare and evaluate the differences between the two treatment group\'s responses based on the original simulations. Similar statistical tests were then performed on the modified simulations. Comparisons of results between the original and modified simulations were created and presented for select "under-reporting percentages".
Conclusions: Utilizing simulated results of ordinal data, we show that for various percentages of under-reporting of extreme values, differences between treatment groups may not be correctly identified. Applied to patient reported outcomes, these results suggest that since individuals, given the freedom to select a response, may be hesitant to use extreme values and thus true differences between treatment groups may not be detected. Furthermore, our simulation illustrates that while an intervention may be truly effective, the reduction in the use of extreme values can obscure the effectiveness of a drug or intervention.
*Note: The views expressed in this poster represent the private views of the presenters and have no endorsement by the U.S. Food and Drug Administration
Noninferiority Hypothesis with Binary Endpoints
Tie-Hua. Ng, FDA, Rockville, MD
The non-inferiority (NI) margin should be a small fraction of the therapeutic effect of the active control as compared to placebo. Such a proposal works well in testing the NI hypothesis of the mean difference with a continuous outcome rather than a binary endpoint. For testing the NI hypothesis of the mean ratio, a similar NI margin on a log scale is proposed. This proposal also works well in testing the NI hypothesis for survival data based on hazard ratio. Some pitfalls of testing the NI hypothesis with binary endpoints based on the difference or the ratio of proportions will be discussed. Testing the NI hypothesis with binary endpoints based on odds ratio is proposed.
A New Graphic Tool for Reviewers to Evaluate Studies for Drug-Induced Liver Injury (DILI)
J. R. Senior, T. Guo, K. Gelperin, CDER, FDA
Almost any drug may sometimes cause serious liver injury in some patients, fortunately rather rarely. The problem for CDER reviewers of clinical trial data under consideration for new drug approval is to decide whether relatively rare incidence of liver injury is serious enough to warrant concern, and whether it is significantly greater than from alternative or no treatments.
Concurrence of both liver injury (raised serum alanine aminotransferase [ALT] activity) and reduced liver function (increased total serum bilirubin [TBL] concentration) in a person is a both sensitive and highly specific test for potentially serious DILI ("Hy's Law"). To translate that idea into a useful tool for reviewers using SAS-derived programs, we have developed a simple graphic display of peak ALT and TBL values observed for each person during a given study, plotting each individual as a single dot on an x-y graph that shows people on study drug with one symbol (∆) and those on control drug with another (o). This allows a reviewer to see at a glance if the incidence of raised ALT or TBL or both is greater on drug than on control treatment. Further, by clicking on any point on the x-y graph, the complete set of data (ALT and TBL, plus other measures) over the course of the study for that individual person is displayed.
Application of this new software tool for CDER reviewers for inspection and analysis of clinical study data may speed and improve review for appreciation of serious DILI.
Power of Simultaneous Test of superiority and Non-inferiority Using Cross-Trial Comparison Approach - A Simulation Study
Y. Tsong, J. J. Zhang, CDER, FDA, Rockville, MD
The topic of switching between superiority test and non-inferiority test has generated many interests in active controlled clinical trials. It has been shown that there is no type I error rate or power change when adopting a two-stage switching procedure using a generalized historical control approach (e.g. non-inferiority margin approach)(Morikawa and Yoshida, JBS,1995). It was shown asymptotically that the same held true when using a cross-trial comparison approach (e.g. percent preservation approach) by Hung and Wang, JBS, 2004). However, Tsong and Zhang, BIMJ, 2004) showed that for a finite sample size, type I error rates changes when an inequality condition involving the variances of the test and active control outcome variable failed. They further proposed to use a simultaneous test for both approaches in order to hold type I error rate constant for both superiority and non-inferiority tests. In this simulation study, we compared power of superiority testing between a stand-alone test and the simultaneous test. It is shown that the power is reduced in the simultaneous testing procedure when using a cross-trial comparison approach.
"Responder analysis" of clinical relevant evidence of trials
Yi. Tsong, CDER, FDA, Rockville, MD
In clinical trial, the benefit of a test treatment needs to be established by showing not only by rejecting the null hypothesis of a regular significance test but also by demonstrating a clinical benefit of a meaningful size. For outcomes of normal distributions of equal variance for test and control treatments, Keiser et al (SIM, 2004) and others in the literature recommended to test if the mean difference is larger than K, a pre-specified size of clinically meaningful benefit. A generalization of the definition to cases with non-normal distribution or unequal variances is to define a responder rate of treatment A (in comparison to treatment B) as PA = Pr(XA - XB > K). A significance test with clinically meaningful benefit K can be performed by test H0: PT = PC versus Ha: PT > PC. An asymptotic test of normal response variables without common variance can be derived with Taylor' expansion (Walter, SIM, 2002). Bootstarp method may be applicable for non-normal response outcome variables. Properties of the proposed approach will be discussed.
Non-inferiority Cardiovascular Device Trial Design with Adaptive Delta to Underlying Control Rate
Y. Xu, L. Yue, CDRH, FDA, Rockville, MD
Many cardiovascular device non-inferiority trials have the adverse event rate as a primary endpoint, and this event rate is usually low. For times when we are quite uncertain about the active control rate, it is very difficult to choose the non-inferiority margin. Therefore, there is increasing demand for adapting the non-inferiority margin to the underlying active control rate. To meet this demand, we describe choosing the non-inferiority margin, delta within a unified framework where delta is a function of the underlying control rate. The traditional fixed value for delta is a special case when the function is constant. Designing the trial with delta as a linear function of the underlying active control rate is another special case. The odds ratio is also examined as a special case of non-linear function for delta. Available tests for the above three special cases are compared with respect to their power characteristics, and appropriate choices of the function for delta are discussed for different situations.
The Application of Propensity Score Analysis to Non-randomized Medical Device Clinical Studies: Regulatory Considerations
L. Yue, CDRH, FDA, Rockville, MD
Key Words: Propensity Score Analysis; Missing Covariates; Treatment Group Comparability
Recently, there is an increased interest in applying propensity score analysis to non-randomized medical device submissions to control for multiple imbalanced baseline covariates in two treatment comparison. There are many practical issues encountered in the application of the methodology, such as handling missing covariates when estimating propensity scores, assessing the success of the propensity score estimation by checking the resulting balance of the distributions of covariates, evaluating treatment group comparability by the distributions of propensity scores, comparing treatment groups by adjusting for the propensity scores, and estimating sample size required for the treatment comparison incorporating the propensity scores. In this presentation, these issues will be discussed and illustrated with regulatory considerations.
New tests for assessing equivalence and non-inferiority from survival data: An intuitive approach
K..M.. Koti, FDA, Rockville, MD
As the median survival time is straightforwardly informative to the clinicians, it has become a common practice in clinical trial study reporting to give point and interval estimates for the median survival times. This motivated us to consider testing for equivalence and non-inferiority of an experimental drug compared to a reference drug in terms of their median event-times. In particular, we consider testing the non-equivalence hypothesis in terms of the ratio of the population median survival times µE and µR, where (0.8, 1.25) is the pre-specified equivalence range. We think of the ratio mE /mR of the sample medians as a point estimate of the ratio µE /µR. We assume that each treatment group has survival curve that is not relatively flat in the neighborhood of 50 per cent survival and use the fact that the median event-time estimate is asymptotically normally distributed. We follow the Schuirmann's "two one-sided tests" approach. We use the Fieller-Hinkley (see Hinkley, 1969) distribution of the ratio of two normally distributed random variables to derive a level-á asymptotic test of each one-sided null hypothesis of non-equivalence in terms of the ratio of median survival times. We also explain how to test the non-equivalence hypothesis using the corresponding two-sided confidence intervals. We point out that it is easy to construct and straightforward to interpret our confidence intervals on the ratio µE /µR. We illustrate our methodology using duration of anemia data from a fictitious equivalence trial in subjects with acute non-lymphocytic leukemia, receiving induction and consolidation chemotherapy. As one of the two-sided 'equivalence' hypotheses has been referred as to the non-inferiority hypothesis, we do not elaborate on testing for non-inferiority. We do not address any clinical, ethical or regulatory issues.
Quantitative Process Characterization - An Essential Step of a Robust PAT Program
P. Cini, J. J. Kamm, Tunnell Consulting
Purpose:
-Present a structured and efficient approach to characterize drug manufacturing processes, in stages as early as process development, using limited and incomplete datasets.
-Illustrate, through several case studies, the power and flexibility of this approach as applied to both optimization and troubleshooting issues typically encountered during drug development, scale-up, setting of specification limits, technology transfer, commercial manufacturing, and in the development of PAT strategies.
Methods:
-A systematic approach combining the use of statistical tools with scientific, process, and engineering 'know-how' that allows for cause & effect hypotheses to be tested analytically, quantified mathematically, and ranked in order of impact and importance. Tools such as capability analysis (CpK), control charts, multiple regression analysis, table of effects, Pareto analysis, contour mapping, as well as innovative variations and combinations of these concepts are applied in this methodology.
Results:
-Case studies will be presented and discussed covering a broad range of scenarios such as slow release rate, excess water content, low potency, poor appearance, and poor stability dissolution profiles.
Conclusions:
-In every case, this methodology provided a significant insight into the process that led to a successful validation and launch or to the resolution of complex manufacturing problems.
A Risk Managed Approach for Implementation of Spectroscopic Techniques in Powder Blend Monitoring
A. El Hagrasy, S. Chang, S. Kiang, Process Research and Development, Bristol-Myers Squibb, New Brunswick, NJ
Purpose:
Assess the capability of spectroscopic techniques in blending process monitoring under varying conditions for optimum utilization of Process Analytical Technology (PAT) in drug product manufacturing.
Method:
Traditionally, the focus of blend uniformity analysis (BUA) has been limited to the assessment of the homogeneity of active pharmaceutical ingredient (API). Excipients play a pivotal role in bringing about the desired performance of the drug product. Spectroscopic techniques allow simultaneous monitoring of API and/or excipients. On-line NIR spectroscopy was used to monitor lubricant blending to enhance the quality and uniformity of the final dosage form. The capability of the technology was assessed by real-time monitoring of blending dynamics under a range of process conditions and raw materials attributes.
Results:
Real-time model predictions correlated well with the expected lubricant concentration during blending, which allowed determination of blend quality.
Conclusions:
The investigation provides an understanding of the system capability to portray process events and the associated risk factors. Furthermore, it highlights the importance of proper selection of processing conditions and raw material attributes to improve process robustness.
Keywords:
PAT, Industrialization, process analyzers, process variation, risk mitigation
Applying Process Analytical Technology to Improve Efficiency and Reliability of Spray Coating
S. Chang1 , A. El Hagrasy1 , S. Kiang1 , S. Kothari2 , S. Paruchuri2 , H. Guo2 , D. Divyakant2 , W. Early2 , H. Stomato2 , 1Process Research and Development, Bristol-Myers Squibb, New Brunswick, NJ, 2Biopharmaceutics Research and Development, Bristol-Myers Squibb, New Brunswick, NJ
Purpose:
Evaluate Process Analytical technologies to characterize spray coating process for efficient development.
Method:
The use of innovative and real-time Process Analytical Technology (PAT) tools can generate process-rich information, which lead to process understanding and therefore can contribute to optimized efficiency and consistency of product quality. Spray coating will be affected by spray nozzle performance, mixing efficiency of tablets and drying conditions. The ultimate goal is to produce uniformly coated products with desired film thickness. Imaging and spectroscopy tools will be assessed to characterize the droplets from spray nozzle and to follow the kinetics of film deposition.
Results:
Real-time imaging data provides characterization of droplets sprayed by nozzles under different operating conditions. Combined with spectroscopic analysis and standard tests on coated tablets, this suite of tools helps to streamline the development process and to generate transferable data for scale up and change control.
Conclusion:
The PAT tools provide performance information of spray nozzle and coating kinetics, which support the optimization of coating uniformity and efficiency.
Keywords:
Industrialization, PAT, process optimization, product consistency, spray coating,
Process Understanding: Relating Scanning Electron Microscopy Studies of Powder Blends with Capsule Dissolution Performance
C. D. Ellison1 , M. L. Hamad1 , E. H. Jefferson1 , W. K. Riemenschneider2 , R. C. Lyon1 , 1DPQR/CDER/FDA, 2DCMS/CDRH/FDA
Background: Effective process analytical technology relies on process understanding. Phenytoin sodium blended with magnesium stearate (MgS) and other excipients exhibits slower dissolution after longer blending time. The current work examines these blends by scanning electron microscopy (SEM) to determine whether MgS is coating phenytoin sodium during blending and acting as a dissolution retardant.
Methods: Binary mixtures consisting of API and MgS (96:4 w/w) were blended in a benchtop V-blender for two hours. Five-component mixtures consisting of API, MgS, sucrose, lactose, and talc were blended in a pilot-scale V-blender for 75mins (Pfizer, NJ). Binary blends were collected at 5 and 120min and five-component blends were collected at 15min and 75min. Blends were encapsulated and dissolved using USP dissolution apparatus I. Collected powders were scanned by SEM and compared with pure components.
Results: All binary blends dissolved rapidly (>90% in 10min). For the five-component blends, substantial retardation of dissolution occurred between 15min blending (99% at 60min) and 75min blending (72% at 60min). Particles in the SEM images were distinguished based on size and distinct morphology. The results suggested that minimal binding of MgS to API occurred with binary blending, insufficient to hinder dissolution. The five-component blends show more extensive aggregate formation occurring with longer blending.
Conclusions: The results suggest that retardation of phenytoin dissolution with longer blending is not a result of API coating by MgS, but due to aggregate formation with other excipients. Such process understanding may be important for effectively modifying the process to control product performance.
Monitoring magnesium stearate coating of particles by Near-Infrared (NIR) chemical imaging
C. D. Ellison1 , R. C. Lyon1 , M. L. Hamad1 , E. H. Jefferson1 , A. S. Hussain2 , 1DPQR/CDER/FDA, 2OPS/CDER/FDA
Purpose: NIR chemical imaging is a promising new tool for process analytical technology. Although magnesium stearate (MgS) is used as a lubricant during blending of dry pharmaceutical powders, excessive coating of the powders can result in retardation of product dissolution. This project examines the utility of NIR chemical imaging for monitoring the coating of powders by MgS during blending.
Methods: Large non-pareils (14-18M), small non-pareils (40-60M), confectioners sugar, and phenytoin sodium (a very fine particulate powder) were each blended with MgS (2% to 4%) for two hours using a Turbula™ benchtop mixer, with an aliquot taken at 10min. Unblended, 10-minute, and two-hour samples were scanned using a Spectral Dimensions (Olney, MD) Sapphire™ NIR chemical imaging system.
Results: Chemical imaging could detect changes in the MgS NIR signal associated with powder blending. Apparent MgS levels increased from 15% at 10min to 24% at 2hr for large non-pareils, and from 1% to 6% for small non-pareils. For confectioners sugar and phenytoin, there was no change in apparent MgS.
Conclusions: This project demonstrated the ability of chemical imaging to monitor the coating of large particles with MgS. Rate and degree of surface coating increased with particle size, likely due to surface area effects. For a given mass, less MgS is needed to coat larger particles. Larger particle size implies fewer particles and smaller total surface area. Monitoring of particle coating by NIR chemical imaging could be used to suggest an appropriate amount of MgS and an appropriate end-point for blending.
Process Analytical Technology (PAT) Tools: Chemometric Evaluation of Furosemide Tablet Formulations
M. L. Hamad, J. T. Wang, A. S. Carlin, C. D. Ellison, E. H. Jefferson, R. C. Lyon, DPQR/CDER/FDA
Background: Near Infrared (NIR) spectroscopy is a commonly used sensor technology for monitoring pharmaceutical manufacturing processes. In this study, NIR spectroscopy was used to evaluate 48 different formulations of furosemide tablets. The resulting data was analyzed with several chemometric algorithms to elucidate relationships between spectroscopic measurements and quality attributes such as tablet hardness, moisture analysis and endpoint dissolution testing.
Methods: A design of experiments (DOE) protocol was developed to produce 48 formulations of furosemide tablets with variations in eight manufacturing variables, including pharmaceutical particle size, tablet compression, lubricant concentration, filler composition, disintegrant composition and concentration, mode of disintegrant addition, and binder concentration. Quality attributes of the tablets were measured with the following analytical methodologies: NIR spectroscopy, hardness, moisture analysis, and dissolution. Dissolution was measured at 5-minute intervals for one hour. Principle Components Analysis (PCA) was used to recognize patterns within the spectroscopic data and Partial Least Squares (PLS) analysis was used to correlate the NIR spectral signals with the measured quality attributes.
Results: PCA of NIR spectra discriminated between formulations based on filler composition, lubricant concentration, hardness, and binder concentration. PLS results showed correlations between NIR spectra and moisture content, dissolution, and tablet composition.
Conclusions: The results from this study demonstrated that NIR spectral data can be used to predict quality attributes in the final product. Because NIR is a fast non-destructive technique, it can be an important tool in DOE by allowing faster, more representative sampling/prediction of products under development.
Preprocessing Method Selection Strategies for Chemometric NIR Spectral Analysis
R. M. Fahmy1 , W. R. Marnane1 , D. Bensley1 , S. Hoag2 , 1CVM, FDA, Rockville, MD, 2University of Maryland, School of Pharmacy, Department of Pharmaceutical Science
Purpose: With NIR* spectroscopy, the spectral signal contains both physical and chemical information. Certain preprocessing methods are designed to remove physical effects from the spectra; however, when analyzing physical properties these procedures may actually remove valuable information from the spectra. In other words, the type of information you are trying to model may affect the preprocessing method choice. Thus, our objective will be to examine the effect a preprocessing method has on the type of information being analyzed.
Methods: Formulations containing sulfamethazine, corn starch and magnesium stearate were wet granulated with a 10% (w/w) starch paste, and compressed on a Stokes B2 rotary tablet press. The data was preprocessed using mean centering, mean centering with unit variance normalization, second derivative with Savitzky-Golay smoothing, and mean scatter correction and wavelength selection based upon the loadings.
Results: Performing PCA* with no preprocessing, mean centered, mean centered with unit variance normalization and second derivative with smoothing had Q residuals of 0.09%, 1.38%, 13.52%, 0.05%, respectively. Using PLS* for content determination the second derivative gave the best results. With physical attributes like dissolution and tablet hardness mean centering gave the best results. Mean centering with unit variance normalization did not improve the model fit and actually amplified the noise, thereby reducing the model predictability. Removing wavelengths that did not contribute significantly to the loading, improved model fit.
Conclusions: It appears that methods which remove baseline shifts also remove physical information from the spectra, which is deleterious when analyzing physical attributes but advantageous for chemical content determination.
Influence of manufacturing changes and formulation excipients on the direct determination of furosemide in solid oral dosage forms using laser-induced breakdown spectroscopy (LIBS)
L. St-Onge1 , M. Tourigny2 , M. Sabsabi3 , R. C. Lyon4 , P. J. Faustino4 , 1National Research Council-Canada, Boucherville, Canada, 2PharmaLaser, Boucherville, Canada, 3NRC-Canada, Boucherville, Canada, 4DPQR, CDER, FDA
Background: To investigate the influence of manufacturing changes and formulation excipeints on the direct determination by LIBS of furosemide in solid dosage forms.
Methods: Sixteen calibration and test formulations (80-mg furosemide 320-mg tablets) manufactured by direct compression or wet granulation differing in their relative excipient content of avicel, lactose and magnesium stearate were evaluated by LIBS. A pulsed Nd:YAG laser (λ=1064 nm) was utilized to produce a gas plasma at the tablet surface, without sample preparation. The emission spectrum was resolved and furosemide was identified unambiguously through emission of atomic chlorine or sulfur.
Results: Furosemide was detected with similar sensitivity using the chlorine line 837.60-nm or the sulfur line 921.29-nm. Internal standardization (IS) using carbon lines at 833.52 nm and 911.18 nm, significantly improved precision. The Cl/C, S/C line intensity ratios and tablet-to-tablet relative standard deviation was below 2.5%. The lactose/avicel ratio significantly affected the furosemide LIBS signals. For a lactose matrix, the signal intensity was found to be approximately 70% of an avicel matrix, even with IS. The magnesium stearate content (0.8 or 6.4 mg), and manufacturing changes such as compression strength (8 or 15 kp) or wet granulation instead of direct compression, affected LIBS signals by less than 10%. Generally, the Cl/C ratio had a weaker dependence on the formulation and manufacturing change than the S/C ratio.
Conclusions: Formulation excipient changes such as the lactose/avicel ratio significantly affect the LIBS signals. A fundamental understanding of formulation matrix effects may facilitate at-line monitoring for product characterization during manufacturing changes.
Impact of Sodium Butyrate Supplementation on Global Gene Expression and Monoclonal Antibody Glycosylation Patterns in Murine Hybridoma Cells
M. A. Hanson1 , N. Rajagopalan1 , S. C. Lute2 , K. R. Brorson2 , A. R. Moreira3 , 1UMBC, Baltimore, MD/CDER, FDA, Bethesda, MD, 2CDER, FDA, Bethesda, MD, 3UMBC, Baltimore, MD
It is well documented that certain mammalian cell culture process changes can modify the protein products of interest. From a product quality and regulatory perspective, it is advantageous to understand the impact of process changes during product development and post-marketing to assure that high quality product is produced at all times. In an effort to understand why and to what extent specific product modifications occur after common cell culture process changes, we are studying the impact of select, industrially relevant mammalian cell culture process changes on global gene expression patterns and product attributes. Our first experimental studies consisted of supplementing media with sodium butyrate to a final concentration of 0.5 mM during growth of IgG3 producing murine hybridoma cells. Class Comparison analysis has revealed differential expression in genes related to apoptosis, translation initiation, signal transduction, and mitochondrial membrane structure. Differences in glycosylation were also studied using the Fluorophore Assisted Carbohydrate Electrophoresis (FACE) analysis kit and densitometry. Lastly, results of a cloning study, carried out for this cell line to increase antibody yield, are presented.
A Comparative Study of Single and Group Assay of Solid Pharmaceutical Dosage Forms to Determine the Potential Sources of Variability Using Near-Infrared Spectroscopy and Chemometric Models
S. Hassannejad Tabasi1 , K. Bakeev2 , R. M. Fahmy3 , W. Marnane3 , D. Bensley3 , S. Hoag1 , 1University of maryland, Baltimore, MD 21201, 2FOSS NIRsystems, Laurel, MD 20723, 3FDA, Rockville, MD 20855
Objective: To use multivariate calibration models as a tool to assess the effects of multiple or single tablet measurements on the variability in Near-Infrared (NIR) spectral data, and the transferability of the calibration models between different NIR spectrometers.
Method: A series of 500mg theophylline tablets were formulated with drug content of 0, 10, 20, 30 and 40% w/w. These tablets were scanned using Foss DS 6500 RCA and Foss XDS RCA NIR spectrometer. After preprocessing, multiple linear regression (MLR) and partial least square (PLS) calibration models were generated to predict the drug content of intact tablets.
Results: For single tablets measurements, both systems provided reliable results in the quantification 0, 10, 20, 30 and 40% theophylline with standard error of prediction (SEP) of 1.27% and 1.51% for the DS and XDS, respectively. Using the XDS system to measure multiple tablets, a SEP of 0.88% was achieved. Comparing external and internal reference standardizations, both (0.93% and 91%) provided close but significantly different SEPs. To test calibration transferability, MLR models developed on the DS system provided significantly smaller SEP (1.27-1.71%) compared to the calibration models generated on the XDS system (3.9-7.5%).
Conclusion: The DS system for individual assay provided satisfactory results over the XDS system in both direct and cross-prediction. However, group assay using either internal or external reference standardization provides better results over individual assay in direct prediction (0.84 and 0.93% vs. 1.5%). Finally, the results of this study show that NIR instrument differences can affect SEP but careful model development minimizes these differences appreciably.
Synergistic antitumor activity of temozolomide and IL13-Pseudomans exotoxin against human glioblastoma in a mouse model
S. R. Husain, R. K. Puri, CBER, FDA, Bethesda, MD
Background:Glioblastoma multiforme (GBM) is an infiltrative brain tumor that poses a major public health concern, causing 14,000 deaths in the US alone. IL13-PE, a chimeric protein composed of human IL-13 and mutated Pseudomonas exotoxin (PE) specifically targets the receptor for the immune cytokine, interleukin-13 (IL-13) over-expressed on glioma cells. Convection-enhanced delivery of IL13-PE is currently being studied in a Phase 3 trial in patients with recurrent GBM. Temozolomide (TMZ), an approved drug for anaplastic astrocytoma, acts by methylating guanosine nucleotides of DNA. The objective is to determine whether the combination of both agents improve the antitumor activity against GBM.
Methods: In vitro antitumor activity of TMZ and IL13-PE was determined by protein synthesis inhibition and clonogenic assays, while in vivo activity was assessed in an immunodeficient mouse model of GBM.
Results: The combination of TMZ and IL13-PE inhibited protein synthesis in U251 and U87 GBM cells by 31% and 50% respectively, however each drug alone inhibited protein synthesis by only <5% in U251 and <35% in U87 cells. Daily administration of one half of the clinical dose of either TMZ (2.5 mg/kg) or IL13-PE (10 or 25 mcg/kg) did not produce any complete regression (CR) of U251 tumors in mice. In contrast, a combination of TMZ with IL13-PE produced CR in about 33% animals. A similar combination of TMZ and IL13-PE produced CR in 40% of animals with established U87 tumors.
Conclusion: Sub-optimal concentrations of TMZ and IL13-PE act synergistically to produce CR and reduce tumor burden in mice. These studies provide unique insight on pre-clinical testing of two unrelated agents for GBM therapy.
Process Analytical Technology (PAT) Tools: Determining Content of Acetaminophen Tablets (APAP) by Attenuated Total Reflectance (ATR) FT-IR Spectroscopy and FT-Raman Spectroscopy
A. S. Carlin, E. H. Jefferson, M. L. Hamad, R. C. Lyon, DPQR, CDER, FDA, Silver Spring, MD
Background: This study assessed the feasibility of predicting active drug content in commercial-grade pharmaceutical tablets by ATR spectroscopy and FT-Raman spectroscopy. This study is part of a comprehensive program to evaluate current sensor technologies, data analysis and regulatory approaches associated with Process Analytical Technology.
Methods: ATR Spectra of tablets were obtained using a Thermo-Nicolet NexusTM 670 FT-IR spectrometer equipped with the Smart Orbit diamond ATR. Raman spectra were obtained using a Thermo-NicoletTM FT-Raman instrument. Nine batches of APAP tablets differing in APAP content (54.2-121.7 mg) were examined. Multivariate analysis was performed using Thermo-Nicolet TQAnalystTM chemometric software.
Results: For each method, a calibration set (n=150 tablet spectra) was used to build a PLS model. The model was then applied to a validation set (n=120) to predict the APAP content of the remaining tablets. The ATR calibration set yielded a correlation coefficient, R = 0.958 and a standard error of calibration, SEC = 5.9. The ATR validation set yielded R = 0.945 and a standard error of prediction, SEP = 5.4. The Raman calibration set yielded a correlation coefficient, R = 0.993 and a standard error of calibration, SEC = 2.4. The Raman validation set yielded R = 0.943 and a standard error of prediction, SEP = 5.4.
Conclusions: BothATR spectroscopy and Raman spectroscopy with multivariate analysis provide an accurate method for predicting the active content of pharmaceutical tablets. Like near-infrared spectroscopy,both techniques have potential as non-invasive sensors for monitoring pharmaceutical processes.
Vibrational Spectroscopy of Coated Tablets
J.F. Kauffman, CDER, Division of Pharmaceutical Analysis, St. Louis, MO 63101
Background: Spectroscopic methods of analysis are particularly useful for process analytical technology because they provide chemically specific information, and are often fast and non-destructive.
Methods: We present a study of the influence of tablet coatings on spectroscopic analysis of tablet core content. We perform near infrared absorbance spectroscopy (NIR) and Raman spectroscopy on a set of acetaminophen tablets whose coating content and thickness have been systematically varied. Simultaneously, we evaluate these methods for measurement of coating thickness. Coating and core content uniformity have been measured using near infrared and Raman chemical imaging. The spectra are analyzed using multivariate statistical methods.
Results: Principal component analysis of NIR spectra reveals separate observables associated with tablet core content, coating thickness and coating composition. Raman spectra of coated tablets provide more detailed chemical information than NIR spectra. For example, Raman spectra can often distinguish different polymorphs of the same drug substance. However, Raman spectra are contaminated by fluorescence from coatings. Photobleaching of coating fluorescence allows measurement of Raman spectra of both tablet core and coating contents. Coatings containing TiO2 exhibit strong, analytically useful Raman bands in the low energy region of the spectrum.
Conclusion: Using suitable chemometric methods, NIR spectroscopy is capable of simultaneously characterizing core content and coating thickness of coated pharmaceutical tablets. Raman spectroscopy of tablets offers greater chemical specificity than near infrared spectroscopy, but suffers from interferences that complicate data analysis. Raman chemical imaging of TiO2 in tablet coatings offers a potential method for evaluating coating uniformity in non-pigmented, coated tablets.
Preparation of Various Insulin Crystals for Long-Acting Insulin Formulation
Y. Vaynshteyn1 , L. X. Yu2 , V. C. Yang3 , J. F. Liang1 , 1Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, 2Office of Generic Drugs, CDER, FDA, Rockville, MD 20850, 3College of Pharmacy, The University of Michigan, Ann Arbor, MI 48109
Background: Recent studies have shown that tight glycemic control (steady glucose levels in blood) can noticeably reduce serious long-term diabetic complications. However, glycemic control cannot be achieved by using current neutral protamine Hagedorn (NPH) insulin because of its short duration of action and rapid release profile.
Methods: Various peptides were designed and synthesized based on sequences of natural protamine. Insulin crystals were prepared in the presence of peptides and zinc irons. Particle size, zeta potential, morphology, and thermal stability of insulin crystals were examined using particle sizer, electron microscopy, and differential scanning calorimetry. Insulin release kinetics from various insulin crystals were measured in vitro using reverse-phase HPLC.
Results: Insulin crystals of narrow size distribution were prepared. Thermal stability of insulin crystal was determined by the sequence of peptides used. Various insulin crystals showed different insulin dissolution rates and profiles. Co-crystals of insulin formed using two or three different peptides could release insulin in a relatively constant rate in vitro.
Conclusion: Sustained release and long-acting insulin can be prepared to achieve tight glycemic control. This project was supported by Technogenesis Inc.
Display contrast changes with viewing-angle in medical AMLCDs: measurement methods and effect on observer detection performance
S-C. Lo, Barr Laboratories, Inc., Pomona, NY
Background: Multivariate data analysis has evolved rapidly and has been broadly implemented in various Process Analytical Technology (PAT) projects. A good process decision relies on correct and validated results from the multivariate data analysis of a PAT measurement system. Therefore, the overall validation issues of software and method development require special attention by PAT developer and users.
Methods: For the assessment of the correctness of the data analysis software, we applied several standard data sets (content uniformity data) to evaluate the outputs of Principal Component Analysis (PCA), Partial Least Squares regression (PLS) using various spectral pre-processing techniques. Various software packages, including Pirouette (v10), Unscrambler (v8.1), PLS-IQ added-in for GRAMS/AI v7, MATALB PLS_Toolbox (v1.5 & 2.0), SIMCA-P (v10), FOSS NIRSystems VISION (v3.20), and Bruker OPUS Quant (v5.0) were investigated. For the PAT method validation, the general elements including the linearity/range, accuracy, specificity, precision, robustness and performance verification were reviewed and compared on both quantitative and qualitative models.
Results: Almost all the software generated identical results (i.e., eigenvalues, loadings, scores, and predicted values) when using the PLS regression on raw spectra, with mean-center and SNV pre-processing methods. However, it was found that some packages generated different PLS predictions when using the Savitzky-Golay derivatives as a pre-processing method. The prediction difference is attributed to the incorrect use of the elimination of data points on each end, during the PLS regression. As reviewed the elements of method validation, the development of qualitative/quantitative technique requires clear understanding of the concept of multivariate data analysis. The model-free methods such as moving-block spectral average for process end-point test need to focus on the reproducibility and sensitivity of the prediction.
Conclusions: From this work, we have learned that the development and application of appropriate multivariate data analysis is much more difficult than we thought. The first observation is that the multivariate data analysis software packages are not fully interchangeable. Therefore, additional algorithm verification should be included in the process of software validation. The second observation is the fact that the users need to pre-define the PAT method validation plan during the development stage and fully understand the functions and limitations of multivariate data analysis.
Process Analytical Technology: FDA Research Activities
R. C. Lyon1 , C. D. Ellison1 , E. H. Jefferson1 , M. A. Khan1 , J. A. Spencer2 , W. H. Doub2 , L. F. Buhse2 , C. Watts3 , H. Wu3 , A. S. Hussain3 , 1DPQR, CDER, FDA, Silver Spring, MD, 2DPA, CDER, FDA, St. Louis, MO, 3OPS, CDER, FDA, Rockville, MD
Background: The FDA's PAT regulatory framework defines PAT as a system for designing, analyzing, and controlling manufacturing through timely measurements (i.e., during processing) of critical quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring final product quality. The FDA's PAT research program is intended to support the implementation and improvement of the PAT guidance. Current laboratory projects are evaluating potential PAT tools for their ability to reliably: 1) monitor physical and chemical attributes of pharmaceutical raw materials and processed materials; 2) provide process understanding and 3) predict process and product performance.
Methods: The presentation highlights PAT applications of several sensor technologies including near-infrared (NIR) spectroscopy, Raman spectroscopy, terahertz spectroscopy, NIR chemical imaging and Raman chemical imaging. These tools have been used at the FDA to study pharmaceutical raw materials, processed blends and finished dosage forms.
Results: Examples illustrate the characterization of both chemical (active content, impurities, blend uniformity) and physical properties (hydration state, particle size) of pharmaceutical materials. Measuring these attributes can provide an opportunity to understand how such characteristics evolve during processing and provide the prospect of quantitatively relating this understanding to product quality and performance.
Conclusions: The overriding goal of PAT is to understand and control the manufacturing process. As more knowledge is obtained and sufficient understanding is achieved, it could then be possible to establish causal and/or predictive relationships between the incoming raw materials, manufacturing process, in-process materials, and final product quality, which could be used for real-time process control.
Monitoring Drug Product Stability by Near-Infrared Chemical Imaging
R. C. Lyon1 , C. D. Ellison1 , E. H. Jefferson1 , E. N. Lewis2 , E. Lee1 , J. K. Drennen III3 , A. S. Hussain4 , 1DPQR, CDER, FDA, Silver Spring, MD, 2Spectral Dimensions Inc., Olney, MD, 3Duquesne University Center for Pharmaceutical Technology, Pittsburgh, PA, 4OPS, CDER, FDA, Rockville, MD
Purpose. This study assessed the feasibility of monitoring the stability of commercial and experimental pharmaceutical products by near-infrared (NIR) chemical imaging. This study is part of a comprehensive program to evaluate current sensor technologies, data analysis and regulatory approaches associated with Process Analytical Technology.
Methods: Reflectance NIR spectra and images were collected using a Sapphire (Spectral Dimensions) NIR chemical imaging system. Commercial theophylline tablets were evaluated before and after exposure to high humidity (90%) in a stability chamber (25oC) for a period up to 10 days.
Results. Exposure to high humidity resulted in the hydration of the active (theophylline) and certain excipients (e.g. lactose, povidone, hydroxypropylmethylcellulose (HPMC)) found in the commercial products. Strong NIR bands at 1972, 1934, 1920 and 1932 nm characterize theophylline monohydrate, lactose monohydrate, hydrated HPMC and hydrated povidone, respectively. Due to spectral overlap, it is difficult to determine which component has been hydrated using traditional NIR spectroscopy. Since chemical imaging provides spatial resolution, the stability profile of each component could be determined. In the most complex product, the hydration of theophylline and the hydration of lactose in the tablet base were distinguished from hydration of theophylline in the embedded, coated beads.
Conclusions. This study demonstrates that NIR chemical imaging is useful for monitoring drug product stability and for root cause analysis. Based on spatial discrimination, this approach can help identify changes in the hydration of individual components within the product. Future studies will determine if these changes in physical stability are responsible for product performance failure.
Rapid Development of a Fragmented Antigen Binding (Fab) Antibody Production Process using Retentate Chromatography - Surface Enhanced Laser Desorption/Ionization - Mass Spectrometry (RC-SELDI-MS)
J. T. Park1 , L. Bradbury2 , F. J. Kragl3 , J. J. Valdes1 , 1US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, 2Ciphergen Biosystems, Inc., Fremont, CA, 3Geo-Centers Inc., APG, MD
Background: An antibody against botulinum toxin (bt-Fab), produced by the bacterium Clostridium botulinum, was cloned in E. coli. The use of bt-Fab specific for botulinum toxin in a biowarfare agent assay requires maintenance of a high quality and economic supply of this critical agent.
Methods: An integrated approach utilizing RC-SELDI-MS and a two-factorial statistical design was employed for rapid development of fermentation conditions and chromatographic purification processes for the bt-Fab production.
Results: A quantitative RC-SELDI-MS was developed using immobilized metal affinity chromatography (IMAC) ProteinChip. The linear range of bt-Fab was found to be 25-200µg/ml. To optimize fermentation conditions, five variables (media type, [glycerol], post-induction temperature, [induction agent], and harvest time) were statistically combined for 16 fermentation conditions using an experimental 25-1 fractional factorial design and were tested for their effects on maximal bt-Fab production using a quantitative RC-SELDI-MS. When the effects of individual variables and their interactions were assessed, media type and post-induction temperature showed statistically significant effects (P<0.05) on the bt-Fab fermentation. The RC-SELDI-MS was also applied to screen various chromatographic adsorbents, using hydrophobic interaction, cationic exchange, and anionic exchange chromatographies (AEC) and IMAC ProteinChips, for bt-Fab purification. We found that bt-Fab in culture lysate can be purified by IMAC followed by AEC steps.
Conclusions: A generic production platform including fermentation conditions and two-step chromatographic process (IMAC-AEC) was developed for bt-Fab production. We also found that RC-SELDI-MS is an invaluable analytical tool (for Process Analytical Technology) to identify, track, and quantify in-process control samples during biopharmaceutical manufacturing process.
Development of Quantitative In-Process Control Affinity HPLC Assays for Production of Four Different Classes of Antibodies: IgG, IgM, IgY, and Fab
J. T. Park1 , F. J. Kragl2 , A. M. Arasteh1 , D. E. Menking1 , J. J. Valdes1 , 1U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, 2Geo-Centers Inc., APG, MD
Background: "Guidance for Industry PAT" recently announced by FDA recommended that the production and purification of therapeutical proteins to be monitored to confirm that processes are functioning within defined boundaries. To apply this we have developed quantitative immunoaffinity HPLC assays, which have desirable characteristics (sensitivity, specificity, precision, reproducibility, speed, and automation), for monitoring antibody production.
Methods: Goat anti-mouse IgG (IgM specific), goat anti-mouse IgG (Fab specific), and rabbit anti-chicken IgG (IgY specific) were covalently linked onto Sepharose resin, respectively. Protein-A and protein-G resins were also obtained for detection of various subtypes of IgG. Validations of these affinity HPLC assays were performed to establish the linearity, precision, accuracy, limit of quantification (LOQ), and specificity. These HPLC assays then were employed for monitoring samples obtained from mammalian culture media, animal sera, fermentation culture lysate, and downstream purification processes.
Results: The affinity HPLC assays developed were found to be acceptable for linearity, precision, accuracy, specificity, and LOQ in the linear range (e.g., 0.05 - 0.8mg/ml for Protein-A). Various in-process samples obtained from mammalian culture media for IgG and IgM production, microbial fermentation culture lysate for Fab production, and chicken sera from immunized chicken for IgY production were analyzed quantitatively by these HPLC assays. In-process samples obtained during downstream chromatographic purification were also analyzed to measure quantity, purity, and yields.
Conclusions: The affinity HPLC assays are invaluable analytical tools for monitoring antibody production and may be modified for continuously monitoring secreted antibodies by automatically drawing samples from bioreactors.
Application of Process Analytical Technology (PAT) To a Commercial Solid Oral Dosage Form
D. N. Shah, D. A. Radspinner, D. Schreiber, Sanofi-Aventis
a. Purpose: The purpose is to share a systematic technical and regulatory roadmap employed by Sanofi-Aventis to successfully apply PAT to enhance the pharmaceutical technology aspects for one of its commercial product. To review and discuss the lessons learned so that PAT could be applied to future products.
b. Methods: An immediate-release solid oral dosage form was selected for this application PAT. The manufacturing scientists identified critical manufacturing steps (e.g. impurity level in the drug substance, specific surface area of the drug substance, moisture content in the tablet granulation at the end of drying, assay and content uniformity of the tablets, etc.) in the manufacture of this drug product. Appropriate PAT tools (e.g. NIR, etc.) were identified for these critical manufacturing steps and preliminary data were generated. A Comparability Protocol was developed which provided the details of the planned PAT-based changes and submitted to the PATRIOT group at the FDA.
c. Results: The Comparability Protocol for the drug substance and drug product was approved by the agency. The company is in the process of making PAT-based changes using this approved Protocol. The details of this approach will be shared in this Poster.
d. Conclusions: Sanofi-Aventis concludes from this first experience in application of PAT that it is a valuable tool, which can be quite useful in optimization of manufacturing cycle of a product, realize real-time release, etc.
Tablet Content using Terahertz Transmission Spectrometry
J. A. Spencer1 , L. F. Buhse1 , A. S. Hussain2 , P. F. Taday3 , D. A. Newnham3 , 1DPA, St. Louis, MO 63101, 2OPS, Rockville, MD 20852, 3TeraView Ltd, Cambridge, CB4 0WG, UK
Background: Practical Terahertz spectrometry has become a reality in the past few years with the introduction of a commercial device that enables non-destructive study of solid samples such as tablets. In this study the content and compression force of a finished dosage form is modeled using chemometrics based on terahertz transmission (approximately 3-300cm-1).
Methods: Acetaminophen tablets were purposely made with nine dosage levels (65 - 135% of label) and three compressions. Terahertz transmission spectra were scanned with a TeraView terahertz pulse spectrometer. An empty beam spectrum was used to calculate individual absorbance spectra for each tablet. Five tablets of each dosage and compression were scanned totaling 135 measurements. Chemometric multivariate analysis was performed with Partial Least Squares (PLS). Various data preprocessing and spectral range limited calculations were tried. Results were evaluated for the three compressions using the predicted vs. concentrations determined from their NIR spectra using a calibration by HPLC analysis.
Results: The terahertz absorbance spectra were featureless. Absorbance exceeded 4.0 at energies above ~45cm-1, which limited the multivariate analysis the range 5 - 45cm-1. PLS was found to be a useful chemometric modeling tool. Mean correlation coefficient for the calculated dosage level in the selected spectral range is ~0.9. PLS models incorporating both dosage and compression data gave lower precision than corresponding models using each of the three compressions individually.
Conclusions: Terahertz transmission spectrometry is able to determine dosage level in formulated acetaminophen tablets made at a single compression with relative standard deviation of <10% using a Partial Least Squares model.
An Integrated Modeling Approach for Studying Coated Tablet Dissolution Process
H. Wu1 , A. S. Hussain1 , M. A. Khan2 , R. C. Lyon2 , R. Voytilla3 , J. K. Drennen3 , 1CDER, Rockville, MD, 2CDER, Silver Spring, MD, 3DCPT at Duquesne University, Pittsburgh, PA
Background: In the current modeling practice of PAT domain, much attention has been paid to the multivariate modeling due to its capability of relating on-line measurement of quality attributes of final dosage forms to some of the formulation and processing parameters. While this may be exciting to the pharmaceutical community due to its convenience and non-destructiveness, the drawbacks of the soft modeling approach are easily overlooked. If not supported by other complimentary modeling approach, soft modeling approach alone could give misleading directions and the risk of extrapolating model for process control could be a reality.
Methods: An integrated modeling approach for studying coated tablet dissolution process was proposed. Soft modeling was applied to relate the tablet NIR spectroscopy data to the coated tablet dissolution data, without considering the complicated physical/chemical phenomena associated with the dissolution process. Then mechanistic modeling was applied to study the coated tablet dissolution process kinetics, taking account of the mass transfer phenomena.
Results: NIR predictions were linearly related to percent released across all studied formulations. The calibration correlation coefficients ranged from 0.87 to 0.99. The prediction errors from independent validation samples were <10% in most cases. However, the prediction quality degrades as dissolution time increases. A linear relationship was observed between the initial dissolution rates and coating levels, indicating Fickian diffusion may dominate the coated tablet dissolution process.
Conclusions: An integrated modeling approach may provide insights about the dissolution process behavior of coated tablets, thus a more reliable process control scheme becomes possible for PAT application.
The segregation propensity of formulations with different drug to excipient particle size ratios in capsule filling performance
L. Xie1 , H. Wu2 , L. L. Augusburger1 , A. S. Hussain2 , S. W. Hoag1 , 1School of Pharmacy, University of Maryland, 20 N. Pine Street, Baltimore, MD 21201, 2CDER, FDA, Rockville, MD 20852
Objective: Capsule formulations with different drug to excipient particle size ratios were examined for segregation propensity.
Methods: Four capsule formulations consisted of 4% aspirin (ASP) (fine or coarse) in combination with 96% microcrystalline cellulose (fine PH-301 or coarse Avicel® PH-200). Capsules were filled using a Zanasi LZ-64 automatic filling machine; 10 capsule samples were collected every 10 minutes throughout the production run. Segregation propensity was measured using the ASTM D 6940 - 04 segregation test method. Particle size and powder flow properties were analyzed by sonic sifter and shear cell. The capsule coefficient of variation (CV) for weight and content were determined for each interval (CVi) and all the intervals combined (CVt).
Results: For weight, the CVi% varied from 0.93% (PH-301+ fine ASP) to 1.46% (PH-200+ coarse ASP). Content uniformity (CVi %) varied from 1.48% (PH-301+ fine ASP) to 13.85% (PH200 + coarse ASP). Weight variation (CVi) of capsules filled with only Avicel® PH-200 varied from 0.9% to 1.6%, and the (CVt) for empty capsule shells was 1.20%. For PH-200, the ASTM segregation test L/F ratio was 0.33 and 0.75 for the coarse ASP and fine ASP, respectively.
Conclusions: Data analysis shows the most significant differences in the weight variation were between PH-301 and PH-200 (p<0.05). For content uniformity, the most significant difference was between fine ASP PH-200 and fine ASP PH-301 (p<0.05). These results are in excellent agreement ASTM segregation test data, which indicates this method has excellent potential to predict weight and content uniformity problems in capsule formulations.
Preclinical Development of Folate Liposomal Doxorubicin
X. B. Zhao, Z. Yang, X. Cao, R. J. Lee, Division of Pharmaceutics, College of Pharmacy, The Ohio State University
Background: Liposomal drug has increasingly been evaluated in clinical trials. Folate liposomal doxorubicin (FLD) has been developed with the intent of treating various malignancies overexpressing folate receptors. Compared with traditional non-targeted formulations, this targeted liposomal drug incorporates many of the inherent advantages of liposomal formulation. In addition, it also improves the therapeutic index by introducing folate receptor-based tumor-selective delivery of the anticancer agent.
Methods: The formulation of FLD was made cost-effective by applying a new composition, incorporating HSPC, cholesterol, folate-PEG-cholesterol, and PEG-cholesterol. A novel processing method of liposome production combining solvent exchange, ultrafiltration, high-pressure homogenization and remote loading was designed and validated for the reliable and scalable production of FLD. In vitro drug release experiment and in vivo pharmacokinetics study were conducted to establish the release profile. Therapeutic effect was evaluated both in a KB oral carcinoma murine xenograft model and an acute myelogenous leukemia model.
Results: Cholesterol as an anchor for PEGlation and folate receptor targeting presented sufficient in vitro stability. However, the in vivo circulation of these vesicles was less than those formulated with DSPE. 3 batches of 400mg (20 vials) doxorubicin each were formulated into sterile folate liposomal suspension, with following parameters: mean particle size 120nm, size distribution 30nm, pH 7.2, drug concentration 2.1mg/mL and encapsulation efficiency 97.3%. In mouse ascites leukemia models generated using L1210JF or KG-1 cells, increase median survival times were obtained with FLD compared to nontargeted formulation. In KB oral carcinoma murine xenograft model, FLD also exhibited greater tumor growth inhibition and a 31% higher increase in lifespan compared to those received control non-targeted liposomal doxorubicin.
Conclusions: The above data from preformulation development, process scaling up and animal studies support further development of this targeted liposomal formulation. With preclinical development approaching completion, we are currently aiming to move FLD rapidly into clinical trial.
Novel Nanopore Structured Glucose Biosensors Promote Reagentless Glucose Concentration Measurements in the Hypoglycemic Range
E. T. Chen1 , J. Thornton2 , 1The Biotechnology Laboratory, the Division of Biology and the Division of Chemistry and Materials Science, Office of Science and Engineering Laboratories, New Hampshire Ave, Silver Spring, MD 20903; Twinbrook Pkwy, Rockville, MD 20852, 2Veeco Metrology Group, Chadds Ford, PA 19317
Background: More than 16 million individuals have diabetes mellitus in the Unites States. Type I diabetes, especially children, are heavily dependent on accurate blood glucose monitoring in the hypoglycemic range. This paper presents a method to build new generation glucose devices that promote direct reagentless glucose measurements based on direct electron transfer (DET) between the nanopore structured biomimetic enzyme (BMZ) self-assembling membrane (SAM) and the electrode. The glucose concentrations are proportional to the measured current.
Methods: Two gold glucose biosensors with nanopore structured SAMs were compared with a gold electrode without a nanopore SAM. Atomic Force Microscopy (AFM) was used to characterize the surface roughness. Cyclic Voltammetry (CV) was used to characterize the DET and monitor the current intensity. Effects of pH on signals were studied.
Results: AFM confirmed the highly ordered appearance of nanopore structures with an average internal nanopore diameter of 19.5 nm (RSM value was 0.55 nm). The DET was observed for both electrodes, not for the control. Linear regression of current vs. glucose concentrations for the new biosensor produced a correlation coefficient of 0.997 in the range from 10 to 205 mg/dL (n=8) and 0.998 from 10 to 90 mg/dL (n=5). The precision at the clinical decision point of 50 mg/dL was 1.4% (n=5).
Conclusion: Nanopore structured SAMs promote DET and make possible reagentless measurements for glucose without the need for adding any native glucose enzymes. Therefore, Nanopore structured SAMs enhanced the glucose sensor performance in the hypoglycemic range and generate less contamination to the environment.
Reducing Risk with Medical Devices Used in the Home
S. N. Gardner, H. B. Albersheim, S. L. Berman, M. W. Brady, J. U. Cope, M. Eakle, P. L. Jahnes, P. L. Jones, A. Pinkos, M. Warner, M. A. Wollerton, A. A. Ciarkowski, Home Health Care Committee
Over the past several years, the use of medical devices in the home has expanded. This trend puts an additional burden on the manufacturer. At one time, it was a safe assumption to design medical devices for trained professionals. Driving forces in the health care industry, however, are shifting the practice and site of patient care. Patients are spending fewer days in a hospital and many procedures are done in clinics. Patients are now expected to continue their care or recovery at home. The impact of this trend is for patients to go home with the medical devices that are part of their care (such as infusion pumps, catheters, oxygen, respirators). Designing a device only for professional use is no longer a valid assumption. Manufacturers need to assess the risks of their device as used in the intended environment, and especially in the home environment where there may be untrained caregivers.
To assess the broad set of risks and recommend procedures to reduce risk, CDRH analyzed MAUDE adverse reports for typical home use devices. The reports were classified into broad risk categories: device performance, hardware, software, device labeling, and use error. Data were viewed from the year 2000 through 2005. The data analysis showed similar risk profiles for devices typically used in a home environment. In reviewing the data, however, there were cases that fell outside of these risk categories. These cases illustrate that good design, excellence in manufacturing, and plain English labeling are not enough to address some of the risks that occur in the home environment. Some examples include:
To reduce risks in these areas, CDRH recommends that medical device manufacturers assess the risks of their devices in the environment where they are intended use. CDRH stresses the need to assess the risk of medical device use in the home. To reduce risks, CDRH encourages manufacturer to consider the following:
In addition the CDRH Home Health Care Committee has prepared a Home Checklist brochure. This checklist will help reduce the unusual adverse circumstances that could arise in the home environment.
Unexpected Genetic Alterations Occur in Nontargeted Regions During the Construction of Viral Vectors
M. J. Dambach, J. Trecki, N. S. Markovitz, CBER, FDA, Bethesda, MD
Background: G207 is a herpes simplex virus vector in clinical trials for the experimental treatment of cancer. G207 and MGH were both made from the R3616 virus that lacks the γ34.5 gene encoding the neurovirulence factor. R3616 was produced from R4002 via the intermediate virus R3617. The UL3 protein is encoded by a gene roughly 11Kbp from γ34.5, and was not intentionally modified during the construction of R3616. However, UL3 from R3616 infected cells migrates faster on SDS-PAGE than UL3 from wild-type infected cells, suggesting unintentional modification.
Methods: We amplified by PCR and then sequenced the UL3 open reading frame from DNA of cells infected with R3616 or other relevant viruses.
Results: Sequence analysis indicated the presence of an in-frame stop codon in the UL3 gene of R3616 that is predicted to encode a truncated UL3 protein, consistent with the increased migration rate. This mutation was present in R3617 and MGH but not in the R3617 progenitor virus R4002.
Conclusion: Our results indicate that large DNA viruses such as HSV can acquire mutations at non-targeted regions during their construction, and suggest that other mutations may exist that either have not been identified or published in the literature. Currently, FDA requires sequencing of only intentionally modified regions of viral vectors larger than 40 kb (e.g. HSV-based vectors) prior to their use in clinical trials. This is one example why sequencing only the intentionally modified regions of large viruses may not provide adequate information on the genotype and phenotype of the virus.
Shielding Small-Field High-Energy Electron Beams in Cancer Treatment
M. Farahani1 , F. C. Eichmiller2 , W. L. McLaughlin3 , 1CDER, FDA, Rockville, MD, 2ADA, NIST, Gaithersburg, MD, 3NIST, Gaithersburg, MD
Background: Conventional preparation of custom anatomical prosthetic radiation shields, which are usually metal alloy masks, has been time-consuming and uncomfortable for the patients. The purpose of this study was to find an effective material that can be prepared quickly and easily prior to small-field electron- beam treatments so that lesions of the head and neck can be treated with minimal irradiation of the surrounding healthy tissue.
Method: New materials, made from light-body ReprosilTM
(L.L. Caulk) filled with fine metal powder consisting of 70% Ag - 30% Cu alloy, can be made by blending 90% (w/w) metal powder with 10% polysiloxane base and adding the polymerization catalyst separately. These combinations were mixed to form comfortably fitted shielding composites of different thicknesses. The electron-beam attenuation properties of slabs of this material were studied by irradiating calibrated Radiochromic film (GafchromicJ) dosimeters behind different thicknesses of composite samples with small-field 13-, 15- and 18-MeV electron beams from a therapeutic linear accelerator.
Results: The results showed that this material can suitably attenuate high-energy electron beams when used in reasonable thicknesses.
Conclusion: The shield thicknesses in the range of 10-14 mm or greater will attenuate 13-18 MeV electron beams to within about 10% or less. Such shields would significantly protect surrounding healthy tissue during small-field radiation therapy with high-energy electron beams.
Development of a FACS based potency assay for immuno-modulatory therapeutics using phospho-specific antibodies
L. J. Graham1 , C. Serrano1 , I. Reischl1 , K. D. DeBell1 , E. Bonvini2 , B. L. Rellahan1 , 1CDER, FDA, Bethesda, MD, 2Macrogenics, Rockville MD
Background: The Division of Monoclonal Antibodies regulates products aimed at modulating lymphocyte activity for the treatment/prevention of graft rejection, autoimmune diseases and cancer. The development and validation of potency assays that accurately reflect the mechanism of activation of such therapeutics is problematic due to the inherent variability of cell based assay systems. The recent development of phospho-specific antibodies raises the possibility that these reagents could be used to quantitate the activity of immuno-modulatory therapeutics in a sensitive, robust assay.
Methods: Site-specific mutations, transient and stable transfections, calcium-mobilization, reporter gene assays, immunoblotting, and fluorescence-activated cell sorter (FACS) techniques were used to identify and characterize phospholipase Cγ1 (PLCγ1) phosphorylation.
Results: The activation of PLCγ1 mediates critical events required for lymphocyte activation. The activity of PLCγ is regulated in part through its tyrosine(Y) phosphorylation. PLCγ1 tyrosine phosphorylation can therefore be used as an indicator of lymphocyte activation status. Our laboratory identified Y775 as a new phosphorylation site required for PLCγ1 activation and confirmed the role of Y783. While mutation of Y775 and Y783 abolished PLCγ1 activation events, mutation of other tyrosine residues had no effect on PLCγ1 activation. These data indicate that Y775 and Y783 are required and sufficient for PLCγ1 activation. A phospho-specific anti-pY775 antibody developed in our laboratory indicates that phosphorylation of Y775 could be used to sensitively quantitate the activation of PLCγ1 by antigen and co-stimulatory receptors.
Conclusion: These data suggest that FACS based assays using phospho-specific antibodies could be developed and validated as potency assays for immuno-modulatory therapeutics.
Carotid Stenting: Real-World Experience during the First Six Months of Marketing.
A. D. Holton, S. E. Rich, T. P. Gross, OSB
Background
Guidant's Carotid Stent System, the first of its kind to receive FDA premarket approval, is used to treat diseased carotid vessels by dilating the lumen to restore blood flow. The device was approved for patients who have had symptoms of stroke or have a carotid artery at least 80 percent blocked, and are not good candidates for surgical repair. The RX Acculink stent is used in conjunction with the RX Accunet carotid embolic protection filter.
Methods
The Manufacturer and User Facility Device Experience (MAUDE) database was queried using manufacturer name (Guidant), and device brand name (RX Acculink and RX Accunet). The analysis covers the time period for reports received since the date of device approval, August 31, 2004 through January 26, 2005. Duplicate reports were eliminated by comparing event date, implant date, patient descriptors and narrative event descriptions.
Results
Of the 33 reports, 32 were submitted by the firm and 1 by a hospital. Individual review of each of the 33 reports identified 28 patient events associated with the carotid stent system. Of these, 5 involve events of death and 23 events of injury, primarily associated with stroke and transient ischemic attack (TIA).
Conclusions
Reported events of stroke and TIA associated with death and serious injury are expected, based on the pre-market experience and are consistent with device labeling, Although expected, the occurrence of these events with this first of its kind device indicates a need for continued monitoring in the larger post-market patient population.
Application of Nanocapsules in Drug Delivery
J. E. Simmons, H. Patel, Y. A.. Hsieh, J. Z. Duan, CDER, FDA, Rockville, MD
Drug therapies affect the body at the molecular level. Most drugs are dispersed throughout the body, rather than targeting to the specific area where they exert an effect, as a result, pharmaceutical effects in other tissues are unavoidable. Targeting drugs using suitable carriers protects them and guides them to specific tissues. A nanocapsule is a molecular level shell with a space into which drugs may be loaded. Nanocapsules were initially developed for the delivery of vaccines and anticancer drugs. Nano- encapsulated drugs can improve drug delivery. They are introduced into the bloodstream by intravenous injection, however, little is known about their kinetics and distribution throughout the vasculature. Covalent attachment of antibodies can allow selective targeting of the drug loaded-nanoparticles. The ability to target particular cell or tissue types and to modulate the release of drugs in response to cellular signals could allow specific interventions into disease pathways while minimizing side effects. This capability may enhance the efficacy and speed of action of a wide variety of therapeutic agents regardless of their aqueous solubility and lipophilicity.
This poster presentation provides a brief overview of the unique features of nanocapsule technology, methods of preparation and drug encapsulation, design of site-specific "smart" nanocapsules and their application in drug delivery including ophthalmic and oral delivery, drug release in vitro and in vivo and nanocapsule stability. Relevant regulatory issues, such as manufacturing and controls, optimal dosing, drug interaction and special population dosing adjustment will be discussed.
A natural anticancer plant drug, b-elemene, sensitizes resistant ovarian carcinoma cells to cisplatin-induced apoptosis
X. Li, G. Wang, J. Zhao, H. Ding, D. C.. Flynn, E. Reed, Q. Q.. Li, West Virginia University, Morgantown, WV
Background: Epithelial ovarian cancer (EOC) is the second most common malignancy of the female genital tract. Resistance to chemotherapeutic drugs is a hallmark of many human cancers including EOC. Therefore, overcoming drug resistance is the key to successful treatment of ovarian cancer. β-Elemene, a new antitumor agent, has multiple anticancer properties and was found to inhibit proliferation, stimulate apoptosis, and induce cell cycle arrest in malignant cells. Our recent studies have shown that β-elemene increases cisplatin cytotoxicity and enhances cisplatin sensitivity in resistant human ovarian carcinoma cells. However, the mechanism underlying this effect of β-elemene is not understood.
Methods: Using several ovarian carcinoma cell lines as models for ovarian cancer, we investigated whether β-elemene augments cisplatin activity in ovarian tumor cells through induction of apoptosis by five different apoptosis assays.
Results: Our study showed that β-elemene triggered apoptotic cell death in cisplatin-resistant ovarian cancer A2780/CP cells in a time- and dose-dependent manner. Morever, our results demonstrated that β-elemene exerted a stronger action in inducing apoptosis in the resistant cells as compared to cisplatin, and a synergistic effect in induction of cell death was seen when cells were treated with both agents. Finally, our results indicated that β-elemene induced increases in caspase-9 activity, but down-regulated the protein expression of Bcl-2 and Bcl-XL in the cisplatin-resistant ovarian cancer cells. β-elemene also reduced the mitochondrial transmembrane potential, and increased the release of cytochrome c into the cytoplasm.
Conclusion: These data indicate that β-elemene strongly sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced apoptosis through a mitochondria-dependent intrinsic cell death pathway.
Beta-elemene, a novel plant antineoplastic agent, enhances chemosensitivity of lung cancer cells through triggering apoptosis
G. Wang, X. Li, J. Zhao, H. Ding, D. C.. Flynn, E. Reed, Q. Q.. Li, MBRCC, West Virginia University, Morgantown, WV
Background: For more than two decades, the most effective systemic chemotherapy for non-small cell lung cancer (NSCLC) was based on cisplatin combinations. However, currently available cisplatin-based therapy for lung cancer is only a narrow therapeutic window between efficacy and unacceptable toxicity. Therefore, new approaches for treatment of this disease need to be pursued.
Methods: Using several NSCLC cell lines as models for lung cancer, we assessed the ability of beta-elemene to augment cisplatin activity as well as the underlying mechanisms in NSCLC cells by five different apoptosis assays and other technology.
Results: Our results showed that beta-elemene enhanced inhibitory effects of cisplatin on cell growth in the human NSCLC cell lines H460 and A549. Beta-elemene was found to augment cisplatin-induced G2-M phase arrest in these cells, which was associated with increases in expression of the checkpoint kinase (Chk2) and decreases in protein levels of cyclin B1 and Cdc25C. We also demonstrated a synergistic effect of beta-elemene with cisplatin on triggering apoptosis in our cell models. This is associated with increased expression of pro-apoptotic protein Bax and decreased expression of anti-apoptotic protein Bcl-2, which lead to the release of cytochrome c from mitochondria, and reduced the protein level of XIAP, a vital apoptotic inhibitor targeting caspase-3, -7, and -9 specifically. Furthermore, beta-elemene promoted cisplatin-induced increases in caspase-9, -3 and -7 activities, as well as in the levels of cleaved caspase-9, -3 and PARP in H460 cells.
Conclusions: These observations indicate that beta-elemene promotes cisplatin-induced cell death in NSCLC and that the effect of beta-elemene is mediated by a mitochondria-dependent apoptotic pathway. These studies provide evidence to exploit the combination of beta-elemene and cisplatin as a potentially effective chemotherapy regimen for treatment of patients with resistant lung cancer.
In vitro combination characterization of beta-elemene with taxanes against human lung cancers
J. Zhao, G. Wang, X. Li, H. Ding, J. E.. Kim, B. Zou, D. C.. Flynn, E. Reed, Q. Q.. Li, MBRCC, West Virginia University, Morgantown, WV
Background: Combination effects of drugs on cytotoxic action include synergistic, additive, or antagonistic. Paclitaxel and docetaxel are two of the most widely used drugs for lung cancer chemotherapy in recent years. However, they have been associated with side effect and drug resistance when used as single agents that often limit their use in the clinic. Beta-elemene, a new anticancer drug isolated from the Chinese medicinal herb Zedoary, possesses antitumor activity in human and murine tumor cells, and may present different anticancer mechanisms and less side-effect to human body when compared with paclitaxel and docetaxel.
Methods: This study evaluated the combination effects of beta-elemene with paclitaxel or docetaxel against human lung cancers, using synergism analysis, micronucleus assay, mitosis arrest analysis, and apoptotic detection. Four human lung cancer cell lines (H460, A549, H23 and H358) were used in this study.
Results: The results of median effect analysis revealed that the interaction effects of beta-elemene with paclitaxel or docetaxel ranged from moderate synergism to additive. Similar combination characterizations were observed in photomicrograph, micronucleus assay, mitosis arrest analysis, and apoptosis induction detection. The mechanisms of apoptosis induced by beta-elemene and taxane may be through attacking different biochemical targets. Beta-elemene seems to trigger cell apoptosis by interaction with cell membrane at early stages. In contrast, taxanes tend to initiate the cell death cascades through nuclear interfering events. In addition, a significant p53 protein expression increment was found in taxane alone and combination treated cells, whereas no alteration in p53 protein expression was observed in cells treated with beta-elemene alone. Furthermore, the expression of the drug resistance-related gene Bcl-XL was strikingly downregulated when cells treated with the combination of beta-elemene and taxanes.
Conclusions: These observations suggest that the ability of combining beta-elemene and taxanes to achieve synergism is due to an increased cytotoxic action and a decreased expression of cell survival genes by the drugs.
Sequence-dependent effect of beta-elemene on 5-fluorouracil-induced cytotoxicity in cyclooxygenase-2-expressing colon cancer cells
H. Ding, G. Wang, X. Li, J. Zhao, D. C.. Flynn, E. Reed, Q. Q.. Li, MBR Cancer Center, West Virginia University, Morgantown, WV
Background: 5-Fluorouracil (5-FU) is the most commonly used drug for the treatment of colorectal cancer, but its effect is affected by cyclooxygenase-2 (COX-2) overexpression. Beta-elemene is a novel plant antitumor agent; however, its effect in colon cancer is not understood.
Methods and Results: We demonstrated that beta-elemene had a greater antitumor activity in colon cancer cells (Caco-2 and Colo-205) expressing endogenous COX-2 than in colon cancer cells (HCT-15 and HCT-116) lacking endogenous COX-2. Our further studies on the interactive effects of 5-Fu and beta-elemene in these cells showed that the combination of beta-elemene with 5-FU resulted in antagonistic effects in all of the four cell lines when concurrently treated with both drugs. Interestingly enough, sequential exposure of the cells to beta-elemene followed by 5-FU produced synergistic effects in Caco-2 and Colo-205 cells, but antagonistic effects in HCT-15 and HCT-116 cells. The induction of apoptosis in Caco-2 and Colo-205 cells, but not in HCT-15 and HCT-116 cells, was also significantly increased following sequential treatment, as determined by TUNEL assay and Annexin-V/PI double staining analysis. Consistent with these results, we found that the sequential treatment with beta-elemene and 5-FU also reduced the mitochondrial transmembrane potential, and increased the release of cytochrome c into the cytosol in Caco-2 and Colo-205 cells but not in HCT-15 and HCT-116 cells. Furthermore, we analyzed caspase activity by Western blotting, and our data revealed that the activation of caspase-3 and -9 was observed in Caco-2 and Colo-205 cells, but not in HCT-15 and HCT-116 cells, following sequential treatment with both agents. Finally, to assess the role of COX-2 in drug effect in colon cancer cells, HCT-15 cells were transfected with the COX-2 cDNA. The overexpression of COX-2 attenuated apoptosis induction by 5-Fu, but significantly increased apoptosis by sequential treatment.
Conclusion: These findings suggest that beta-elemene may enhance the effect of 5-Fu in colon cancer cells through a COX-2-dependent, mitochondria-mediated death pathway.
EVALUATING SELF-MICROEMULSIFYING DRUG DELIVERY SYSTEM (SMEDDS) FOR POORLY WATER-SOLUBLE DRUGS (I):THE OIL EFFECT ON SOLUBILIZING FLURBIPROFEN BY OIL-IN-WATER MICROEMULSIONS
P. Li, R. F. Wagner, H. Lee, A. Ghosh, A. Serajuddin, Novartis Pharmaceuticals
Purpose: This study is to evaluate the oil effect on the solubilization capacity of oil-in-water microemulsions consisting of different oil and surfactant compositions. Methods: Based on phase-diagram, a number of preconcentrates were selected and were emulsified into oil-in-water microemulsions. In these systems, the oil was either Capmul® PG8 or Captex® 8000, and the surfactant was a mixture of Tween® 20 / Cremophor® EL in constant 1/1 ratio (w/w). The emulsified solutions or the oil-in-water microemulsions were used as vehicles to evaluate their capacity in solubilizing poorly soluble drug flurbiprofen. The phase inversion temperature (PIT) of microemulsions of different compositions was also measured in relating to the oil effect.
Results: The solubility of drug flurbiprofen increased in the oil-in-water microemulsions as the preconcentrate percentage increased. The solubility increase was also found to be more significant when Capmul PG8 was used as the oil, as compared to when Captex 8000 was used as the oil. Similar results were obtained for different oil/surfactant ratios: 0/10, 1/9, 2/8, and 3/7 (w/w). The phenomenon was correlated to the effect the oil might have on the microemulsion structures: Capmul PG8, a single chain oil (propylene glycol monocaprylate), has a small molecular volume (calculated as 240 cm3/mole). This makes it possible for the oil to penetrate into the surfactant interfacial layer. While for Captex 8000, a capric triglyceride, it has a relatively large molecular volume (572 cm3/mole), and is primarily swelled in the oil center of the droplet. Investigation on microemulsions' phase inversion temperature (PIT) was found to be consistent with this explanation: the PIT decreased as the oil composition increased with the use of Capmul PG8, but being increased with the use of Captex 8000. Further studies on solubility comparison in microemulsions obtained experimentally and by estimation also lent support to different oil effect on microemulsion structure: the Capmul PG8 is primarily a penetrating effect while Captex 8000 is the swell effect.
Conclusions: This study indicates that oils of different molecular volume may have different effect on the oil-in-water microemulsion structures, and thus directly influence the drug flurbiprofen solubility. The finding has important implications in future optimising oil component in preconcetrate/microemulsion dosage development.
EVALUATION OF X-RAY IRRADIATED PNEUMOCOCCAL 7-VALENT CONJUGATES AND POLYVALENT PNEUMOCOCCAL VACCINE POLYSACCHARIDES AND X-RAY and GAMMA RAY IRRADIATED ACELLULAR PERTUSSIS COMPONENT OF DTaP VACCINE PRODUCTS
J. C. May1 , L. Rey2 , C. J.. Lee1 , J. Arciniega1 , 1CBER, FDA, Rockville, MD, 2Conseiller Scientifique,Chemin de Verdonnet 2, CH-1010, Lausanne, Switzerland
Samples of Pneumococcal Vaccine Polyvalent and Pneumococcal 7-valent Conjugate Vaccine were irradiated with X-rays. IgG and IgM antibody responses in mice (ELISA) are given for types 9V, 14, 18C and 19F pneumococcal polysaccharides and pneumococcal conjugates in the irradiated and corresponding control samples. Acellular pertussis vaccine antibody response in mice (ELISA) are presented for samples of 3 lots of two manufacturer's Diphtheria and Tetanus Toxoids and Acellular Pertussis Vaccine Adsorbed irradiated by gamma rays (25 kGy) at room temperature, and irradiated by X-rays (25 kGy and ~12 kGy) while frozen at liquid nitrogen temperature. The antibody response in mice was run for the detoxified pertussis toxin (PT) antigen and the filamentous hemagglutinin antigen (FHA) as well as PRN (pertactin) and FIM (fimbriae types 2 and 3) antigens for the appropriate vaccine type. The antibody response was not significantly changed in the X-ray irradiated vaccines frozen in liquid nitrogen compared to the control vaccines.
| Sigma Xi Outstanding Poster Award - 2005 FDA Science Forum | |
Breakage of Blood Bags Used Off-Label to Store Hematopoietic Stem Cells
M. K. McDermott, D. M. Saylor, C. N. Witkowski, C. M. Williams, T. M. Thomas, A. S. Lee, J. C. Hutter, CDRH, FDA, Rockville MD
Background: In hematopoetic stem cell (HSC) therapy, the patient's blood is removed and centrifuged to separate out HSCs. The HSCs are stored in polymer blood bags at -195°C. The patient then undergoes chemotherapy which reduces the white blood cell count (WBC) to near zero. HSCs are thawed and reintroduced into the patient to restore their WBC count. If a bag fractures during storage or rewarming, HSCs are lost; so fewer HSCs would be available for the patient, resulting in a lower WBC and increased risk of sepsis.
Methods: We identified potential causes of blood bag fracture, measured blood bag structure by scanning electron microscopy and cryogenic properties by dynamic mechanical analysis;and developed a mathematical model related to strain energy in blood bags to evaluate the mechanisms of fracture.
Results: Cracking occurred along welds in the blood bags where high stresses were calculated by computer modeling. Brittle delamination and cracking occurred in the presence of stress risers such as pores. Mechanical testing and the presence of brittle fracture surfaces indicated that the elastic bag material becomes brittle at low temperatures. Computer modeling suggests that the bag may fracture either during freezing when ice in the bag expands more than the bag or during thawing when gas expands and fractures the brittle bag.
Conclusions: A combination of cryogenic material properties, design and physical changes in the bag and its contents may contribute to blood bag fracture.
Fabrication Variability and Consistency of Controlled Release Technologies
M. K. McDermott, T. M. Thomas, A. S. Lee, C. N. Witkowski, D. M. Saylor, J. C. Hutter, FDA, CDRH, Rockville MD
Background: Conventional drug delivery by oral routes or other methods is often limited by metabolism or absorption, toxicity, or unsteady dosing dynamics. This may limit the effective dose to target organs. By incorporating drug in a polymer matrix, drug release at a controlled rate is possible. Recent advances in controlled release coatings for use on medical devices resulted in an increased interest in fabricating and testing these products to assure quality. R. Langer and others first demonstrated the link between structure and release rates in the early 1980's.
Methods: We set up a spray coating apparatus in our laboratory to fabricate different variations of controlled release coatings. We tested variations in spray pressure, flow, temperature, atmospheric conditions (partial pressures of solvents and humidity) in the chamber, composition, and post coating processing. The resulting coatings were characterized for structure and release kinetics. Numerical models of the coating process and release kinetics were developed.
Results: Control of structure of the coatings was possible depending on the fabrication method. Evaporation rates of the solvent, initial compositions, and temperature were found to be the critical parameters in the fabrication process.
Conclusions: Connected porous microstructure results in enhanced release rates relative to diffusion through a polymer film. These structures can be controlled by the conditions and post processing of the coatings.
| Clear Science Communication Award - 2005 FDA Science Forum |
Characterization of the Structure and Properties of Authentic and "Suspect Counterfeit" Polypropylene Surgical Meshes
M. K. McDermott, A. S. Lee, T. M. Thomas, I. S. Isayeva, A. D. Lucas, C. N. Witkowski, J. C. Hutter, FDA, CDRH, Rockville MD
Background: In 2003, patients undergoing tension free hernia repair were implanted with a "suspect counterfeit" polypropylene mesh. After discovering this, the FDA issued a public health notice recommending the removal of the mesh from the market place. Initial testing of the "suspect counterfeit" mesh indicated that the sterility was compromised.
Methods: Meshes from five different manufacturers were tested along with the "suspect counterfeit" product. We compared the structural, physical, chemical and mechanical properties as well as extractables and cytotoxicity of the "suspect counterfeit" mesh to polypropylene meshes previously cleared by FDA.
Results: Samples of the "suspect counterfeit" were found to be non-sterile. The mesh fibers for all products tested were found to have similar chemical and physical properties (e.g., molecular structure, crystallinity). The mechanical properties were related to the weave structure (e.g., loop size, repeat distance, fabric tightness) and porosity. The structure and mechanical properties of the "suspect counterfeit" mesh were in the range of other meshes cleared by FDA. Extracts from the "suspect counterfeit" mesh passed cytotoxicity screening tests.
Conclusions: The differences in the mechanical and chemical properties of the "suspect counterfeit" and the authentic meshes measured in this study were not significant. However, the safety of the "suspect counterfeit" was compromised because it was found to be non-sterile.
Observations on the Morphology and Growth of Microvacuoles in Foldable Intraocular Lenses
B. J. Dair, D. V. Patwardhan, L. W. Schroeder, D. C. Richardson, FDA/CDRH, ROCKVILLE, MD
Background: "Glistenings" in rigid intraocular lenses (IOLs) were first reported in '84. Since FDA approval of a foldable hydrophobic acrylic lens, microvacuoles have been confirmed in these materials. Our concern is that, in some patients over time, the vacuolar spatial density could increase to where light scattering impacts contrast sensitivity or night vision, necessitating IOL removal.
Methods: Vacuoles were induced in vitro in foldable IOLs by annealing at elevated temperatures in phosphate-buffered saline. Vacuole morphology was observed using differential interference contrast and polarized light microscopies. Liquid nuclear magnetic resonance (NMR) was used to confirm presence and type of water within IOLs. Accelerated time course experiments were conducted to study growth of vacuoles, while different saline concentrations were used to study the osmotic gradient effect.
Results: Vacuoles were observed, in this instance, to be flat discs, formed within the bulk of the IOL rather than at the surfaces, and randomly oriented. Polarized light microscopy reveals that the strain field of material around a vacuole is higher perpendicular to the plane of the vacuole. Vacuole size is found to depend on saline concentration and annealing time with an upper limit to both. Liquid NMR spectra are consistent with the presence of both free water, presumably within the vacuoles, and bound water within the bulk polymer.
Conclusions: Preliminary results support the osmotic gradient hypothesis of vacuolar growth and an intrinsic material driving force for vacuolar formation. Observed morphology and birefringence characteristics may be related to mechanisms of stress distribution within the material.
In Vitro Clustering of Microvacuoles in Foldable Intraocular Lenses
E. T. Chen, D. C. Richardson, FDA/CDRH, ROCKVILLE, MD
Background: "Glistenings" have been observed in intraocular lenses (IOLs) since 1984. The public health concern is: Will vacuoles continue to appear and enlarge after implantation until their frequency and spatial density induces clinically significant light scattering? As part of a collaborative effort to characterize microvacuoles and their development, this study focuses on the effect of the immersing medium choice on morphology and distribution after incubation and annealing.
Methods: Vacuoles were induced in vitro in foldable IOLs by incubation at elevated temperature, and controlled annealing, in deionized water (DI), buffered saline solution (BSS) or bovine serum. Vacuolar morphology was observed via differential interference contrast microscopy. After photodocumentation, samples were returned for additional incubation.
Results: As expected, incubating IOLs in DI leads to larger (up to 80%) median vacuolar diameters than in BSS, for the same duration. Vacuolar clustering is well established in serum after 14 days of incubation, with greater frequency than in BSS. Cluster diameter and frequency in serum after 21 days exceeded that for DI or BSS.
Conclusions: Preliminary results confirm the osmotic gradient hypothesis. Vacuolar clustering is worse in serum than in BSS or DI; both in time to onset and cluster density. This effect suggests a mechanism where patients with compromised eye-blood barrier, e.g., diabetes mellitus, would be at greater risk of visual disturbance, as compared with healthy patients.
Improving drug infusion safety with a microfluidic sensor
D. R. Sparks, N. Najafi, R. Smith, ISSYS
Background: Drug infusion errors are known to cause numerous injuries and deaths each year. The types of errors include improper dose, dose rate, volume, wrong drug type or concentration as well as problems due to IV line occlusions and air bubbles.
Methods: New micromachined devices have been developed that can accurately measure the mass flow rate, specific gravity and temperature of a fluid. This technology is being applied to the area of medication infusion.
Results: Testing of the micromachined Coriolis mass flow sensor developed at ISSYS indicates that volumetric flow rates of common intravenous solutions can be measured below 0.1mL/hr and IV line occlusions can be detected. At the same time the specific gravity of the drug can be measured with better than 3 digits of accuracy enabling the user to distinguish the majority of common IV solutions from each other and measure drug concentration. This function also enables air bubble detection. Coupled with a timing input the device can simultaneously measure dose, dose rate and volume infused.
Conclusions: ISSYS has developed and demonstrated new technology to improve the safety of mediation infusion. A single disposable chip can be used to monitor flow rate, dose, dose rate, volume infused, drug type, drug concentration and detect complete or partial occlusions and air bubbles. This technology is flexible enough to be used with virtually any conventional infusion product such as gravity IV bags, syringes and syringe, peristaltic and pressurized pumps and other liquid delivery systems to reduce the incidence of infusion errors.
EFFECT OF CYCLODEXTRINS ON THE PERMEABILITY OF MODEL DRUGS THROUGH CACO-2 CELL MONOLAYERS
D. A. Volpe, R. L. Hunt, DPQR/CDER/FDA, Silver Spring
Background: Cyclodextrin excipients may alter a drug's intestinal absorption and solubility properties. This study examines whether an in vitro cell culture model can detect permeability differences of model drugs in the presence of hydroxypropyl cyclodextrins (HP-CDX).
Methods: HP-β-CDX and HP-α-CDX were used with theophylline, piroxicam, atenolol, furosemide. An MTT assay measured potential toxic effect of the cyclodextrins and drugs to Caco-2 cells. Caco-2 monolayers were cultivated on polycarbonate filters for transport studies and measurement of TEER. Model drug transport was conducted in the absence and presence of HP-α-CDX and HP-β-CDX at 1:1 or 1:10 (drug:CDX) molar ratios.
Results: HP-β-CDX and HP-α-CDX (10-150 mg/mL) were not cytotoxic to the Caco-2 cells after a 2-hour incubation. There were no incidences of significant toxicity for the drugs alone or in combination with either HP-CDX. A decrease in TEER at 100 and 150 mg/mL HP-α-CDX was noted during a 2-hour incubation. For HP-β-CDX, 150 mg/mL reduced TEER values. There were no significant differences in the Papp for atenolol or furosemide when assayed with either HP-CDX. For theophylline, there was a decrease in Papp with 1:10 HP-β-CDX. There was a significant decline in Papp for piroxicam with 1:1 or 1:10 HP-α-CDX or HP-β-CDX.
Conclusions: The cyclodextrins were not toxic to the Caco-2 cells in the microtiter assay. Alterations in TEER occurred at the highest concentrations indicating some effect on the monolayer. The HP-CDX altered the absorption of the two high permeability drugs in the in vitro assay.
EFFECT OF TEMPERATURE AND pH STRESS ON LIPOSOME CYTOTOXIC ACTIVITY
D.A. Volpe, DPQR/CDER/FDA, Silver Spring
Background: There is a regulatory concern regarding the stability and integrity of liposome drug products (LDP). For this purpose an in vitro cell assay was evaluated to determine whether temperature or pH stress conditions affect the cytotoxic activity of LDP.
Methods: SKOV-3 (human ovarian) and J774A.1 (murine macrophage) cell lines were used in the microtiter tetrazolium cytotoxicity assay. Two LDP (Doxil®, DaunoXome®) and their parent drugs (doxorubicin, daunorubicin) were stressed for 6 days by temperature (22°C, 50°C) and pH (pH 3, pH 8). The LDP and drugs were then added to adhered cells in a microtiter plate for a 48-hour exposure and assessed for toxic effects.
Results: The J774A.1 cells were more sensitive to the untreated LDP and drugs than the SKOV-3 cells. Exposure for 6 days to 22°C or 50°C reduced the cytotoxic effect of doxorubicin in both J774A.1 and SKOV-3 cells. For Doxil® and daunorubicin, decreased toxicity to both cell lines occurred only with the 50°C exposure. When doxorubicin was exposed to pH 8 conditions its cytotoxicity was reduced in J774A.1 and SKOV-3 cells. Doxil® toxicity was also diminished at pH 8 in J774A.1 cells only. The cytotoxic activity of DaunoXome® was not decreased by temperature or pH in these experiments.
Conclusions: These results demonstrate a 6-day stress exposure to elevated temperature conditions or acid/base changes altered LDP and drug cytotoxicity to SKOV-3 and J774A.1 cells in a microtiter assay. Further development of cell-based assays may offer a method to assess the biological quality of LDP.
CpG independent synergistic induction of beta-chemokines and a dendritic cell phenotype by orthophosphorothioate oligodeoxynucleotides and GM-CSF in elutriated human primary monocytes
J. Wang1 , R. Alvarez1 , G. Roderiquez1 , E. Guan1 , Q. Caldwell1 , M. Phelan1 , J. Wang2 , M. A. Norcross1 , 1DTP, CDER, FDA. MD, 2DMA, CDER, FDA. MD
Chemokines attract leukocytes bearing the relevant chemokine receptors and regulate innate immune responses. CpG oligodeoxynucleotides (ODN) and GM-CSF are potent vaccine adjuvants and in combination induce enhanced Th1 responses by mechanisms yet to be determined. We have examined combinations of CpG or non-CpG ODN and GM-CSF for effects on the production of chemokines and the differentiation of monocytes to dendritic cells. High levels of the Th1-attracting, HIV-1 inhibitory chemokines, CCL3/MIP-1α and CCL4/MIP-1β, were induced in human primary monocytes when CpG or non-CpG ODN was combined with GM-CSF, but not with interleukin (IL)-4 or IFN-γ The synergistic induction of β-chemokines by non-CpG ODN was phosphorothioate (PS) chemistry dependent and inhibited by blocking endosome maturation/acidification and ERK1/2 activation. Chemokine and TLR9 mRNAs were induced by PS-ODN. Cells treated with non-CpG PS-ODN and GM-CSF expressed dendritic cell marker CD83+, high levels of HLA-DR and costimulatory molecules, and were CD14- or CD14dim, consistant with monocytes differentiation into a dendritic cell phenotype. The induction of CD83 and β-chemokines was tyrosine phosphorylation dependent. Secreted CCL3 and CCL4 were detected as a heterodimer. Our results indicate the CpG independent synergy between PS-ODN and GM-CSF mediated through chemokine and dendritic cell induction. In addition, our observations suggest that PS-ODN plus GM-CSF may be useful as potent ex vivo DC differentiation/maturation agents for dendritic cell therapy and as vaccines adjuvants for tumor and infectious micro-organisms, including HIV-1.
Characterization of differences in isoelectric focusing behavior of plasma-derived alpha-1-proteinase inhibitor products
E. Marszal, A. Shrake, CBER, FDA, Bethesda, MD 20892
Purpose: In response to reports from a physician and a patient advocacy group, we investigated isoelectric focusing (IEF) properties of licensed alpha-1-proteinase inhibitor (a1-PI) products, which are used to treat a1-PI deficiency. Methods: IEF Results: Products A and B showed IEF patterns similar to that of normal a1-PI in plasma, whereas the pattern of product C suggested that the major part of the protein carries an additional negative charge. The products were desialylated to eliminate heterogeneity caused by the presence of sialic acid capping N-linked oligosaccharides. IEF patterns of desialylated products showed that each product consists of two major and some minor populations of a1-PI. Products A and C appear to contain: the a1-PI form dominant in plasma; a form bearing an additional negative charge; and at least two other forms, which may be present in plasma at low levels. The a1-PI dominant in plasma appears to be the major form in product A and minor form in product C. The major form of a1-PI in product B is slightly more negative than the dominant a1-PI form in plasma. The minor form in product B appears similar to the form with an additional negative charge in products A and C. Analysis of current and historical lots suggests that the IEF pattern has been consistent for all three products. Conclusions: The products contain atypical forms of a1-PI, which may result from chemical modifications and/or conformational changes. Further analysis will be performed to identify the modifications and to establish if these forms also occur in vivo in trace amounts.
In-vitro test method development for determining Transdermal Drug Delivery Systems (TDDS) adhesion
A. M. Wokovich1 , S. A. Brown2 , 1FDA/CDER/OPS/DPA/St. Louis, MO, 2FDA/CDRH/OSEL/DSFM/Rockville, MD
Background: Although skin adhesion is an important functional property for Transdermal Drug Delivery Systems (TDDS), there currently is no generally accepted method for evaluating adhesion-to-skin performance for these products. Even though in-vivo human skin testing is the most reliable method for evaluation of TDDS adhesion, it is not a cost-effective method for quality control.
Methods:UsingASTM D3330 as a starting point for method development, medical tapes were used as controls to establish the effect of various parameters on release properties. Parameters evaluated include substrate, substrate cleaning solvents, substrate cleaning procedures, sample size, application technique, dwell time, peel angle and peel speed.
Results: Our studies show that all of these parameters have some affect on peel strength. Substrate, peel speed, peel angle, and substrate preparation (cleaning solvent and cleaning method) are some of the critical parameters that gave rise to differences in peel force for a given sample.
Conclusions: By careful control of the critical in vitro peel parameters, the developed method was reproducible and easily distinguished between the test tapes. Future work will determine the applicability of this method to TDDS.
Impact of correction formulae on the QTc interval with a drug suspected to prolong the QT interval
C. R. Bonapace, V. R. Jarugula, CDER, FDA, Rockville, MD 20850
Background: Drug Y is an investigational agent from a class of drugs known to prolong the cardiac repolarization (QT interval). In order to compare on-treatment and off-treatment ECGs, the QT interval should be corrected for heart rate (HR). However, numerous correction formulae exist and the results obtained from these formulae often differ.
Methods: 68 healthy subjects were enrolled in a randomized, double-blinded, multiple-dose, four-period crossover study. Each subject was to receive placebo, X mg BID, 2X mg BID, and 3X mg BID of Drug Y for 5 days in one of four sequences.
Results: The study was interrupted due to non-cardiac adverse events and only 27 subjects completed the study. Among the four regimens, the QT interval did not exceed 470 msec. The difference in Δ QTc between the correction formulae increased as the dose increased and ranged from 1.4 msec with placebo to 6.8 msec with the 3X mg BID regimen. Using Bazett's formula, the QTc values demonstrated the widest range throughout the study (331 to 476 msec). The mean Δ QTc increased with increasing dose for each method of correction, although the mean values were the greatest using the formula of Bazett followed by the formula of Fridericia and then individual regression correction.
Conclusions: Consideration should be given to the formula that adequately corrects the QT interval for changes in HR. The individual regression correction appeared to best account for changes in HR on the QT interval in subjects not receiving drug and should be considered in analysis of thorough QT studies when possible.
Intra-island horizontal exchange drives genetic diversity within the SPI-1 pathogenicity gene cluster of group I Salmonella pathogens
E. W. Brown, A. W. Shifflet, A. Perlloni, J. E. LeClerc, T. A. Cebula, CFSAN, FDA, Laurel, MD
Background: Salmonellosis is a serious public health concern in the U.S. Salmonella persistence in unique niches such as the small intestine is attributed in part to specific virulence determinants encoded on pathogenicity islands. Understanding the evolutionary mechanisms for acquisition of pathogenicity islands by horizontal gene transfer (HGT) is essential for more accurate risk assessment of this pathogen.
Methods: Salmonella pathogenicity-island 1 (SPI-1) is necessary for host cell invasion. To determine the extent that HGT may have disrupted SPI-1 evolution across human pathogenic salmonellae, a cladistic analysis was conducted of nine SPI-1 invasin loci sequences from the SARB (group I) reference strain collection, which comprises strains of human Salmonella pathogens.
Results: Comparison of invasin gene phylogenies with whole-chromosome phylogeny revealed numerous examples of HGT for all nine loci. spa genes M, N, O, P, Q and S and inv genes A, B and F retained alleles that had recombined in their entirety. Nearly every gene analyzed was evolutionarily discordant with other SPI-1 loci and the adjacent mutS gene, suggesting that most members of this gene cluster were acquired independent of one another. Additionally, a novel SPI-1 insert, first detected in S. Enteriditis, varies in size and structure among group I strains.
Conclusions: We conclude that the SPI-1 genomic region is evolutionarily unstable and prone to HGT. The data demonstrate a key role for HGT in configuring a mosaic structure within a significant pathogenicity determinant of Salmonella, suggesting that recombination is pivotal in enabling Salmonella to adapt to host challenges.
Use of Standards-Based Data and Tools to Improve the Efficiency of the NDA Safety Review
C. K. Cooper1 , J. G. Levine1 , J. M. Tonning1 , D. Fram2 , J. Millstein2 , G. Rochester1 , A. Szarfman1 , 1CDER, FDA, Rockville, MD, 2Lincoln Technologies
Background: The adoption of a common data format standard for New Drug Application (NDA) submissions allows for the opportunity to develop web-based safety assessment tools that are reusable across applications. FDA has collaborated with the Clinical Data Interchange Standards Consortium (CDISC) in the development of a new standard (STDM) for representing clinical trial data in NDA submissions. Sponsors and FDA have begun to work with that new standard.
Methods: We transformed Integrated Summary of Safety datasets submitted for a recent NDA into Clinical Data Interchange Standards Consortium (CDISC) Standard Data Tabulation Model (SDTM) version 3.1 format. This enabled us to load the NDA data into a software program developed under a Cooperative Research and Development Agreement. The Web-based software combined with CDISC's standardized data definitions and variable names allowed us to use tailor-made, reusable, tables and graphs of patient data designed to enhance the interactive analysis of the clinical trial data in the NDA.
Results: The use of specialized safety assessment tools and CDISC data allowed us to identify and assess potential safety signals in a more rapid and efficient manner. The gain in efficiency was represented by a significant reduction in time necessary to identify and review potential signals. This process involved the interpretation of numerous tables and graphs, some predefined, and some designed by us using customizable report generators. Adverse events were analyzed using a variety of sortable tables, and graphics display, including a "Sector map" that provided a visual presentation of clinical adverse event data for each System Organ Class (SOC), with tiles representing Primary Terms (PTs), High Level Term (HLTs), or High Level Group Terms (HLGTs). The colors and patterns in the sector maps provided a "big picture" overview of the adverse event profile of the study drug vs. the controls. Laboratory data was analyzed using various tabular and graphical displays, including distribution plots of changes from baseline over time.
A key feature is that the system automatically performed a variety of pre-specified safety analyses, and ranked the results according to the strength of the signal. Inspection of the ranked results combined with review of tabular and graphical displays enables the rapid identification of potential safety concerns. Once potential signals (or alerts) were identified, we were able to quickly drill-down to examine supporting data and move quickly from "big-picture" overviews to "fine-detail" views consisting of Patient Profiles with narratives as well as other source data.
Difficulties encountered included a) inconsistent formatting of values in the original data which required additional effort in the data transformation process and b) unclear definitions of safety outcome variables required for data transformation and review.
Conclusions: CDISC's standardized data definitions and variable names allowed us to rapidly apply ready-made, reusable tools embodying tables and graphs of patient data designed to enhance the interactive analysis of the clinical trial data in the NDA. The use of these review tools resulted in less time spent by medical officers and statisticians on data management and analysis and provided more time for the interpretation of results. This strategic approach has great promise for improving the quality, speed, and transparency of the NDA safety review at FDA which can be realized through wide-scale adoption of these emerging standards at FDA.
Modeling the Interaction of the Physiological State of the Inoculum and CO2 Atmosphere on the Lag Phase and Growth of L. monocytogenes
A. J. De Jesús, R. C. Whiting, FDA/CFSAN, College Park, MD
Several studies have modeled the growth of L. monocytogenes under different CO2 headspace concentrations but they used inoculum cells that were in the stationary phase. In this study, the growth of L. monocytogenes under different CO2 concentrations as affected by physiological state of the cells was investigated. Exponential growth phase, stationary phase, desiccated and starved cells were inoculated into BHI broths at 5°C that were pre-equilibrated under the following atmospheres: 0%, 20%, 40% and 80% CO2 (balance N2). Lag phase duration times (LDT) and exponential growth rates (EGR) were determined by enumerating cells at appropriate time intervals and by fitting the data to a two-phase linear function that has a lag period before the initiation of exponential growth. Longer LDTs were observed as the CO2 concentration increased, with no growth observed at 80% CO2. With the 40% CO2 atmosphere, for example, the LDT's for exponential, starved, stationary and desiccated cells were 6.5, 9.8, 18.4 and 19.3 days, respectively. In general, exponential growth cells had the shortest LDTs followed by starved cells and stationary phase cells. Desiccated cells had the longest LDTs. Exponential growth rates decreased as the CO2 concentrations increased. Once exponential growth was attained, no retained differences among the different initial physiological states of the cells for any of the atmospheres were observed in the EGR's. The EGR's for 0, 20, 40, and 80% CO2 averaged 0.39, 0.37, 0.19 and 0.0 logs/day, respectively.
The Most Common FDA-Regulated Foods Linked to Foodborne Illness Outbreaks, 1990-2003
C. Smith DeWaal, G. C. Hicks, Center for Science in the Public Interest, Washington, DC 20009
BACKGROUND. There are an estimated 76 million cases of foodborne illness each year. Only a small proportion of illnesses are associated with an outbreak, and of those, only a small proportion have an identified food source.
METHODS. CSPI maintains a unique listing of foodborne illness outbreaks, categorized by food. CSPI's data is compiled from various sources, including the CDC, state health departments, and scientific and medical journal articles. The database contains only those outbreaks with known or suspected etiology and an identified food source. The database is useful in identifying the most common foods associated with foodborne illness outbreaks, the most common food/hazard combinations, and provides better information for food safety resource allocation.
RESULTS. Between 1990 and 2003, there were 2,958 outbreaks including 83,097 individual cases linked to FDA-regulated foods, listed in the CSPI database, which constituted 66% of the outbreaks in the CSPI database, and 60% of the cases. The single-food categories most commonly linked to foodborne illness outbreaks were seafood (n=904), produce (n=554), and eggs (n=329). Seafood outbreaks are predominantly caused by chemical hazards, while produce outbreaks are generally linked to norovirus and Salmonella.
CONCLUSIONS. Seafood and produce have consistently remained the foods most commonly linked to outbreaks in the past few years. More than 80% of the seafood, and 20% of fresh produce, consumed in the United States is imported. These findings suggest that import inspections alone are not adequate to control hazards entering the United States on imported foods.
Prevalence and Antimicrobial Susceptibility of Campylobacter Isolated from Retail Meat and Poultry in the United States, NARMS 2003
L. L. English, D. G. White, E. Hall-Robinson, S. L. Ayers, S. K. Hubert, R. D. Walker, FDA, Laurel, MD
Background - To better understand the contribution of the food supply to antimicrobial resistance, the National Antimicrobial Resistance Monitoring System (NARMS) was expanded in 2002 to include surveillance of retail meats for antimicrobial resistant enteric bacteria. In 2003, NARMS was further enhanced by including more state sampling sites and retail meat samples.
Methods - In 2003, Campylobacter were recovered from chicken breast, ground turkey, ground beef, and pork chops purchased from grocery stores in eight FoodNet sites (CA, CT, GA, MD, MN, NY, OR, and TN). Isolates were speciated using PCR and tested for susceptibility to five antimicrobials using the CLSI/NCCLS agar dilution procedure.
Results - Thirteen percent of 3523 samples were contaminated with Campylobacter (50.7% of chicken breast; 0.1% of ground beef; 0.6% of ground turkey; and 0.3% of pork chop samples). C. jejuni was the predominant species recovered (70.3%), followed by C. coli (29.3%). Of the C. jejuni, 14.4% had an MIC > 4 µg/mL for ciprofloxacin, 22.1% had an MIC > 16 µg/mL for doxycycyline, and all were susceptible to erythromycin. Of the C. coli, 14.7% had an MIC > 4 µg/mL for ciprofloxacin, 47.1% had an MIC > 16 µg/mL for doxycycyline, and 10.3% had an MIC > 8 µg/mL for erythromycin. All Campylobacter were susceptible to meropenem, and with the exception of one C. jejuni isolate, all were susceptible to gentamicin. Twenty-seven isolates showed resistance to multiple antimicrobials.
Conclusions - Campylobacter, including antimicrobial resistant strains, contaminate retail chicken and can serve as a reservoir of resistant strains in the food processing chain.
Identifying Public Food Safety Information by analyzing the contacts with the Food Information Center (FIC)
A. Fooladi Moghaddam, FIC, Office of Applied Research, FDA, MOHME, IRAN
Introduction: The recent study was performed to identify the public food safety information by analyzing the calls to FIC.
Methods: In a study all the contacts with FIC were recorded during six month then all the information was analyzed. The collected information were: demographic information; how they got informed of the FIC Hotline; their previous calls; the purpose of the calls; the food groups concerned; the replying and the references .Two nutritionists and one consultant answered the questions and when they were not able to answer they searched the references .The excel software was used for data recording and analysis.
Results: In the six month period from the 21st of March to the 21st of September the FIC Hotline received a total of 147 calls. Most of the contacts were from the 21st of June to the 21st of July. The majority of the contacts were from Tehran (135 calls) and most of the people in contact were graduated from college (30%). 71% of the total cases were women, 33% were housewife, and 28% of the total were retired cases .The purpose of the contacts in 110 cases was food safety, in 20 of the cases was nutrition and 17 contacts had either advice or complaints. Among 110 food safety inquiries 82 cases fell under one of the top five categories .These top five categories were :the meat group (19%), food safety (general), canned food, grains and legumes and finally the dairy products. Each of the groups had 21, 18, 17, 15, 11 questions respectively. Hazard was the most common kind of concern (30%), the remaining 70% of inquiries reflected other concerns including storage, handling and consumption, contamination, diet, nutrient value, cooking, license controlling, nutritional behavior, and poisoning.
Conclusion: Food safety information activities should better be based on the acceptance of the target groups and relevant to the group's knowledge of food safety and should provide the information they need. An educating program for housewife about the general food safety issues and some tips on meat, grain and dairy safety would be successful .These findings will not be accurate for all the time periods so in order to understand the main concerns taking advantage of other methods like focus groups, interviews or surveys, knowledge assessment and controlling information sources are recommended.
Drug Interaction Studies -- Study Design, Data Analysis, and Implications for Dosing and Labeling: Current Opinion
S. M. Huang1 , K. S. Reynolds1 , J. M. Strong2 , S. Nallani1 , L. J. Lesko1 , R. Temple3 , S. Abraham1 , S. Alhabet1 , R. Baweja1 , S. M. Chung1 , P. Colangelo1 , J. M. Collins2 , D. Frucht2 , M. D. Green4 , P. Hepp1 , R. Kavanagh1 , H. S. Ko5 , P. Marroum1 , J. Norden3 , W. Qiu1 , A. Rahman1 , S. Sobel2 , T. Stifano5 , X. Wei1 , S. U. Yasuda1 , L. K. Zhang1 , J. H. Zheng1 , 1OCPB, CDER, FDA, 2OPS, CDER, FDA, 3OMP, CDER, FDA, 4OND, CDER, FDA, 5CBER, FDA
Purpose: Creation of a concept paper that reflects the Agency\\\'s current view that the metabolism of a new drug should be defined and its potential to interact with other drugs should be explored as part of the assessment of safety and effectiveness.
Methods: Latest scientific consensus on drug metabolism, drug transporters and pharmacokinetics was considered, based on discussions at conferences, advisory committee meetings, and consultation with experts.
Results: Evaluation of drug interactions involves an integration of in vitro and in vivo methods, as indicated by the decision tree in the concept paper.
In vitro methods: Concept paper describes principles of study conduct, including up to date information on in vitro induction studies. A list of probe CYP substrates, inhibitors and inducers is provided. Paper indicates specific results from in vitro studies that warrant in vivo investigation. In vitro studies that evaluate the potential for interactions related to drug transporters, particularly P-glycoprotein, are described.
In vivo methods: Concept paper includes details of study design and a list of probe CYP substrates, inhibitors and inducers. The paper also includes recommendations on the conduct of in vivo P-glycoprotein inhibition related interactions. Appropriate labeling language is discussed. The labeling discussion includes a list of CYP3A sensitive substrates and strong and moderate inhibitors, to help make labeling recommendations more consistent.
Conclusions:
The concept paper includes current recommendations for in vitro and in vivo drug metabolism and drug interaction studies performed during drug development. The decision tree emphasizes the integrated approach to evaluation of drug interactions
Reference: http://www.fda.gov/ohrms/dockets/ac/04/briefing/2004-4079b1.htm (topic 2)
Stability screening of in vitro stock drug solutions
R. L. Hunt, P. J. Faustino, D. A. Volpe, DPQR/CDER/FDA, Silver Spring, MD
Background: The in vitro stability screening of drugs solutions utilized as membrane markers, internal cellular standardization and permeability class markers in typical biopharmaceutical applications is essential for accurate drug exposure and cellular response. The stability of the stock drug solutions were tested by high-performance liquid chromatography (HPLC) under various storage, preparation and standard experimental temperature conditions and timepoints used for biopharmaceutical studies.
Methods: Atenolol, FITC-dextran, furosemide, piroxicam, theophylline were evaluated in pH 6.8 and 7.4 buffers at 22°, 37°, 4°, -20° or -80° over 7 days. Drug stock solutions samples were tested was on an Agilent 1100 HPLC with UV and fluorescence detection. Separation was obtained on Phenomenex Luna C18 (Torrance, CA) column with phosphate buffers (pH =3.0, 6.9, 7.0) and acetonitrile (10-30%).
Results: Furosemide and theophylline were stable at all temperature conditions. FITC-dextran decreased 7% after 2 weeks at -80°. Piroxicam at -20° showed 3% decrease after 3 weeks. At -20°, atenolol had a 2% decrease after 2 weeks. All drugs at 37° were stable for 6 hours. There was less than 10% variation for each drug at all conditions. There was no difference between conventional biopharmaceutical cellular test pH's of 6.8 and 7.4 at all temperature.
Conclusion: A standard screening protocol was developed and tested for commonly used in vitro drug stock solutions. Screening data indicated that avoidance of drug loss or degradation, usage must occur in 1 week. Efficient screening protocols are essential for the application of regulatory risk management tools such as the Biopharmaceutical Classification Guidance.
Quality review of congenital anomaly reports sent to the FDA
D. L. Kennedy, A. Erickson, K. Uhl, R. Goetsch, CDER, FDA, Rockville, MD
Background: Knowledge of teratogenic potential is a critical part of a drug's benefit/risk profile. Most human teratogens are identified through postmarketing case reports. To be useful, a report on a drug-induced fetal effect must contain certain critical information.
Methods: We identified all adverse event reporting system (AERS) reports received by FDA in CY2003 with the "congenital anomaly" outcome box checked. Domestic reports were reviewed for the presence of critical information needed to evaluate the report.
Results: A total of 632 congenital anomaly (C.A.) reports were reported in CY2003: 286 domestic and 346 foreign. After identifying duplicate reports, 261 unique domestic cases remained: 93 direct, 11 manufacturer (mfr) periodic, and 157 mfr expedited. 73% of direct reports were from consumers and 83% of direct reports did not involve pregnancy or a C.A. 94% of mfr expedited reports involved pregnancy and a C.A. 35% of mfr expedited reports came from a pregnancy registry. Only 6% of mfr expedited reports contained complete information for evaluating the case; of these most were from a pregnancy registry (an epidemiology study).
Conclusions: The quality of congenital anomaly reports in AERS is generally poor, lacking critical information needed to assess the relationship between drug exposure and pregnancy outcome. Reports from pregnancy registries are relatively much better, but given the purpose of a registry is to actively gather data prospectively, there is still room for improvement. Consumers don't know what "congenital anomaly" means; "birth defect" may be a better term to use.
Testing for Hypersensitivity Reactions to Intravenous Drug and Biologic Agents: Recommendations in Product Labels
H.S. Ko, CBER, FDA, Rockville, MD
Purpose: Administration of intravenous agents may be associated with serious allergic reactions. It would be important to examine the recommendations on testing for hypersensitivity reactions in the use of available intravenous products that carry such risks.
Methods: Package inserts for marketed intravenous drug and biologic products were examined for information on the immunogenicity of the product, precautions before and after dosing, and recommendations on testing.
Results: The evaluation included 22 package inserts of blood-derived products (polyclonal antibodies and coagulation factors), monoclonal antibody products, volume expanders (dextrans), pharmaceuticals (iron, insulin, penicillin), and radiographic contrast media. Among the recommendations in these labels, skin testing is the most common, and appears in five product labels. Three labels recommend administering test doses prior to full dosing. Two package inserts advocate conjunctival testing, two propose screening for antibodies and two other labels state that skin testing is not suggested or required prior to dosing. The remaining labels are silent on testing for reactions. There is no uniform description of what constitutes a "positive" test and when a product should be avoided if it is observed. Despite "negative" tests, some patients may still experience reactions to the products. Desensitization procedures are described in three labels.
Conclusions: Currently there is much variation in the recommendations on hypersensitivity testing for intravenous agents that carry reaction risks. Formulating recommendations for testing should be based on an evidence-based approach.
Mechanistic Information & Regulatory Decisions
S. Kozlowski, CDER, FDA, Bethesda, MD
Despite tremendous advances in the understanding of molecular and cellular biology, the ability to utilize this information in predicting clinical outcomes is questionable. Clearly, the low percentage of drug candidates making it to market suggest that presently used molecular information is not predictive in a reliable manner. However, the proposed mechanism(s) of a new molecular entity may suggest appropriate pre-clinical and clinical study design. In addition, there are situations where an empirical approach is not possible. For example, the relevance of a product safety signal to other related products may be difficult to evaluate. There may not be sufficient data with the related products to assess this potential safety concern although rapid decisions are needed. These products may be related in terms of structure, molecular targets or impurity profiles. The biologic context of these relationships may help inform the risk-benefit analysis for important regulatory decisions. Such an approach must utilize mechanistic information in combination with clinical and toxicology data to make risk-based decisions.
Trends in Use of Clotrimazole/Betamethasone Dipropionate combination products in pediatric patients
L. A. La Grenade, D. Moeney, M. R. Pitts, CDER, FDA, Rockville, MD
Background: Topical products containing clotrimazole, an antifungal agent, and betamethasone dipropionate, a corticosteroid, include Lotrisone® and generic products. They are not recommended for children under age 12 years. However, a 1999 review indicated substantial off-label use of Lotrisone® cream and adverse events in children under age 12 years. Therefore when Lotrisone® lotion was approved for use in the US in December 2000, the sponsor committed to an educational campaign aimed at reducing use of Lotrisone® in the pediatric population. Generic combinations of topical clotrimazole/betamethasone dipropionate (CBD) were also introduced into the US in 2000.
Method: We obtained data on age, indication and estimated total prescriptions dispensed in the US for all CBD combination products from databases available to FDA for 1996 - 2004. We also searched FDA's Adverse Events Reporting System (AERS) for adverse event reports associated with CBD products in pediatric patients. We compared results before and after the Lotrisone® educational campaign in 2001.
Results: The results show a marked decline in the total number of Lotrisone® prescriptions dispensed overall and in children since 2001. However use of generic CBD increased in inverse relationship to the decrease in Lotrisone®. Overall, there has been a modest decline in use of all CBD products. Adverse event reports with Lotrisone® use in children also declined after 2000.
Conclusion: The educational campaign designed to reduce off-label use of Lotrisone® products in children may have had some effect but this may have been offset by use of generic products.
Development and Use of the Aquaculture Risk Information System (AQRIS) and Risk Ranking Tool (AQRRT) for Prioritizing Import Sampling and Other Activities
J. P. Laurenson1 , B. L. Jones1 , R. M. Kauffman2 , J. C. Cleland3 , R. A. Schnick4 , 1ICF Consulting, Fairfax, VA, 2ICF Consulting, Lexington, MA, 3ICF Consulting, Saunderstown, RI, 4Michigan State University, La Crosse, WI
Introduction. World fisheries stocks and harvests are rapidly declining and being replaced by aquaculture products. In a few years, aquaculture production is expected to exceed wild caught. Furthermore, fish as a source of protein continues to increase and replace other sources. Aquaculture imports to the U.S. far exceed domestic production, and the vast majority of these imports are from countries with little or no government oversight of the use of drugs and chemicals in aquaculture production. To date, approximately 70 to 80 drugs and 20 to 30 chemicals (including pesticides) are known to have been used in aquaculture worldwide, yet most are not approved for use in the U.S. nor in products imported to the U.S. Genetically modified fish -- regulated as drugs in the U.S. -- also are being developed worldwide. Given the thousands of potential country, species, and drug or chemical combinations that exist, a risk-based strategy is needed to help prioritize drugs/chemicals, countries, and species for monitoring purposes, among other needs.
Approach. The U.S. Food and Drug Administration, Center for Veterinary Medicine (FDA/CVM), which regulates the use of drugs for animals, is funding and providing direction for the development of the Aquaculture Risk Information System (AQRIS) and the Aquaculture Risk Ranking Tool (AQRRT). AQRIS houses data on drug identity and regulatory status, chemical and toxicological properties, conditions and patterns of drug use, occurrence of drug residues in edible tissues, and methods of residue analysis. This information will be used in AQRRT to develop relative risk scores that will allow FDA to (1) prioritize residue monitoring needs; (2) prioritize development of new analytical methods; and (3) promote international discussion regarding hazard concerns identified by the risk assessment. The scoring method is based on two primary criteria: toxicity (TOX) and population exposure (EXP). The TOX and EXP ratings are summed to yield the RISK rating (reflecting the multiplicative relationship between toxicity and exposure, and logarithmic scaling). TOX is based primarily on a quantitative measure of a drug's toxic potency for chronic exposures via the oral route. EXP utilizes two main factors: annual U.S. population consumption of contaminated aquaculture product from a given country (CONS) and average drug residue at time of consumption for a given food product/country (RES). The scale for CONS is based primarily on two subfactors: annual aquaculture import quantity to the U.S. for a given food product/country and percentage of the export crop treated with a given drug for a given food product/country.
Status. This project is nearing the end of the main data gathering (AQRIS) phase and is in the middle of the risk ranking (AQRRT) development and testing phase. Full use of AQRRT is expected by fall 2005.
Communication of Drug Risk Information to Pharmacists
P. Nourjah, L. Lee, C. Kortepeter, CDER, FDA, Rockville, MD
Pharmacists need accurate, concise, and practical drug information in order to effectively communicate important drug risks to patients.
Objective: To learn how pharmacists view the availability and the usefulness of Dear Healthcare Professional (DHCP) Letters in communicating drug risks.
Methods: State Boards of Pharmacy in the U.S. were contacted to obtain lists of registered pharmacists from which 5000 pharmacists were randomly selected from 4 geographical regions to receive a survey questionnaire.
Results: Approximately 50% of selected pharmacists responded to the mailed questionnaire and 89% of respondents were employed in a position requiring an active pharmacist license. Drug manufacturers sometimes send "Dear Healthcare Professional" (DHCP) letters to communicate drug risks and adverse events to pharmacists. Eighty one percent of respondents received DHCP letters, of which 39% always read the letters, 32% often read the letters, and 7% rarely or never read the letters. Among those who always read the DHCP letters, 54% believed that the letter was very effective in communicating drug risks to pharmacists. The top four recommendations for improving the drug risk communication between drug manufacturers and pharmacists were: disseminating via email/list serve, providing brief and concise information, distinguishing DHCP from advertisements, and sending a manufacturer representative to the pharmacist.
Conclusion: There is not only a need to educate pharmacists about drug information tools but also current tools can be modified for improving risk communication.
A National Survey of Pharmacists to Assess Drug Risk Communication Tools for Patients
P. Nourjah, L. Lee, C. Kortepeter, CDER, FDA, Rockville, MD
Pharmacists play an important role in communicating drug risks to patients through the use of medication guides and patient package inserts.
Objective: To obtain insight of licensed pharmacists' views of the availability and usefulness of drug information tools for communicating drug risks to patients.
Methods: State Boards of Pharmacy in the U.S. were contacted to obtain lists of registered pharmacist from which 5000 pharmacists were randomly selected from 4 geographical regions to receive a survey questionnaire.
Results: 2,052 registered pharmacists responded to the mailed questionnaire. 70% of respondents were familiar with the Medication Guide (MG), of which 30% stated that the MG was very effective in communicating drug risk. However, only 26% of respondents correctly answered that MGs are required to be dispensed with both new and refill prescriptions. Among pharmacists who have dispensed medications requiring a medication guide, 23% reported that patients have complained that the material is not understandable. 19% of the pharmacist respondents were not aware that package inserts undergo revisions. 20% of pharmacists felt that Patient Package Inserts (PPIs) were very effective in communicating drug risks to patients while 18% stated that the PPIs were not effective.
Conclusion: There is a need to educate pharmacists about drug information tools. In particular, special effort is needed to ensure that the contents of patient education materials are comprehensible for the targeted population, particularly when direct consultation by a pharmacist is optional.
Acute renal failure in patients using sodium phosphate tablets as bowel preparations
A. C. Mackey1 , L. Green2 , L. A. La Grenade2 , 1CDER, FDA, Rockville, MD, 2CDER, FDA, Rockville, MD
Background: Sodium phosphate tablets are indicated as bowel preparations in patients before undergoing colonoscopy.
Methods: Cases of acute renal failure (ARF) associated with sodium phosphate tablets and oral solution and polyethylene glycol were sought in the Adverse Event Reporting System (AERS) and the medical literature. AERS is a voluntary surveillance system (mandatory for manufacturers holding New Drug Applications); it is subject to under-reporting. There is no certainty that sodium phosphate caused the event, it could be due to any number of factors; however, the contribution of sodium phosphate products could not be excluded.
Results: We identified 6 cases of ARF associated with sodium phosphate tablets. Most of these patients experienced dehydration due to nausea/vomiting or diarrhea and electrolyte imbalance. The patients were older and had underlying vascular diseases and were taking medications that could have predisposed them to develop ARF. Two patients took the product incorrectly. A total of 13 cases of ARF associated with sodium phosphate oral solution were identified (cases similar in presentation to those described above); some patients' biopsies noted calcium-phosphate precipitation in their renal tubules. No cases of ARF were identified for polyethylene glycol products.
Conclusions: Most patients who use sodium phosphate products as bowel preparations do not experience serious outcome. A combination of factors (e.g., advanced age, underlying vascular disease, concomitant medications, dehydration, electrolyte imbalance) may have led to the patients' ARF; patient biopsy reports provided evidence of direct toxicity (i.e., calcium-phosphate precipitation) to the renal tubules. The importance of taking sodium phosphate products correctly should be stressed.
Genetic homogeneity and evolutionary discordance implicate horizontal gene transfer in the evolution of the polA active site in Salmonella enterica
T. J. Mays, E. W. Brown, J. E. LeClerc, T. A. Cebula, FDA, CFSAN, OARSA, DMB, Laurel, MD
Background: Errors in DNA replication and repair accelerate adaptive evolution of foodborne bacterial pathogens. DNA polymerase I, encoded by the polA gene, is essential for proper repair function. Despite its critical role, polA retains a highly plastic active site, capable of tolerating numerous amino acid substitutions in vitro. There is near sequence uniformity, however, across the active site of polA alleles from feral Escherichia coli strains and Salmonella enterica subspecies I strains. One explanation for this result is that horizontal gene transfer (HGT) has homogenized polA active site sequences to the few alleles observed in nature.
Methods: We tested this hypothesis by genetically and phylogenetically analyzing polA sequences in Salmonella reference collection C (SARC). SARC is composed of strains from eight diverse subspecies of S. enterica, representing the breadth of species diversity.
Results: Our analysis revealed evidence for HGT of polA sequences within Salmonella. Analysis of SARC polA clades showed at least five strains that appeared to be recipients of horizontally transferred sequences from disparate Salmonella subspecies. Only seven of 39 polA active site positions were variable, however, of which only a single change resulted in an amino acid substitution.
Conclusions: Lack of sequence variation among SARC strains that diverged millions of years ago, coupled with cladistic evidence for HGT, suggests that polA active sites have been homogenized throughout the evolution of S. enterica subspecies. These data lend support to the HGT hypothesis for polA evolution and suggest that, like E. coli, Salmonella has been subject to the 'cleansing' effects of recombination.
A New Face on an Old Problem: Excipient Exposure in the Neonatal Intensive Care Unit
S. K. McCune, S. Y. Buckman, W. J. Rodriguez, OCTAP, CDER, FDA, Rockville, MD
In 1937, sulfanilamide was compounded with diethylene glycol to improve solubility; 107 patients, many of them children, died due to diethylene glycol toxicity. In 1981, the "neonatal gasping syndrome" secondary to benzyl alcohol toxicity was reported. Extremely low birth weight (ELBW) infants receive significant numbers of medications, putting them at risk for acute and chronic exposure to single or multiple excipients.
A literature review of common excipients led to a focused examination of drug labels for some commonly used drugs in the neonatal intensive care unit (NICU). Also, accepted standards of excipient exposure were examined. Up to 90% of NICU patients are exposed to drugs that are either unlabeled or used in an off-label manner. This study does not endorse the off-label use of these drugs but only examines the potential exposure to excipients during a NICU admission.
Benzyl alcohol, propylene glycol, polyethylene glycol, sodium benzoate, and polysorbate are excipients found in drugs commonly used in the NICU. Aminophylline, Vitamin K, lorazepam, bumetanide, dexamethasone, diazepam, doxapram, pancuronium, phenobarbital, hydrocortisone, methylprednisolone, succinylcholine, enalapril, midazolam and Vitamin E preparations contain benzyl alcohol. Lorazepam, phenytoin, digoxin, MVI-12®, phenobarbital, and diazepam contain propylene glycol. Lorazepam contains polyethylene glycol, diazepam contains sodium benzoate, and MVI® pediatric contains polysorbate. Excipient concentrations and potential exposure will be discussed.
It will be important to document individual neonatal excipient exposures compared to known thresholds. Future trials need to determine the metabolism of excipients in ELBW infants and the potential long-term effects of chronic excipient exposure.
The Food Safety Risk Analysis Clearinghouse: Practical Uses in Risk Analysis.
J. Hinton1 , C. F. McLaughlin2 , H. Patel1 , 1University of Maryland, 2FDA, CFSAN
Food Safety Risk Analysis is a multidisciplinary field with multiple dimensions. The Food Safety Risk Analysis Clearinghouse is an invaluable resource to professionals in food safety risk analysis, as it hosts information across the relevant dimensions within the Risk Analysis framework. The Goal of the Clearinghouse is to encourage the use of risk analysis in the development of food safety policy by facilitating access to information that might otherwise be difficult to find. Reasons that enable Clearinghouse users access to more relevant data include the way it categorizes information and the two different search engines it uses. The Clearinghouse hosts data, tools, reports, select Federal Register documents, and presentations, and serves as a vast and authoritative portal to online resources that support the work of food safety risk analysis professionals. Its domain includes both chemical and biological hazards in foods as well as nutrition and weight management as related to food. Using both biological and chemical hazard examples, this poster provides an introduction to the Clearinghouse and demonstrates how to effectively use the Clearinghouse to find resources and data that will support all steps of the risk analysis process.
EVALUATING THE TERATOGENIC POTENTIAL OF ANTIBIOTICS IN PREGNANT WOMEN
G. G. Nahum, K. Uhl, D. Kennedy, CDER, FDA, OND, Rockville, MD
Background: Approximately 10 million women are either pregnant or lactating in the U.S. at any particular time. These women constitute a vulnerable population for whom the risks of drug use must be assessed differently than for the general population. Besides the physiologic changes that alter the pharmacokinetics of drugs during pregnancy and lactation, there is the added concern of possible teratogenic and toxic effects that drugs may have on the developing fetus and newborn. Antibacterial agents are widely used drugs during pregnancy and lactation and have potential uses for bioterrorism agents. Our objective is to review current information concerning the risks of antibiotic use during pregnancy and lactation.
Methods: Information from the package inserts, TERIS, REPROTOX, Shepard's Catalog of Teratogenic Agents, and the published literature were reviewed for eleven common antibiotics.
Results: The teratogenic potential of these antibiotics during human pregnancy ranged from "none" (penicillin G and VK) to "unlikely" (amoxicillin, chloramphenicol, ciprofloxacin, doxycycline, levofloxacin, and rifampin) to "undetermined" (clindamycin, gentamicin, and vancomycin). These assessments were based on "good data" (penicillin G and VK), to "fair data" (amoxicillin, chloramphenicol, ciprofloxacin, doxycycline, levofloxacin, and rifampin), to "limited data" (clindamycin and gentamicin), to "very limited data" (vancomycin).
Conclusions: There is interest in the potential risks associated with anti-infective drug use during pregnancy and lactation. Healthcare professionals should be familiar with the safety profiles of antibiotics to help make prescribing decisions during pregnancy and lactation, especially if anti-infective countermeasures are needed to protect the health, safety, and survival of individuals exposed to bioterrorism agents.
The Annual Health Cost of Food-Related Illness
A. Ritzert, D. Zorn, K. Klontz, C. Nardinelli, FDA
Background
According to the Centers for Disease Control and Prevention, approximately 76 million cases of food-related illness occur in the United States in a typical year. As a first step toward a measure of the direct and indirect health costs associated with these illnesses, we have developed a model to estimate the health costs of illness for the 14 million food-related illnesses caused by known viruses, bacteria, and parasites.
Methods
We estimate the costs of acute foodborne illnesses as the sum of medical expenses plus the implicit monetary costs of reduced functional status and pain and suffering. For those acute illnesses that can lead to chronic complications, we add the costs of those chronic complications. We estimate the implicit costs of functional disability and pain and suffering by first calculating the quality-adjusted life years lost due to the illness. We then estimate monetary values by multiplying the value of a quality-adjusted life year by the number of quality-adjusted life years lost due to the illness. For fatal cases, we estimate the health costs by multiplying the estimated number of deaths by the monetary value of a statistical life.
Results
The model can be used to estimate the total costs for a particular illness, the total costs associated with outbreaks of one or more illnesses, or the total costs of all 14 million food-related illnesses caused by known viruses, bacteria, and parasites. We present the model results for two recent outbreaks: one involving Norovirus and one involving non-typhoidal Salmonella.
Effect of ambient temperature/atmospheric pressure ammoniation of aflatoxin-contaminated dairy ration cottonseed on the levels of aflatoxin M1 in milk
H. Njapau1 , S. Trujillo1 , N. Fico2 , B. J. Canas1 , D. L. Park1 , P. J. Cotty3 , L. Antilla4 , R. Webb4 , 1OPDF, FDA, College Park, 2JIFSAN, FDA, College Park, 3USDA, Univ. Arizona, Tucson, AZ, 4ACRPC, Phoenix, AZ
Background: Milk is widely consumed by children. Cottonseed, a desirable feed ingredient, can be contaminated with a toxic mold product, aflatoxin B1 (AFB1). Milk from cows fed AFB1-contaminated cottonseed may contain the AFB1 metabolite aflatoxin M1 (AFM1), which is also toxic to the consumer. Atmospheric pressure/ambient temperature (AP/AT) ammoniation is a process that can degrade AFB1 in contaminated cottonseed. This phase of the study determined the effect of AP/AT ammoniation on aflatoxin B1 in cottonseed and subsequent aflatoxin M1 in milk.
Methods: Aflatoxin B1-contaminated (700 ppb) and aflatoxin-free (<10 ppb) cottonseed were ammoniated using the AP/AT process and incorporated (~6%TMR) into rations of lactating dairy cows. The cottonseed and milk were analyzed for aflatoxin B1 and aflatoxin M1, respectively.
Results: Temperature (26-80°C) and moisture (12-30%) gradients, and consequently an initial stratification in the reduction (70-99%) of aflatoxin B1, occurred within a pile undergoing AP/AT ammoniation. Mixing all the cottonseed plus a further 7-10 days treatment yielded an overall ~98% reduction in aflatoxin B1 content. No aflatoxin M1, above baseline levels, was detected in milk from cows fed rations containing AP/AT-treated-AFB1-contaminated cottonseed. Excess aflatoxin M1, (x = 1.75 ppb) was detected within 24 hours in milk from cows fed rations containing untreated cottonseed but dropped to baseline levels within 36-48 hours of withdrawal of the contaminated feed.
Conclusions: The AP/AT ammoniation process degrades aflatoxin B1 in cottonseed. Milk from cows fed aflatoxin-contaminated-ammoniated cottonseed will not contain excess aflatoxin M1. The study will, once completed, provide important information on the efficacy and safety of the AP/AT process.
Evidence for type I restriction-modification as a porter for extensive recombination among closely related Salmonella strains
A. Perlloni, E. W. Brown, J. E. LeClerc, T. A. Cebula, CFSAN, OARSA, DMB, Laurel, MD 20708
Background: Our laboratory previously documented the prevalence of horizontal gene transfer (HGT) among strains of Salmonella enterica. In comparing subspecies, a recombination gradient was noted wherein the incidence of HGT is inversely correlated with the genetic diversity separating individual strains. The compatibility of restriction-modification (R-M) systems among strains was proposed as one explanation to account for the contrasting recombination rates.
Methods: hsd genes, R, M, and S (composing the type I R-M system in Salmonella enterica), from closely-related strains of the highly homogenous subspecies I Typhimurium complex were subjected to DNA sequence analysis. Cladistic comparisons were conducted to investigate whether HGT of hsd alleles among strains occurred.
Results: Several Salmonella strains were found to be evolutionarily discordant when hsd gene trees were compared to known markers of S. enterica chromosome evolution (e.g., mdh). Additionally, several distinct clusters of mdh and mutS alleles were coalesced into single hsd clades for the three type I R-M loci. Analyses of congruence among hsd genes showed nearly unanimous discordance, the only exception being the hsdM/S2 comparison (p = 1.0).
Conclusions: These findings suggest that the type I R-M operon in Salmonella is an evolutionary mosaic, subject to numerous HGT events. These data demonstrate that HGT has been a common occurrence in hsd gene evolution and point to a genetic compatibility among closely-related salmonellae for exchange of hsd alleles. This may explain in part why Salmonella known to share homologous genomes and common niches are permitted to exchange DNA more freely.
Effects of Androstenedione on Total Free Fatty Acids in Pregnant Rats.
I. A. Ross, P. P. Sapienza, W. D. Johnson, R. L. Sprando, K. R. O'Neill, S. C. Sahu, T. J. Flynn, P. L. Wiesenfeld, T. F. Collins, C. S. Kim, FDA
Background: Androstenedione is used as a dietary supplement to enhance athletic performance.
Methods: Rats were treated with androstenedione (60 mg/kg/day) from 2 weeks before mating to gestation day (GD) 19. On GD 20, maternal blood, brain, and livers and fetal brain were analyzed for free fatty acids (FFAs).
Results: Androstenedione produced changes in the level of selected FFAs in the target organs. The concentrations of palmitic acid and stearic acid for the plasma were 34.82 ± 2.85 µg/ml and 15.96 ± 1.38 µg/ml, respectively, in controls and 39.90 ± 4.03 µg/ml and 22.60 ± 4.02 µg/ml, respectively, in rats treated with 60 mg/kg. The results were not significantly different between the controls and treated rats. However, oleic, linoleic, and docosahexaenoic acids (DHA), were 17.94 ± 2.06 µg/ml, 24.23 ± 2.42 µg/ml, and 4.08 ± 0.53 µg/ml, respectively, in controls and none of these were detectable in treated plasma. Palmitic, stearic, oleic, linoleic, and DHA were present in both control and treated livers. In liver, linoleic and DHA were 87% (0.05 < p < 0.1) and 169% (p < 0.05) increased, respectively, over controls. Palmitic, stearic, and oleic acids were not significantly affected by the treatment. Palmitic, stearic, and oleic acids were present in treated maternal and fetal brains while linoleic acid was found only in control fetal brain. DHA was present only in the control maternal brain (0.02 ± 0.02 µg/mg protein) and fetal brain (0.24 ± 0.15 µg/mg protein).
Conclusions: The results indicated that androstenedione exhibits different effects on the FFA composition of target organs during pregnancy.
Hearing Loss in Patients Treated with Itraconazole Standard and Pulse Therapy: A Review of FDA's Adverse Event Reporting System (AERS).
A. M. Rothstein, M. M. Truffa, DDRE/ODS/FDA, Rockville, MD
Purpose: Cases of hearing loss temporally related to use of standard and "pulse" itraconazole have been reported. These events may be due to direct ototoxicity of itraconazole, drug interaction, saturable metabolism, autoimmunity reaction, or microvascular pathology.
Methods: AERS was searched using the MedDRA grouping terms of Hearing Disorders and Hearing Losses for reports of hearing loss with itraconazole.
Results: We identified 30 cases of hearing loss in patients receiving itraconazole from 1993 to 2004; 21 from domestic and 9 from foreign sources. Fifteen patients were receiving "pulse" itraconazole therapy (400 mg daily x 1 week, 3 weeks off itraconazole). In 13 cases, quinidine was given concomitantly. The majority of cases (73%) occurred in males with a mean age of 66 years (range 38-82). There was an apparent dose effect with 18 patients (60%) receiving a daily dose of 400 mg or more. Only 3 patients reported a previous history of hearing loss. There was a positive dechallenge in 27 cases and a positive rechallenge in 7 cases when patients resumed "pulse" itraconazole. After discontinuation of itraconazole, 21 patients had a complete recovery, 4 had improvement in hearing loss, and 1 had permanent deafness; outcome was unknown in 4 cases.
Conclusions: Although this analysis was based on spontaneously reported AEs with known limitations, these data suggest that hearing loss may be related to itraconazole, especially with "pulse" therapy and/or use with quinidine.
Disclaimer: Views expressed in this abstract are those of the authors and not necessarily of the FDA.
Foodborne Outbreaks and Latinos: 1973-2004 A Retrospective
L.A. Solorzano, San Francisco District
In the United States (US) food-borne illnesses have been described as affecting an estimated 76 million individuals yearly, requiring 325,000 hospitalizations, and resulting in 5,000 deaths (Mead et al). Summaries correlating epidemiological data to such illnesses in the US Latino population are sparse. Pathogens including Listeria monocytogenes, Brucella melitensis, Brucella abortus, Mycobacterium bovis, (cheese); Non-typhoid Salmonella (soft cheeses and mangoes); Vibrio vulnificus (raw oysters); and Salmonella typhi (imported mamey), have been associated with foodborne illness in the US Latino population. Many more unreported foodborne illness cases exist for this population, which are not reported. From 1973 - 2004 there are reports of high morbidity and mortality implicating foodborne pathogens affecting the US Latino population. Results of a review indicates that food preference, social and cultural behaviors, pre-existing conditions, and demographic factors, place this population at high risk for foodborne illness.
Minimizing hypoglycemia attributed to an interaction between icodextrin peritoneal dialysis solution and blood glucose test strips: Utility of a U.S. postmarketing safety program
M. R. Southworth, PharmD, C. Kortepeter, PharmD, S. Lu, RPh, Office of Drug Safety, CDER, FDA, Rockville, MD
Background
The FDA approved icodextrin peritoneal dialysis solution (Extraneal, Baxter Healthcare Corporation) on December 20, 2002. The presence of maltose, a metabolite of icodextrin, can falsely elevate blood glucose readings in patients utilizing blood glucose monitors/test strips with nonglucose-specific, glucose dehydrogenase pyrroloquinolinequinone (GDH PQQ) technology. Falsely elevated blood glucose readings may cause patients to receive more insulin than is necessary with resultant hypoglycemia. Actions taken by the FDA and manufacturer to minimize this risk include healthcare provider (HCP) education, patient education and training (patient package insert, written safety information, MedicAlert, wallet cards), appropriate product labeling, HCP and patient surveys, expedited adverse event reporting, and communication with glucose monitor/test strip manufacturers via collaboration with CDRH. The purpose of this study was to assess the number and severity of adverse events relating to hypoglycemia and icodextrin post-approval.
Methods
A review of hypoglycemic events associated with icodextrin was performed using the FDA's Adverse Events Reporting System database to capture cases during the 2 years post-approval.
Results
Eighteen cases involving hypoglycemia were contained in the database (13 foreign; 5 domestic). Of the 5 domestic cases, one had a serious outcome that involved an emergency room visit. The adverse event in the remaining cases was limited to erroneous blood glucose readings, with symptoms (2) and without (2).
Conclusion
Erroneous blood glucose readings leading to hypoglycemia is a potentially lethal adverse event related to icodextrin use. It is encouraging that the number of reported hypoglycemic adverse events is low and these educa tional programs should be continued.
Radiation Safety in Medicine: Radiation Dose and Risk Assessment
O.H. Suleiman, CDER, FDA, Rockville, MD
In FDA many disciplines deal with assessing the safety associated with ionizing radiation, a carcinogen. Although the medical benefits are almost always greater than the radiation risk, society is generally more sensitive to the risks associated with radiation than other comparable risks. Understanding and communicating these risks, in a way understandable by society, is essential in the overall assessment of the safety of FDA regulated medical products.
The increasing complexity and frequency of new imaging procedures has increased the need to better understand the radiation doses patients and research subjects receive. The dose from a fused positron emission tomography (PET)/computed tomography (CT) procedure may be equivalent to 1000 chest x-rays, 6.7 years of natural background radiation, or the risk of lifetime cancer mortality of 1 in 1000. Such radiation doses will be compared along with a discussion of relative risk. The concept of organ dose will be presented along with the International Council on Radiation Protection (ICRP) term "effective dose", which allows partial body irradiations to be compared with whole body irradiations.
Lactic Acidosis: Unraveling the Individual Toxicities of Drugs Used in HIV and Diabetes Polytherapy by Hierarchical Bayesian Logistic Regression Data Mining
A. Szarfman1 , W. DuMouchel2 , D. Fram2 , J. M. Tonning1 , J. Almenoff3 , R. D. Fleischer1 , J. G. Levine1 , 1CDER, FDA, Rockville, MD, 2Lincoln Technologies, Wellesley Hills, MA, 3GlaxoSmithKline, Research Triangle Park, NC
Background: FDA has been utilizing the Multiple Item Gamma Poisson Shrinker (MGPS) algorithm in its data mining analyses of its Adverse Events Reporting System (AERS) database containing reports of adverse drug events. MGPS utilizes disproportionality analysis, combined with Bayesian shrinkage, to identify drug-event combinations that appear with higher than expected frequency among the reports in the database. MGPS includes a Maentel-Haenszel style approach for adjusting the expected counts for potential strata heterogeneity; we routinely stratify by over 900 categories derived from different combinations of age, sex, and year of report.[i],[ii]
However, in situations where multiple drugs are often used in combination, results from MGPS analysis may be subject to "signal leakage,"a phenomenon whereby a safety signal truly associated with Drug A may manifest itself as an apparent signal with Drug B simply due to the fact that the two drugs are frequently co-prescribed.[iii] MGPS may also be subject to "signal masking" whereby a signal for Drug A can be diminished in magnitude if Drug B has a very dominant signal in the database. This issue is more common in small, non diverse databases.[iv]
Herein, we examine the utility of an empirical Bayes method, the Hierarchical Bayesian Logistic Regression (HBLR), that effectively adjusts both for signal leakage and for masking.[v] HBLR uses a prior distribution (estimated from the data) to improve the modeling of the joint associations for up to hundreds of drugs with a logistic regression response variable.
Clinical context: Lactic acidosis is a serious toxicity known to be associated with the use of some of the nucleoside reverse transcriptase inhibitors (NRTIs) and is believed to result from mitochondrial DNA depletion, and downstream effects on oxidative metabolism. Since 1998, FDA has required the labeling of all HIV nucleoside and nucleoside analogs to carry a warning to alert clinicians and patients to this serious therapeutic complication. In contrast, the protease inhibitors (PIs), and non-nucleoside transcriptase inhibitors (NNRTIs) have not been independently associated with lactic acidosis. NRTIs are commonly co-prescribed with PIs and NNRTIs in treating HIV/AIDS. Phenformin was withdrawn from marketing due its association with lactic acidosis. Metformin's lactic acidosis appears to develop in patients with underlying medical conditions, most notably renal insufficiency. Lactic acidosis with phenformin may be due to disruption of oxidative phosphorylation in mitochondria. Lactic acidosis with metformin is probably due to back-up of lactate from reduced gluconeogenesis. These two mechanisms are not mutually exclusive.
Methods: We conducted an initial MGPS analysis using the AERS database to identify drugs which demonstrated at least a moderately strong association for lactic acidosis. We then performed an HBLR analysis that included all of these drugs in the model. Some of the nucleoside anti-HIV drugs and the biguanide anti-diabetes drugs (phenformin and metformin) have been shown historically to be associated with the development of lactic acidosis as an adverse drug reaction. We then used our external scientific knowledge to validate the results of the HBLR; in doing this, we focused specifically on anti-HIV and anti-diabetes drugs because some, but not all of the drugs to treat these conditions are known to be associated with lactic acidosis.
Results: With MGPS the signal scores with the NRTIs stavudine, lodenosine, and didanosine are higher than with other NRTIs, as well as with PIs and NNRTIs. However, the PIs and NNRTIs do show false positive signals for lactic acidosis with MGPS, based on our external scientific knowledge of these products. Using HBLR, scores for the NRTIs and analogue NRTI drugs (e.g., stavudine, lodenosine, didanosine, tenofovir, zidovudine, and lamivudine) remained high, while those for the PIs and NNRTIs were markedly diminished. This is consistent with signal leakage from the NRTIs that is adjusted with HBLR.
MGPS also shows signal leakage with antidiabetic drugs; moderate signals seen with MGPS for antidiabetic drugs other than biguanides were markedly decreased after adjustment with HBLR. Scores were higher for phenformin and metformin than for the other anti-diabetes drugs studied, with phenformin showing the highest scores across all drugs analyzed. Although MGPS scores for phenformin and metformin were high, these values increased with HBLR, suggesting that the magnitude of these signals is "unmasked" though the use of HBLR.
Conclusions: HBLR applied to AERS data showed a more discriminating detection of lactic acidosis associations than MGPS for drugs that are commonly used in polytherapy regimens for both HIV and diabetes. These findings are consistent with our current clinical knowledge of the associations of lactic acidosis with NRTIs and biguanides. These data suggest that HBLR may be a useful adjunct to MGPS in post-marketing safety assessments, especially in polytherapy regimens.
[3] GlaxoSmithKline manufactures some of the products described in these analyses
References:
PROJECTING THE NUMBER OF EXCESSIVE ADVERSE EVENTS DUE TO A TREATMENT
J. J. Zhang1 , Y. Tsong1 , R. T. Oneill2 , 1CDER, FDA, Rockville, MD, 2CDER, FDA. Rockville, Md
The projection of the number of a specific excessive adverse event due to a treatment is important in the assessment of the risk impact of the treatment on the patient population. The projection may be carried out at three stages: (1) pre-approval stage (2) early-postmarketing stage and (3) postmarking stage with observational studies. At the pre-approval stage, the number of the excessive events may be projected into the patient population for the label usage as well as for the simulated labeled and off-label usage. This projection may be helpful in risk-benefit decision making as part of the drug approval process. At the early-postmarketing stage, the drug usage is monitored but with no available proper observational studies for risk update, the estimate of the number of the excessive events can be projected into the patient population after adjusting the frequency distribution derived from the observed usage data set. At the postmarketing stage with available observational studies, the projection can be made by combining the estimate derived from clinical studies and the estimate derived from observational data. The approach is illustrated with published vioxx data, Caremark and IMS data .
Consumer Reactions to Modifications of Allergen Information on the Food Label: Focus Group Results
L. A. Verrill, Ph.D., A. L. Lando, M.P.P., C. J. Choiniere, Ph.D., OSAS/DMS/Consumer Studies
Background: Food allergies affect approximately 4 percent of Americans. Each year about 30,000 individuals are treated in emergency rooms and about 150 individuals die because of allergic reactions to food. The food label is the major purveyor of information about allergenic substances in food. The Food Allergen Labeling and Consumer Protection Act of 2004(FALCPA) (Public Law 108-282) requires that food labels include more information, and in a more consumer-friendly form, concerningabout concerning allergenic ingredients in food.
Methods: Focus groups with food-allergic, adult consumers in four separate U.S. regions were used to assess the utility of food allergen labeling for food allergic individuals. The participants reacted to a variety of source labeling formats and a number of advisory statements for allergenic ingredients.
Results: Focus group participants would search the food ingredients list if they did not see their particular allergen listed in an allergen source labeling "Contains" statement. Participants also reacted to advisory statements. For example, participants said the statement "This product was produced on a line that also produces products that contain peanuts" was more informative than the statement "May contain peanuts." Participants with less severe allergies said they might eat an allergenic food if they "wanted it badly enough."
Conclusion: The focus groups provided important data about allergic consumers' information needs. The results will help inform the content of a nationally representative survey on food allergen labeling and will contribute to the design of an experiment to assess the effectiveness of different allergen labeling formats.
POST-LICENSURE SAFETY SURVEILLANCE FOR RECOMBINANT HUMAN COAGULATION FACTOR VIIa
J. J. Wood, K. A. O'Connell, R. P. Wise, CBER, FDA, Rockville, MD
Background: FDA licensed recombinant human coagulation Factor VIIa (rFVIIa) in 1999 for bleeding in hemophilia A or B patients with inhibitors to Factors VIII or IX. Post-licensure, there has been increasing use of rFVIIa in patients without hemophilia.
Methods: We reviewed thromboembolic adverse events (AEs) reported to FDA's Adverse Event Reporting System for rFVIIa through 2004. Manufacturer reporting is mandatory, but reporting by clinicians and others is voluntary. Therefore, AERS is likely to under-represent actual events.
Results: A total of 185 thromboembolic AEs among 168 patients receiving rFVIIa were reported to FDA; 59 of the 168 patients were enrolled in a post-marketing study. Unlabeled indications accounted for 151 patients, including surgical procedures, intracranial hemorrhage, and others. Most patients were actively bleeding (n=115). Reported AEs were thromboembolic cerebrovascular accident (n=39), myocardial infarction (n=34), other arterial thromboses (n=26), pulmonary embolism (n=32), other venous thromboses (n=42), and clotted devices (n=10). In 36 of 50 deaths, the probable cause (from autopsy or clear clinical data) was the thromboembolic event. In 144 patients with timing information, 73 events occurred in the first 24 hours (30 within 2 hours). Pro-coagulant concomitant medications were identified in 64 patients.
Conclusions: Most reported thromboembolic AEs followed the use of rFVIIa for unlabeled indications, often resulting in serious morbidity and mortality. However, evaluation of the reports is confounded by underlying diagnoses, concomitant medications, pre-existing conditions, and inherent limitations of passive surveillance. Randomized clinical trials designed to establish the safety and minimum effective dose in patients without hemophilia are needed.
UV Doses Worldwide.
D.E. Godar, OSEL, CDRH, FDA, Silver Spring, MD
UV affects human health. Human exposure to UV causes a few beneficial health effects, like vitamin D3 formation, while it causes many detrimental health effects: sunburn, ocular damage, photoaging, immune suppression, DNA damage, and skin cancer. In countries with fair-skinned populations, skin cancer is the most diagnosed of all cancers. In the U.S. in 2002, there were over one million new skin cancers. That means 1 out of every 285 people got skin cancer. Skin cancer of fair-skinned individuals is increasing at an alarming rate (4-6% per yr) around the world and has now reached so-called "pandemic" proportions. Thus, it is important to know what UV doses people around the world get throughout their lives. This review covers how the outdoor UV doses are weighted for different biological effects, the most commonly used measuring devices for terrestrial and personal UV doses, the natural and other effects on terrestrial and personal UV doses, the time people spend outside, their ambient exposures, and the terrestrial and personal UV doses of adult outdoor and indoor workers as well as children and adolescents around the world. Overall, outdoor working adults get about 10%, while indoor working adults and children get about 3% (2-4%) of the total available annual UV (on a horizontal plane). People's UV doses increase with increasing altitude and decreasing latitude; most indoor working adult Europeans get 10,000-20,000, Americans get 20,000-30,000, and Australians are estimated to get 20,000-50,000 J/m2 per yr (excluding vacation, which can increase the dose by 30% or more).
A Melanoma Hypothesis: The Paradox of Outdoor and Indoor Solar UV Exposures.
D. E. Godar1 , J. C. Dowdy2 , S. G. Coelho3 , R. J. Landry3 , R. M. Sayre2 , J. C. Van der Leun4 , 1OSEL, CDRH, FDA, Silver Spring, MD, 2Rapid Precision Testing Laboratories, Cordova, TN, 3OSEL, CDRH, FDA, Rockville, MD, 4ECOFYS, Utrecht, The Netherlands
Melanoma has been increasing at a steady logarithmic rate in fair-skinned, indoor workers since the mid 1930's. A paradox exists between indoor and outdoor workers because indoor workers get three to nine times less solar UV (290-400 nm) than outdoor workers, yet have a higher incidence of melanoma. Thus, another factor is involved that increases the melanoma risk for indoor workers. We hypothesize this factor involves indoor workers getting exposed during work to only UVA (321-400 nm) passing through windows and, unlike outdoor workers have lower levels of vitamin D3 in their skin because only UVB (290-320 nm) can make vitamin D3, while UVA can break it down. Skin cells can convert vitamin D3 to the hormone, 1, 25-dihydroxyvitamin D3, or calcitriol, which causes growth inhibition and apoptotic cell death of melanoma cells in vitro and in vivo. We measured and calculated the outdoor and indoor solar UV contributions toward different biological endpoints by weighting the emission spectra by the action spectra: erythema, squamous cell carcinoma, melanoma (fish), and previtamin D3. We found indoor solar UVA irradiances are about 60 times greater than fluorescent lights and represent about 25 percent (or 5-10 W/m2) of the outdoor UVA irradiances. In addition, we find that people cannot produce vitamin D3 inside, only outside where there is UVB. We agree that intense, intermittent outdoor UV exposures and sunburns initiate melanoma; we now propose that inadequately maintained levels of vitamin D3 in the skin and indoor UVA exposures that break it down promotes melanoma.
Post-Marketing Reports of Serotonin Syndrome Associated With Linezolid
A. Sorbello, R. Wassel, J. Soreth, S. Nambiar, CDER, FDA, Rockville, MD
Background: Serotonin syndrome, a potentially life-threatening condition, is frequently related to drug exposure(s) causing excessive serotonin neurotransmission. In the original NDA for linezolid, an oxazolidinone antibiotic and weak monoamine oxidase inhibitor, serotonin syndrome was not observed among the 3,347 patients in the safety population. We report serotonin syndrome in 50 linezolid-treated patients who were treated with concomitant serotonergic agents based on a post-marketing safety analysis.
Methods: Post-marketing adverse event (AE) reports among linezolid-treated patients submitted to the FDA Adverse Event Reporting System from July, 2000-November, 2004 were reviewed.
Results: A total of 55 AE reports were retrieved involving 50 patients (28 with serotonin syndrome; 22 with suggestive symptoms). The 28 patients with serotonin syndrome consisted of 26 adults and 2 children (age range 4-85 years). There was one death; the remaining patients recovered within 1-9 days following drug discontinuation. Two patients required treatment with cyproheptadine. The 22 patients with suggestive symptoms were adults (age range 28-88 years). There were no deaths; 8 patients recovered within 1 week following drug discontinuation; outcome was not reported for 14 individuals. Drugs that were administered concomitantly with linezolid included selective serotonin reuptake inhibitors (SSRIs) (65.5%), tricyclic antidepressants (8.6%), mixed norepinephrine/SSRI agents (8.6%), dopaminergic agents (6.9%), CNS stimulants (5.2%), and others (5.2%).
Conclusion: Our post-marketing safety analysis indicates that concomitant administration of linezolid with serotonergic agents is associated with the subsequent development of serotonin syndrome. Linezolid's product label was updated to reflect this potential drug interaction. Data was insufficient to definitively assess the strength of the association.
LC-Visible Analysis of Malachite Green and Leucomalachite Green (LMG) Residues in Salmon, Catfish, and Trout with In-Situ LMG Oxidation
W. C. Andersen, S. B. Turnipseed, J. E. Roybal, Animal Drugs Research Center, FDA, Denver District
Background: Malachite green (MG) is an effective topical fungicide used by the aquaculture industry. MG and the metabolite, leucomalachite green (LMG), are suspected mutagens. For this reason, MG is not approved as an aquaculture veterinary drug in many countries including the United States, Canada and the European Union. A liquid chromatographic method was developed for the determination of malachite green in salmon, catfish, and trout.
Method: MG and LMG residues were extracted from tissues with ammonium acetate buffer and acetonitrile, and then isolated by partitioning into methylene chloride. LMG was quantitatively oxidized to the chromic malachite green by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Samples were cleaned-up by solid phase extraction with alumina and propylsulfonic acid phases. Extracts were analyzed for MG by LC with visible detection at 618 nm, using isocratic elution and a C18 column.
Results: The method was validated for tissues fortified with LMG at 1, 2, 4, and 10 ng/g (ppb) with average recoveries of 95.4 % and an RSD of ± 11.1 % for salmon, 83.1 ± 11.4 % for catfish, and 86.9 ± 4.7 % for trout. Salmon exposed to water with 10 µg/L MG were extracted and found to contain 34 and 46 ng/g total MG and LMG residues 2 and 4 hours after exposure, respectively. Residues in tissue extracts were confirmed by LC-MSn.
Conclusions: This method provides a reliable LC-visible method for the determination of LMG and MG in fish that is suitable for routine regulatory analysis with a detection limit of 1.0 ng/g.
Multiclass Confirmation of Veterinary Drug Residues in Fish by Liquid Chromatography-Ion Trap Mass Spectrometry
S. Smith, C. Gieseker, R. Reimschuessel, M. C. Carson, Office of Research, CVM, FDA, Laurel, MD
Background. The FDA is responsible for ensuring the safety of seafood, but currently lacks methods to efficiently monitor aquacultured fish for all the diverse residues that could be present.
Methods. We used the selective power of LC-ion trap mass spectrometry to detect 35 compounds from a variety of drug classes in a single analysis. Finely ground fish muscle was extracted with acetonitrile and hexane. The acetonitrile phase was evaporated, redissolved in water and acetonitrile, and washed again with hexane. A portion was analyzed by gradient chromatography on a phenyl column. MS2 or MS3 spectra were monitored for each compound. Method performance was evaluated by the analysis over several days of replicate samples of control salmon, salmon fortified with a drug mixture at 1, 0.1 and 0.01 ppm, and salmon dosed with a representative from each drug class (lincomycin, florfenicol, ampicillin, cephalexin, metronidazole, albendazole, oxolinic acid, malachite green, tylosin, Romet).
Results. More than half (20) of the 35 drugs were detectable at 0.01 ppm, the lowest fortification level. This included all of the quinolones and fluoroquinolones, the macrolides, malachite green, and most of the imidazoles. Florfenicol amine, metronidazole, sulfonamides, tetracyclines, and most of the betalactams were detectable at 0.1 ppm. Due to lower instrument sensitivity, ivermectin and penicillin G were only detectable in the 1 ppm fortified samples. With the exception of metronidazole and tylosin, residue presence was confirmed in all the dosed salmon.
Conclusions. This method can enable FDA to efficiently monitor fish for the presence of veterinary drug residues.
Probing Alterations of the Mitochondrial Membrane Proteome in a Murine Model of Parkinson's Disease using Liquid Chromatography-Tandem Mass Spectrometry.
D. C. Cawthon1 , J. A. Gantt2 , M. B. Goshe2 , Z. A. Xu1 , W. Slikker, Jr.1 , S. F. Ali1 , 1NCTR - Neurotoxicology, 2North Carolina State University
Background: An important murine model of Parkinson's Disease utilizes the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Both mitochondrial inner and outer membrane proteins are directly involved in the hypothesized mechanisms of MPTP toxicity. Additionally mitochondrial membrane proteins are likely to be affected by oxidative damage, post-translational modifications, and/or altered production/degradation in this model.
Methods: A method was developed to analyze mitochondrial membrane fractions by multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Enriched mitochondrial fractions were isolated from murine forebrains following homogenization and centrifugation through Percoll™ gradients. Mitochondrial membranes from these fractions were further enriched using a sodium carbonate procedure followed by organic-aqueous extraction of the membrane proteins.
Results: Protein profiles were obtained for both control and treatment groups. Based on the collected MS/MS spectra, 853 proteins (2276 unique peptides) were detected in the sample obtained from the MPTP-treated mice whereas 784 proteins (2334 unique peptides) were identified in the control. A total of 422 proteins were common to both samples, which included numerous proteins comprising complex I of the respiratory chain and the subunits of complexes II, III, IV, and V (F0F1 ATP synthase). Analysis of the proteins unique to the control or the treated samples revealed alterations in the mitochondrial membrane proteome following MPTP administration.
Conclusions: The method reported here utilizing mitochondria enrichment and organic-aqueous extraction, solubilization, and proteolysis permits both highly hydrophobic integral membrane proteins and membrane associated proteins to be effectively identified from brain tissue using multidimensional LC-MS/MS.
Liquid Chromatography/Tandem Mass Spectrometry for the Determination of Bound Residues of Nitrofurans in Channel Catfish
P. Chu1 , M. Lopez1 , A. Abraham2 , K. R. El Said2 , S. M. Plakas2 , 1CVM, FDA, Laurel, MD, 2CFSAN, FDA, Dauphin Island, AL
Background: Nitrofurans are antibacterials commonly used in aquatic species and terrestrial animals for treatment and control of bacterial and protozoan infections. However, nitrofurans have been banned for use in food-producing animals in most countries because of their mutagenicity and potential carcinogenicity. Analytical methods are, therefore, needed for both research and monitoring purposes.
Methods: An LC/MS/MS method is described for the quantitation and confirmation of bound residues of nitrofurans at 2 ppb in channel catfish. The method involves prewashing 2 g of channel catfish tissue sequentially with 5 mL each of 70% aqueous methanol, ethyl acetate, and ethanol. To the remaining pellet is added 10 mL of 0.125 M HCl followed by 400 :L of 50 mM 2-nitrobenzaldehyde in DMSO. The mixture is placed in an incubator at 37oC overnight with gentle shaking, during which the side-chains of the bound nitrofuran residues are released through hydrolysis and simultaneously derivatized with 2-nitrobenzaldehyde. After liquid-liquid extraction cleanup, the derivatives are detected and quantitated by LC/MS/MS.
Results and Conclusions: The method was validated at 1, 2, and 4 ppb using fortified tissues and tissues of channel catfish treated with nitrofurans.
Development of a high-performance liquid chromatography (HPLC) stability-indicating method for gabapentin
A. B. Ciavarella1 , A. Gupta1 , D. A. Volpe1 , M. A. Khan1 , V. A. Sayeed2 , A. S. Hussain3 , P. J. Faustino1 , 1Division of Product Quality Research, 2Office of Generic Drugs, 3Office of Pharmaceutical Science
Background: Gabapentin, an anticonvulsant drug is used in the treatment of epilepsy. Few analytical methods are present in the scientific literature for the chromatographic analysis of gabapentin. Gabapentin (pKa = 3.7) is a challenging drug to chromatograph since it's a small, highly polar molecule poorly retained on most reversed phase (RP) HPLC column chemistries. Additionally, its main degradation product (3,3-pentamethylene-γ-butyrolactam), is very non-polar, making it difficult to efficiently analyze both compounds. As part of a series of stability and forced degradation studies, a novel stability-indicating method for gabapentin API and drug product was developed.
Methods: Samples were analyzed on a HP 1050 HPLC (Wilmington, DE) with UV detection at 210 nm. Separation was obtained on a Brownlee (Boston, MA) Spheri-5 RP cyano column with phosphate buffer (pH=6.2), acetonitrile, tetrahydrofuran (92:5:3%) mobile phase delivered isocratically (1 ml/min.). The method was validated according to USP <1225> Validation of Compendial Methods. The API and drug product were tested using standard forced degradation conditions for 24 hrs: 0.1N HCl, 0.1N NaOH, and 50 oC.
Results: Gabapentin and its major impurity (RT 4.2 and 9.6 min.) were well resolved from co-eluting peaks resulting from API and DP samples subjected to forced degradation conditions. The total analysis, equilibration and recovery time was 15 minutes
Conclusions: A simple and selective RP stability-indicating HPLC method for gabapentin was developed using a cyano-chemistry sorbent that efficiently analyzed compounds of significantly different polar selectivities. This novel analytical tool will efficiently allow for additional product characterization to ensure drug product quality.
Detection of Penicillin Residues on Environmental Swabs by Liquid Chromatography and Mass Spectrometry
S. B. Clark1 , C. M. Karbiwnyk1 , M. R. Madson1 , J. A. Hurlbut2 , J. N. Sofos3 , 1FDA, Denver, CO, 2Western Washington University, Bellingham, WA, 3Colorado State University, Ft. Collins, CO
Background: Based on the Code of Federal Regulations, a zero tolerance exists for penicillin cross-contamination of non-penicillin products. To date, there are neither official nor unofficial methods for the identification and/or confirmation of penicillin residues in non-penicillin finished bulk- or dosage-form products or environmental samples from related industries.
Methods: A liquid chromatographic (LC) method is described for the detection of four penicillin residues (amoxicillin, penicillin G, penicillin V, and ampicillin) on environmental swabs collected from a pharmaceutical firm which compounds and repacks penicillin and non-penicillin products. The method includes extraction of the swabs with a phosphate buffer followed by concentration of the penicillin residues on a Waters extraction cartridge. The cartridge is eluted with acetonitrile and the eluate is evaporated to dryness. The residue is reacted with benzoic anhydride at 50oC for 5 min and with 1,2,4-trizole and mercury (II) chloride solution pH 9 at 65oC for 30 min. The derivatized compounds are eluted on a Symmetry C8 column with a mobile phase containing acetonitrile, phosphate buffer, and sodium thiosulphate. In addition, the LC/mass spectrometer (MS) conditions are described for the confirmation of amoxicillin residue from the swab extracts.
Results: Amoxicillin residue was detected in seven of the 25 swab extracts and confirmed in five by LC/MS. Positive presence of amoxicillin was confirmed in a sample if the MS ion chromatogram showed a peak retention similar to that of a standard, and the secondary MS showed product ions of 114, 208, and 234. Recoveries of buffer samples fortified at approximately 100 to 25 ng/mL with the four standards of interest were approximately 50%.
Conclusion: Identification and confirmation of individual penicillin residues is possible at the needed low part-per-billion levels to determine the extent of cross-contamination in repacking pharmaceutical establishments.
Analysis of Penicillin Residues on Environmental Swabs by Liquid Chromatography and Mass Spectrometry
S. B. Clark1 , C. M. Karbiwnyk1 , M. R. Madson1 , J. A. Hurlbut2 , J. N. Sofos3 , 1FDA, Denver, CO, 2Western Washington University, Bellingham, WA, 3Colorado State University, Ft. Collins, CO
Background: Based on the Code of Federal Regulations, a zero tolerance exists for penicillin cross-contamination of non-penicillin products. To date, there are neither official nor unofficial methods for the identification and/or confirmation of penicillin residues in non-penicillin finished bulk- or dosage-form products or environmental samples from related industries.
Methods: A liquid chromatographic (LC) method is described for the detection of four penicillin residues (amoxicillin, penicillin G, penicillin V, and ampicillin) on environmental swabs collected from a pharmaceutical firm which compounds and repacks penicillin and non-penicillin products. The method includes extraction of the swabs with a phosphate buffer followed by concentration of the penicillin residues on a Waters extraction cartridge. The cartridge is eluted with acetonitrile and the eluate is evaporated to dryness. The residue is reacted with benzoic anhydride at 50oC for 5 min and with 1,2,4-triazole and mercury (II) chloride solution pH 9 at 65oC for 30 min. The derivatized compounds are eluted on a Symmetry C8 column with a mobile phase containing acetonitrile, phosphate buffer, and sodium thiosulphate. In addition, the LC/mass spectrometer (MS) conditions are described for the confirmation of amoxicillin residue from the swab extracts.
Results: Amoxicillin residue was detected in seven of the 25 swab extracts and confirmed in five by LC/MS. Positive presence of amoxicillin was confirmed in a sample if the MS ion chromatogram showed a peak retention similar to that of a standard, and the secondary MS showed product ions of 114, 208, and 234. Recoveries of buffer samples fortified at approximately 100 to 25 ng/mL with the four standards of interest were approximately 50%.
Conclusion: Identification and confirmation of individual penicillin residues is possible at the needed low part-per-billion levels to determine the extent of cross-contamination in repacking pharmaceutical establishments.
Analysis of Ginkgo Marker Compounds in "Functional Food" Products
L. S. DeJager, G. A. Perfetti, G. W. Diachenko, CFSAN, FDA, College Park, MD
Introduction: Recently, several conventional food products containing botanical dietary supplement ingredients have appeared on the market. Because most dietary supplements are not approved food additives nor have they been determined to be GRAS, these so called "functional" food products may be subject to regulatory action. Ginkgo biloba has been used in traditional medicine for centuries as a treatment for circulatory diseases and memory loss resulting from advancing age. Ginkgo is one of the top selling herbal dietary supplements in the US and a number of foods containing ginkgo have been introduced on the domestic market. This research focused on the development of analytical methodology for the determination of ginkgo marker compounds in beverages and dietary supplements.
Methods: A large number of compounds have been isolated from ginkgo leaves. The terpene lactones and flavonoid compounds are thought to be the components with pharmacological activity. In this study, four diterpene lactones and bilobalide were used as characteristic marker compounds for the presence of ginkgo. Solvent extraction and solid phase extraction methods were developed for sample clean-up and preconcentration prior to APCI-LC-MS analysis.
Results: Thirteen drinks, two teas and five dietary supplements were purchased and analyzed using the optimized method. Total marker compound concentrations in the drink products ranged between 546 and 21ng/mL while tea concentrations ranged from 25.8 to 11.6µg/mL.
Conclusions: Ginkgo marker compounds were found in all beverage products tested which listed ginkgo as an ingredient. Concentrations found in the teas were significantly higher than those measured in the beverage products.
Monitoring Deaeration of Aqueous Media for Dissolution Testing by Total Dissolved Gas Pressure Meter
Z. Gao, T. Moore, CDER, FDA, St. Louis
Background: The USP states that if dissolved gasses in a dissolution medium affect the dissolution results for a product then the medium must be degassed. Several articles have been written about the use of Oxygen meters to determine the extent of media degassing but PhRMA and USP do not consider these meters to be acceptable for testing dissolution media because they do not detect total gasses.
Method: We test the hypothesis that measurement of both total gas pressure and oxygen concentration will allow correlation between deaeration method and dissolution results. Various deaeration methods are evaluated. Correlations are made between the extent of degassing and dissolution results using an in house calibrator tablet (NCDA#2).
Results: The USP method, with or without heating to 41°C, and a simpler degassing method developed in-house both show low total dissolved gasses and yield equivalent dissolution results. Both helium and nitrogen sparging reduce dissolved oxygen to very low levels; however, dissolution results for these two degassing methods were quite different from one another.
Conclusions: This study confirms that use of an oxygen meter alone is not a reliable method for monitoring dissolved gases for purposes of dissolution testing. The Total Dissolved Gas Pressure Meter does provide the information necessary to determine if a dissolution medium has been degassed sufficiently.
Evaluation of Commercial ELISA Assays for the Detection of Egg in Food
E. A.E. Garber, V. A. Brewer, CFSAN, FDA, College Park, MD
It is estimated that 6% of children under the age of 3 are allergic to some form of food with 1.3% allergic to eggs. Currently, the only method available for individuals allergic to a specific food to avoid an allergic reaction is avoidance of the allergenic food. To address the need for validated methods to test food products for the presence of allergens, the FDA has initiated studies to evaluate commercial immunology based assays for the detection of allergenic foods. Seven commercial ELISA-based assays for the detection of egg proteins were evaluated with six different matrices representative of various forms of processing. The matrices examined included baked goods, pasta -analyzed before and after boiling, vanilla ice cream, salad dressing (no processing), and phosphate buffered saline. Each of the matrices were spiked with either 0, 2, 5, 10, 25, or 100 ug/g of the NIST whole egg powder standard reference material (SRM) # 8415. Six of the ELISA kits relied on washing/partitioning to extract the antigenic biomarker into an aqueous buffer. The seventh kit was unique in that it employed reducing-denaturing conditions to extract the antigenic proteins. All seven kits readily detected egg spiked into foods which were subjected to none or minimal heating. However, the assays displayed significant differences in ability to detect egg in foods that were exposed to heat. Only the kit that employed reducing-denaturing conditions to extract the antigenic biomarkers detected egg in all matrices spiked with > 2 ug/g of the NIST SRM.
Evaluation of Commercial ELISA Kits for Wheat Allergens
V. A. Brewer1 , E. A.E. Garber1 , S. P. Amato2 , G. Orlowski2 , 1CFSAN, FDA, College Park, MD, 2JIFSAN, University of Maryland, College Park, MD
The incidence of wheat sensitivity has increased greatly in recent years. Individuals suffering from wheat sensitivity can be divided into two classes, those displaying a true IgE allergic response and those suffering from an intolerance. The most common form of wheat intolerance is celiac sprue disease. In either case, avoidance of the offending food is currently the only method to manage the problems. Enzyme-linked immunosorbent assay (ELISA) kits provide an effective and reliable method for the detection of wheat in foods. To protect sensitive individuals, the FDA has initiated a program to evaluate and validate test kits for the detection of food allergens such as wheat. The wheat kits detect gliadin, the prolamin portion of gluten, which compromises approximately 50% of the gluten. It is known that the kits will nonspecifically detect the presence of rye and barley prolamins too. Two commercial gluten kits have already been evaluated. Currently we are evaluating four more kits which have recently been developed for gluten. Transia Plate Prolamins and Transia Plate Gluten by Diffchamb SA (Lyon, France), Veratox Quantitative Gliadin Test by Neogen Corporation (Lansing, MI), and Wheat Protein ELISA Kit by Morinaga Institute of Biological Science, Inc. (Yokohama, Japan). The evaluation of these kits, which is still in progress, consists of three phases. The first is to determine the specificity of the assays (cross-reactivity) to other cereals and foods. The second is to determine the limits of detection and recoveries using spiked samples which are exposed to various forms of processing, such as baking and boiling. The third phase is a survey of commercially available commodities known to contain wheat and wheat products.
Evaluation of Commercially Available ELISA-Based Kits for Wheat Allergen Analysis in Malt Beverages
J. R. Ammann1 , M. A. Mabud1 , S. M. Dugar1 , E. A.E. Garber2 , D. L. Park2 , 1Alcohol and Tobacco Tax and Trade Bureau, US Treasury Dept., Beltsville, MD, 2CFSAN, FDA, College Park, MD
Background
Gluten is a group of cereal proteins comprised of approximately 50% glutelins and 50% prolamines. It is not known if gliadin, a wheat prolamine known to be involved in food allergies and intolerances, is present in malt beverages at concentrations high enough to elicit food sensitivities. An evaluation of commercially available methods for reliable gliadin detection in malt beverages was initiated.
Method
The evaluation utilized commercially available ELISA-based kits including R-Biopharm's RIDASCREEN® FAST Gliadin, Tepnel Biosystems' Gluten Assay Kit, and Neogen Corporation's Veratox® Quantitative Gliadin Test. Malt beverages tested include domestic, import, light, lager, stout, ale, non-alcoholic, flavored and wheat-labeled beers.
Results
A total of 18 commercially available malt beverages were used in the evaluation. Degassing and ethanol extraction of samples yielded no significant difference in gluten concentrations compared to non-prepared samples using the Tepnel and R-Biopharm kits. Hence, all analyses of malt beverage samples in the study were carried out without sample preparation when using the aforementioned kits. The Tepnel kit showed all non-wheat labeled beer gluten concentrations at or below the lower limit of quantitation for the kit, which is 10 ppm. The highest concentration of gluten in a non-wheat labeled beer was found in a lager at 10.4 ppm (+/- 0.6 ppm for eight runs). The Tepnel kit also showed gluten concentrations in the wheat-labeled beers above the saturation point of the kit (>200 ppm gluten). The spiking of a gluten-free malt beverage using Durum wheat (Sigma) resulted in gluten detection with good correlation to kit standards, indicating that the malt beverage matrix does not interfere with the ELISA-based analysis. The spiking experiment also demonstrated that the kit reliably detected gluten in a beer matrix within the linear range of the calibration curve (10-200 ppm gluten).
Conclusion
Our evaluation demonstrated that commercially available ELISA-based kits may be used to quantitate gluten in malt beverage matrices at levels as low as 10 ppm.
Evaluation of Actuation Parameters That Affect Nasal Spray Characteristics
C. Guo, DPA,CDER,FDA, St. Louis, MO
Background:Nasal spray drug products are normally characterized via measurement of shot weight, spray pattern, plume geometry and droplet size distribution (DSD). In this project, the actuation parameters, stroke length, hold time, inter-actuation delay time (IAD), actuation velocity and actuation acceleration were investigated to ascertain how they affect nasal spray characteristics. Previous published studies have dealt only with the affects of controlling the applied actuation force.
Methods: Pfeiffer nasal spray pump units filled with water were used in the study. Actuation parameters were adjusted using an automated actuation system, SprayVIEW NSx. Spray pattern and plume geometry measurements were carried out using a high speed optical spray characterization system, SprayVIEW NSP, and droplet size distribution analysis was performed using a Malvern 2600 laser diffraction system.
Results: Different actuation parameters affected the nasal spray characteristics in different ways and to different degrees. Among the five actuation parameters studied, stroke length and actuation velocity had a significant effect on the nasal spray characteristics, while the other actuation parameter had little, if any, effect. Compared to spray pattern, plume geometry and DSD, shot weight provided very little characterization information..
Conclusions: The findings from this work suggest that, for in-vitro BA and BE studies of nasal spray products, the actuation parameters, stroke length and velocity must be carefully selected. Spray pattern, plume geometry and DSD appear to provide critical data for assessment of nasal pump performance.
NMR Spectroscopy in Herbal Drugs and Dietary Supplements
G..M.. Hanna, NERL, ORA, Jamaica, NY
Background: Fatal toxicity, adulterations with prescription medicines, misidentifications, substitutions or false labeling are some of the problems with herbal drugs and dietary supplements.
Methods: We developed NMR spectroscopic methods permitting characterization and differentiation between isomers of aristolochic acid, (S)-S-adenosyl-L-methionine, and melatonin. The methods are able of assaying each component, contaminant or impurity in the respective herbal and dietary supplements.
Results: All NMR resonances were assigned on the basis of chemical shifts, spin multiplicities and decoupling experiments. The assignments were facilitated by comparing model compounds. Accuracy of NMR methods was established by analyzing sets of 10 synthetic mixtures containing selected internal standards and components of dietary supplements. Excellent agreement was found between assay results and quantities in mixtures. Lots from different commercial sources were assayed by the developed methods and the amounts of aristolochic acid I, aristolochic acid II, (S)-S-adenosyl-L-methionine, (R)-S-adenosyl-L-methionine, and melatonin were within the following ranges: 45.3-97.1%, 0-15.4%, 0-110%, 0-82.3%, and 89.9-109.8%, respectively.
Conclusions: The developed NMR methods greatly simplified characterization and simultaneous accurate quantification of the injurious isomers of aristolochic acid, the active (S)-S-adenosyl-L-methionine and its inactive (R)-diastereomer, and melatonin and its precursors L-tryptophan and tryptamine.Tighter controls of herbal and dietary supplements for consumer protection are greatly facilitated by the developed approaches.
Analysis of Consumer Cosmetic Products for Phthalate Esters
J. C. Hubinger, D. C. Havery, CFSAN, FDA, College Park
Background: Phthalate esters are present in many consumer products, and are used in cosmetics, as solvents for fragrances, suspension agents for solids in aerosols, and skin emollients. Animal studies showing reproductive and other toxic effects have raised a concern about the safe use of phthalate esters.
Methods: A rapid and sensitive reverse_phase HPLC method with UV detection was developed for the quantitation of dimethyl phthalate (DMP), diethyl phthalate (DEP), benzyl butyl phthalate (BBP), dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) in cosmetic preparations.
Results: Average recoveries of the phthalate esters added to a variety of cosmetic products were greater than 90%. In a survey of 48 consumer cosmetic products, including hair care, deodorants, lotions and creams, nail products, fragrances, and body washes, most products were found to contain at least one phthalate ester. DEP was detected most frequently at concentrations up to 38,663 ppm. DBP was found in fewer products, but at levels up to 59,815 ppm.
Conclusions: Phthalate esters were found in most cosmetic products. The FDA will use the survey data to assist in the evaluation of the safe use of phthalate esters in cosmetics.
Testing the Test Kits: An LC/MS/MS method for penicillin in positive control milk
D. N. Heller1 , S. E. Cullison2 , 1CVM Office of Research, Laurel, Maryland, 2University of Montana, Missoula
Purpose
Every tanker truckload of raw milk shipped in the United States is tested for presence of beta-lactam antibiotics before processing. The FDA Center for Veterinary Medicine, Office of Research, evaluates the rapid test kits which are used in this screening program. The test kits are required to include a negative and positive control sample for quality control purposes. The Pasteurized Milk Ordinance (PMO) of the National Conference on Interstate Milk Shippers (NCIMS) sets strict limits on the concentration of the positive control included with each test kit. The concentration of penicillin G must be 5.0 ng/mL with an allowable error of 10%, i.e., 5 ± 0.5 ng/mL. To date, regulatory agencies have not had a chemical method for verifying the concentration and stability claims for these test kit positive control samples. This method development project aimed to develop and apply such a method based on liquid chromatography-tandem mass spectrometry.
Methods
Test kit positive control samples were prepared according to label instructions, by diluting with water or raw control milk, depending on manufacturer. A standard curve was prepared by fortifying raw control milk with penicillin G at levels ranging from 1 - 20 ng/mL. Phenethecillin was used an an internal standard. The samples were extracted twice with acetonitrile. The combined extracts were evaporated and diluted with water. Extracts were further cleaned up on C-18 bonded silica solid phase extraction cartridges. Analysis was carried out on a benchtop triple quadrupole mass spectrometer. On-line liquid chromatography was performed on a short C-18 column, and data were collected using selected reaction monitoring of two product ions from penicillin and one from phenethecillin.
Results
After testing a variety of approaches, the final form of the method was set as described above. Positive control samples from a variety of test kits were analyzed with the provisional method. There was no evidence of any serious problem needing immediate attention; all results fell within the ± 10% tolerance, or nearly so.
Conclusions
This important aspect of the United States' milk safety program was evaluated successfully. The provisional method appears satisfactory for its purpose. Preliminary results with the provisional method do not indicate any major problem with test kit controls. The next step will be to perform a second analyst check and complete the method validation process. At that point the final method will be reapplied to the test kit positive controls, to verify concentration claims and to test the stability of their concentrations over their useful life under recommended storage conditions.
Acknowledgments
Sarah Cullison served as an intern in the FDA's "Windows to Research and Regulatory Science" program during 2004. She is presently a graduate student in chemistry at the University of Montana, Missoula.
Cold Vapor Atomic Absorption Determination of Mercury in D&C Black No. 2 (High Purity Furnace Black)
N.M. Hepp, OCAC, FDA, College Park, MD
Background: D&C Black No. 2, a high purity furnace black, was recently listed as a color additive subject to batch certification. A method was needed to enforce a 1 ppm limiting specification for mercury in certified lots of the color additive (21 CFR part 74.2052).
Methods: A simple procedure was developed to determine mercury in D&C Black No. 2 by cold vapor atomic absorption by adapting a method used for other color additives. The samples were treated with a mixture of nitric and hydrochloric acids and heated by microwave in sealed Teflon® vessels. The resulting solutions, which were stable to mercury loss for at least one week, were diluted and analyzed for mercury by cold vapor atomic absorption spectrometry (CVAAS). The technique avoids the use of extremely hazardous hydrofluoric or perchloric acids.
Results: Method validation was performed by spiking high purity furnace black with inorganic mercury (HgNO3) at levels from 0.1 to 1.5 µg/g and comparing the results with those for standard reference materials. At the specification level of 1 ppm Hg, the 95% confidence interval was ± 0.01 ppm.
Conclusion: A method was developed that accurately measures mercury at the specification level in D&C Black No. 2 samples. By eliminating volatility and adsorption factors through the formation of HgCl4-2 complexes, one can avoid using hydrofluoric and perchloric acids, which are extremely hazardous.
Highly Optimized strategy for expression and purification of two biologically active tumor specific cytotoxins consisting of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli
B. H. Joshi, R. Puri, P. Dover, R. K. Puri, CBER, FDA, Bethesda, MD
Background: We have previously reported that over expression of IL-13R on a variety of solid human tumor cell lines can sensitize them to a chimeric fusion cytotoxin consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). However, critical steps involved in the production of these therapeutic cytotoxins at high yield and effects of mutations to increase cytotoxicity have not been determined.
Methods: We produced two chimeric constructs consisting of human IL-13 and 38kDa truncated PE (PE38) and IL-13 and PE38QQR, in which 3 lysine residues at position 590, 606 and 613 were substituted with two glutamine and one-arginine residues. These constructs were used to express and purify proteins in prokaryotic expression systems.
Results: Our protocol resulted in 14-22 mg and 21-28 mg of highly pure and biologically active proteins from one-liter batch of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture system, respectively. The cytotoxic activities of both cytotoxins were found to be similar in IL-13 receptor positive renal cell carcinoma (RCC) and bladder carcinoma cell lines. IL-13-PE38 and IL-13-PE38QQR mediated similar cytotoxicity with IC50 (concentration of protein causing 50% inhibition of protein synthesis) in the range of <0.1 ng/ml and <5 ng/ml in both RCC and bladder carcinoma cell lines.
Conclusions: Improved protocol for producing tumor-targeted cytotoxins will not only enhance the yield but also allow safety testing of these molecules in various tumor model of human disease.
Determination of Sildenafil in Tablets by Liquid Chromatography with Tandem Mass Spectrometry Detection Method
C.M. Karbiwnyk, FDA, Denver, CO
Background: Counterfeit tablets of the prescription drug Viagra® (sildenafil) may be obtained without a physician's prescription and thus contraindications are not monitored, posing a danger to consumers. In the interest of public safety, the FDA needs to analyze certain products for the illegal presence of sildenafil.
Methods: In order to determine the presence of sildenafil in adulterated products, an LC/MS/MS detection method was devised. Sample and standard solutions were separated on a Zorbax SB C-18 (2.1 x 150 mm x 5 µm) column with an isocratic flow of 10mM ammonium acetate with 0.1% acetic acid-acetonitrile (1:1, v/v). The mobile phase was at a flow of 200 µL/min, the column oven was at 35°C and the autosampler made 5 µL injections. Using the ESI in the positive ion mode, the source voltage was 5.0 kV; capillary voltage was 36.0V; capillary temperature: 350°C; and collision energy 55%.
Results: Positive identification of sildenafil was based on the product ions: m/z 377, 311, 283 and 163 in the MS2 spectra of m/z 475. Two sets of blue tablets were collected and analyzed for the illegal presence of sildenafil. The counterfeit tablets were found to contain 123.34 mg and 128.48 mg of sildenafil per tablet based on composite sample analysis. Pfizer formulates its Viagra® prescription drug to contain 25 mg, 50 mg, and 100 mg of sildenafil per tablet.
Conclusion: This LC/MS/MS method may be used to screen for sildenafil in suspected products or to confirm the presence of sildenafil following LC/UV analysis.
Confirmation of Oxolinic Acid and Flumequine Residues in Shrimp by Liquid Chromatography with Tandem Mass Spectrometry Detection
C. M. Karbiwnyk1 , L. E. Hibbard2 , R. H. Lee2 , L. L. Murphy2 , C. L. Burns2 , M. R. Madson2 , 1ADRC, FDA, Denver, CO, 2FDA, Denver District Lab, Denver, CO
Background: Oxolinic acid and flumequine are structurally related antibacterial agents known as quinolones. Quinolones are administered to aquaculture species for their broad spectrum of activity against bacterial pathogens. Drug residues are a potential hazard for consumers and are a concern due to the emergence of drug resistant bacteria. Quinolones are not approved for use in food fish.
Method: Recently an LC-fluorescence method was developed with a simplified extraction scheme. Based on that sample prep, an LC/MS/MS method using positive ion ESI is being developed to confirm the presence of oxolinic acid and flumequine drug residues in shrimp found positive by the LC/FL analysis. Sample and standard solutions are separated on a Zorbax SB C-18 (2.1 x 150 mm x 5 µm) column with an isocratic flow of 1% acetic acid:49.5% water:49.5% acetonitrile (v/v). The mobile phase flow is 200 µL/min, the column oven set at 25°C and the autosampler generates 20 µL injections. An MS3 scan, m/z 262 at 30% collision energy isolating m/z 244 with 40% collision energy, is used for both oxolinic acid and flumequine.
Results: Positive identification of oxolinic acid is based on the product ions: m/z 216, 200 and 158. Positive identification of flumequine is based on the product ions: m/z 234, 219, and 202. Spike recoveries for the extraction scheme developed for the LC/FL method are approximately 85% for oxolinic acid and 75% for flumequine.
Conclusion: This method will replace the GC/MS confirmatory method which required derivitization of the quinolones before analysis.
Quantification and Activity of Human Alpha-1-Proteinase Inhibitor in Complex Biological Mixtures: Assay Development
E. Karnaukhova, Y. Ophir, B. Golding, A. Shrake, CBER, FDA, Bethesda, MD 20895
Three plasma-derived (pd) alpha-1-proteinase inhibitor (alpha-1-PI) products are licensed for augmentation therapy of deficient patients. Nevertheless, reliable, standardized assays for quantifying alpha-1-PI in complex biological mixtures, e.g., in plasma samples or recombinant human alpha-1-PI (r-alpha-1-PI) in fermentation mixtures, are required for research and development.
As a part of our multi-step, long-term project focused on alpha-1-PI structure-function relationships, we have established a sandwich ELISA with high specificity and a low limit of detection (2.6 ng/ml), which is now routinely used for quantification of pd-alpha-1-PI and r-alpha-1-PI in complex multi-component mixtures.
Currently, to complement the ELISA, we are developing high-throughput (HTP) assays to measure inhibitory activity, particularly for screening r-alpha-1-PI at low and extremely low concentrations in complex mixtures such as raw fermentation samples. Sensitivity of an assay involving inhibition of the steady-state activity of trypsin by alpha-1-PI has been increased by using substrates with different Km and kcat values thereby allowing accurate determination of inhibition in a concentration range comparable to that developed for the ELISA (2.6-200 ng/ml). Also, at higher alpha-1-PI concentrations (1.04-8.8 mg/ml), direct titration of trypsin with an active site titrant before and after inhibition by alpha-1-PI has been utilized. Assessment of key analytical characteristics of these assays has allowed their partial validation. These functional and antigenic assays will be used to determine level of inhibitory potential of extremely low concentrations of alpha-1-PI in various multi-component mixtures.
Recombinant Glycosylated Human Alpha-1-Proteinase Inhibitor Expression in a Filamentous Fungus, Aspergillus niger
Y. Ophir1 , E. Karnaukhova1 , L. Trinh2 , A. Shrake1 , B. Golding1 , P. Punt3 , J. Shiloach2 , 1CBER, FDA, Bethesda, MD 20895, 2NIH, NIDDK, Bethesda MD 20892, 3TNO, Zeist, The Netherlands
Human alpha-1-proteinase inhibitor (alpha-1-PI) is one of the major protease inhibitors in human plasma. Its deficiency is associated with development of progressive emphysema. Three licensed plasma-derived alpha-1-PI products are available for replacement therapy. Robust viral inactivation steps clear known viruses, but emerging viruses pose a theoretical risk. Recombinant alpha-1-PI (r-alpha-1-PI) provides an attractive alternative. Although r-alpha-1-PI has been produced in several hosts, protein stability has been an issue, primarily due to lack of glycosylation (in prokaryotes) or modified glycosylation patterns (in eukaryotes). We have explored the possibility of expressing the human alpha-1-PI gene in Aspergillus niger, a system reported to provide "mammalian-like" glycosylation. Our expression strategy features fusion of alpha-1-PI with a strongly expressed, secreted leader protein (glycoamylase), separated by a KEX2-like processing sequence that allows in vivo cleavage. SDS-PAGE, Western blot, ELISA, and activity assay enabled us to select the transformant(s) secreting glycosylated, biologically active r-alpha-1-PI with yields up to 50 mg/L. Currently we are working to achieve enhanced production via multiple insertions of the expression cassette. In the future, we plan to use this expression system for other recombinant therapeutic glycoproteins.
Lead in Pharmaceutical Dosage Forms
J. F. Kauffman, B. J. Westenberger, CDER, Division of Pharmaceutical Analysis, St. Louis, MO 63101
Background: Few pharmaceutical substances have specified limits for heavy metals, and consequently, little is known about heavy metal contamination in pharmaceutical products. Furthermore, there are currently no general limits for heavy metal contamination in pharmaceutical products.
Methods: We have recently surveyed the lead content in 27 oral pharmaceutical products including 13 different prescription and over-the-counter products in several dosage forms and strengths using inductively-coupled plasma mass spectrometry.
Results: Most of the drug products exhibited lead concentrations of under 20 parts per billion, and provide an estimate of environmental background lead levels. Four drug products showed elevated lead levels, the highest having a concentration of 500 parts per billion.
Conclusion: This survey indicates a low level of lead contamination in the nation's pharmaceutical supply. However, certain products exhibit relatively high lead levels, and it appears that products containing calcium and other metals are especially prone to lead contamination. Following presentation of the experimental results, we survey recent literature on lead toxicity in order to initiate a discussion of the appropriate regulatory level for lead in pharmaceutical products.
Microarray Detection of Multiple Antibiotic Resistance Markers by the Use of RNA Obtained by a Commercial Kit and Triton X-100 Boiling Method
S. A. Khan, K. Sung, M. S. Nawaz, A. A. Khan, NCTR, FDA, Jefferson, AR 72079
Background: Disk-diffusion and broth-dilution assays are the most common methods of determining the antibiotic resistance/sensitivity of bacteria to different antibiotics. However, none of these assays provide any information regarding the presence/absence of resistance markers.
Methods: An oligo-based microarray method was developed for the screening of 131 antibiotic resistance markers associated with most of the antibiotics used by the National Antibiotic Resistance Monitoring System (NARMS). Specificity of the probes was tested by Cy3/Cy5-labeled antisense probing. RNA preparations from multidrug-resistant Salmonella, Staphylococcus and Enterococcus spp. were made by i) a commercial RNA preparation method and ii) a Triton X-100 boiling procedure and reverse-transcribed to make Cy3-labeled cDNA for hybridization and detection of resistance markers.
Results: Antisense probing suggested probe-specific hybridization with little or insignificant background. Resistance markers were successfully detected in multidrug-resistant bacteria mentioned above. A comparison of the data indicated that only 1 µg of RNA obtained by the Triton-X-100 boiling method was required to generate reproducible microarray data as compared to 20 µg of RNA prepared by the commercial method. Moreover, the RNA obtained by Triton X-100 boiling procedure yielded better results in terms of signal quality, strength and correlation with disk-diffusion data than the RNA prepared by a commercial kit.
Conclusion: The whole process from RNA isolation to hybridization and detection of resistance markers by microarray method takes almost the same time (20-24 h) as required by disk-diffusion and broth-dilution assays but provides far more information than above techniques. The method has a potential for use in the detection of resistance markers in food-borne human pathogens and/or bio-terror agents.
Determination and Confirmation of 17alpha-Methyltestosterone in Rainbow Trout
M. Lopez, P. Chu, R. Reimschuessel, S. Serfling, C. Gieseker, Office of Research, CVM, FDA
Background: In fish farming, 17alpha-methyltestosterone (MT) is used to induce sex reversal. After MT is administered to fish, most of the parent compound and its metabolites are excreted through the gills, feces, and urine. Residues remaining in edible tissues are expected to be diluted through fish growth. MT has been reported to produce a significant increase in fish weight; therefore, analytical methods are needed to monitor the misuse of MT as a growth promoter.
Method: In this poster, we present an LC/MS/MS method for the quantitation and confirmation of MT in rainbow trout tissues at 0.8 ppb.
Conclusion: The method was validated using fortified and incurred tissues.
Release of the plasticizer di-2-ethylhexyl phthalate (DEHP) into normal saline stored in heated and nonheated PVC bags
A. D. Lucas, R. P. Brown, CDRH, FDA Silver Spring MD
Background: Warming intravenous crystalloid solutions (normal saline; NS) before administration to patients is commonplace in some clinical settings. However, concern has been expressed that heating crystalloid solutions in PVC bags will increased the amount of di-2-ethylhexyl phthalate (DEHP) released from the bag. A study was conducted to compare DEHP release into NS stored in heated and nonheated PVC bags.
Methods: PVC 1L saline bags were either microwaved for 2.5 minutes with outer covering on (mean temp. 41.6oC) or kept at room temperature (22.7oC). A 10 ml aliquot was taken from each heated bag 60 minutes after microwaving; an aliquot was taken from the non-heated bags at the same time. DEHP was extracted from NS with hexane and concentrations were determined using the HPLC method described by Kambia et al. (J. Chromatog. B, 755: 297-303) with minor modifications. The isocratic mobile phase was 90:10 (v:v) acetonitrile:aqueous buffer at a flow rate of 1.5 ml/min. Separation was performed on a C18 column and absorbance measured using a photodiode array detector (202 nm).
Results: Recovery of DEHP standards spiked into normal saline was good (96-102%). Levels of DEHP released into NS from heated and non-heated PVC bags were below the limit of quantitation (approximately 0.1 ug/ml).
Conclusion: The dose ofDEHP received by patients following administration of NS from heated and nonheated PVC bags is well below the parenteral Tolerable Intake for this compound established by CDRH. Concerns about the release of DEHP from heated PVC bags into crystalloid intravenous solutions appear unfounded.
Bleach Tampering Investigation of Two Fruit Flavored Beverages
S. E. McMullen, D. S. Jackson, J. R. Urban, D. F. Crockett, FDA
The US Food and Drug Administration is responsible for protecting public health by assuring that the safety of our nation's food supplies are maintained. Every year, numerous consumer complaints related to foods and beverages are logged and investigated by the FDA to assure that our nation's food is safe. Numerous household cleaning products have been used to adulterate foods and beverages. Household bleach has been identified as the adulterant in numerous tampering investigations of beverages at the FDA. Household bleach contains 4-6% sodium hypochlorite, a caustic and strong oxidizer. It is inexpensive and readily available in most households. Two beverages(fruit punch and lemonade) suspected of being adulterated with bleach were analyzed by the Southeast Regional Laboratory and Forensic Chemistry Center in a collaborative investigation. Several analyses were performed on the beverages including: ion chromatography, pH, spot tests for oxidizing agents, static headspace gas chromatography-mass spectrometry analysis for chloroform, and Inductively Coupled Plasma-Atomic Emission Spectrometry for sodium. This poster describes the findings of this collaborative investigation which confirmed that the suspect beverages were consistent with bleach adulteration.
Are Homogenizers Extinct?
S. E. McMullen, V. A. Vega, F. J. Schenck, FDA
Homogenizers have been considered necessary equipment for residue extraction for many years. Recently, many methods have been developed and evaluated replacing homogenization during the extraction process with vortex mixing and/or mechanical shaking. Residues studied were pesticides, aflatoxins and veterinary drug residues. Incurred pesticide residues in eleven produce samples were extracted and analyzed by official methods of the US and Canadian government that utilized mechanical homogenization [Luke et al (1975) JAOAC Int 58:1020-1026; and Fillion et al (2000) JAOAC Int 83:698-713] and by a method utilizing vortex mixing. Recoveries using the vortex mixing method were comparable to the two traditional pesticide methods. Incurred aflatoxin residues in peanut butter in an AOCS proficiency sample were extracted using the official AOAC method (utilizing mechanical homogenization) and using a method where extraction was accomplished by vortex mixing. Results from both methods were comparable. Consistent results between both methods were also obtained with fortified samples. Ivermectin, a veterinary drug residue, was extracted from salmon tissue by vortex mixing and good recoveries (>80%) were obtained for a range of fortification levels. It has been demonstrated that vortex mixing/mechanical shaking can replace homogenizers in the extraction process for a variety of matrices and residues.
Dissolution Testing of Mesalamine Delayed Release Tablets
T. Moore1 , Z. Gao1 , B. J. Westenberger1 , L. F. Buhse1 , R. Lionberger2 , H. Kwon2 , L. X. Yu2 , 1CDER, FDA, St. Louis, MO, 2CDER, FDA, Rockville, MD
Background: Mesalamine is intended to provide topical anti-inflammatory action in the colon but is rapidly absorbed in the intestine. Asacol tablets are coated with an acrylic-based resin that dissolves at pH 7 or greater and delays the release of mesalamine until it reaches the terminal ileum and beyond. The current USP dissolution method is a multistage method (pH 1.2 for 2 hours, pH 6.0 for 1 hour, pH 7.2 for 1.5 hours) that attempts to correspond to physiological conditions. However, gastro-intestinal pH can vary and thus additional pH conditions were evaluated to develop a dissolution method suitable for comparing dissolution profiles of two products across the physiologically relevant pH ranges.
Method: The final pH was varied (6.5 to 8.0) and an initial pH of ~2 was compared with the USP method initial pH of 1.2. High variability in some of the results also prompted investigation of basket vs. paddle and techniques for handling tablets.
Results: Certain combinations of initial and final pH result in high tablet to tablet variability. Visual examination of the tablets during dissolution suggests that the coating may be the cause of this variability.
Conclusions: The USP method uses conditions that minimize the tablet to tablet variability observed at other pH conditions and thus is not sufficiently discriminating to distinguish differences between tablets. The variability in tablet dissolution at other pH conditions may explain the high inter and intra subject variability observed in Asacol pharmacokinetics.
Separation and Quantitation of Arsenic Species in Food and Dietary Supplements by HPLC-ICP-MS
S. Nam, J. Cheng, S. G. Capar, CFSAN, FDA, College Park, MD
Background
Naturally occurring arsenic is found in foods in both organic and inorganic forms. Because of the differences in toxicity, meaningful assessments of arsenic exposure require knowledge of the amounts and of the species present.
Methods
Various extraction procedures were investigated using reference materials and samples to evaluate extraction efficiency and effectiveness. ICP-MS (Inductively Coupled Plasma Mass Spectrometry) was used to measure total arsenic and coupled to an HPLC (High Pressure Liquid Chromatography) to quantitatively determine arsenic species.
Results
Accelerated solvent extraction (ASE) with water/methanol mixtures was used to extract rice flour (NIST 1568a). Total arsenic extraction efficiency was 64, 65, 58, and 42 % with water, 25, 50, and 100% methanol, respectively. Trifluoroacetic acid (TFA) was used to extract spinach (NIST 1570), a freeze-dried apple sample, and rice flour. Total arsenic extraction efficiency was 90% for spinach, 75% for freeze-dried apple, and 83% for rice flour when an extraction temperature of 100¡ÆC was used. Enzymatic extraction with alpha-amylase and sonication resulted in extraction efficiencies of 104% for rice flour, 98% for freeze-dried apple, and 7% for spinach. Chromatograms of arsenic species extracted by the optimum extraction methods were obtained, and the species quantified. We found arsenite (As (III)), arsenate (As (V)), dimethylarsinic acid (DMA), and monomethylarsonic acid (MMA) in the apple sample, and DMA and As (V) in the rice flour sample. In an herbal dietary supplement, we found MMA and As (V).
Conclusions
The selection of extraction method is very important for different types of foods. Enzymatic extraction was effective for rice flour and freeze-dried apple, but was not adequate for spinach. For spinach, extraction with TFA provided the best efficiency.
 | Sigma Xi Outstanding Student Poster Award - 2005 FDA Science Forum - Vicky Hsu |
The Determination of Semicarbazide (N-aminourea) in Commercial Bread Products
G. O. Noonan1 , C. R. Warner1 , W. Hsu2 , T. H. Begley1 , G. A. Perfetti1 , G. W. Diachenko1 , 1CFSAN, FDA, College Park, MD, 2JIFSAN, FDA, College Park, MD
Recently semicarbazide has been found in food in jars sealed with cap liners that were manufactured using azodicarbonamide as a blowing agent. These reports raised the concern that the use of azodicarbonamide-an approved dough conditioner-may result in semicarbazide residues in bread. To answer this question, a method based upon the previously reported HPLC/MS determination of the semicarbazone of o-nitrobenzaldehyde (NBA) was utilized. The method adopted for this work includes an extensive cleanup and reaction with NBA at pH 3.5, rather than with the widely used 0.1 M HCl (HCl), to form the semicarbazone derivative. Stable isotope dilution assay (SIDA) was used to determine the free semicarbazide present in the bread products. Levels of semicarbazide ranged from 10 ppb to 1200 ppb in commercial bread products with azodicarbonamide listed among their ingredients.
Determination of Furan in Foods
P. J. Nyman, K. M. Morehouse, T. P. McNeal, G. W. Diachenko, CFSAN, FDA, College Park, MD
Recently, furan, an animal carcinogen, was found in a number of foods that were heat-treated during production. In order to evaluate this potential problem, a headspace gas chromatography/mass spectrometry method for the determination of furan in foods was developed. An in-house method validation was conducted that included the determination of limits of detection and quantitation (LOD/Qs), linearity, repeatability, within laboratory precision, and ruggedness testing in apple juice, chicken broth, infant formula, green beans, and peanut butter. The method of standard addition with d4-furan as internal standard was used to quantify furan. The LOD/Qs ranged from 0.21 and 0.64 ng/g in apple juice to 0.94 and 2.9 ng/g in peanut butter. Recoveries were conducted at 0.5, 1, 2, and 3 times the LOQ. At 1, 2, and 3 times the LOQ, the recoveries ranged from 89.4 to 108 percent and the relative standard deviations ranged from 3.2 to 17.5 percent for all the matrices. For apple juice, chicken broth, and infant formula, the averaged coefficients of determination from the linear regression analyses were greater than 0.99 with calibration standards fortified at ½, 1, 2, and 3 times the LOQ. The coefficients of determination were greater than 0.99 for green beans and 0.96 for peanut butter with calibration standards fortified at 1, 2, and 3 times the LOQ. Within laboratory precision was determined by comparing the amount of furan found in 18 samples by two analysts on different days with different instruments. For the majority of the foods, the difference between the amounts found by each analyst was less than 20 percent.
Separation and Identification of Pigments found in Permanent Cosmetic/Tattoo Inks using Reversed-phase High Performance Liquid Chromatography and Photodiode Array Detection.
B.R. Petigara, CFSAN, FDA, College Park, MD
Background: Reports of adverse events from permanent makeup/tattoos and from the effects of tattoo removal have recently increased. Color additives permitted for use in cosmetics are listed in the Code of Federal Regulations, but these approvals do not include injection into the skin. Historically, inorganic pigments and carbon black were used for tattooing. The expanded use of organic pigments in tattoo inks provided a wider range of shades and better color stability, but the safe use of these pigments is in question. The Agency has initiated studies on the identification and safety of organic and inorganic pigments used in permanent makeup/tattoo inks. This work describes the identification of organic pigments in selected commercial inks.
Methods: An extraction procedure followed by qualitative HPLC analysis was developed to separate and identify organic pigments present in permanent makeup/tattoo inks. Analyses were performed using reversed-phase chromatography with photodiode array detection. Organic pigments were separated from the inks and from one another, and identified by comparison of their UV/visible spectra and HPLC retention times with those of reference standards.
Results: This study determined that most of the inks examined contain mixtures of organic pigments from several chemical classes. Insoluble ink residues indicated the additional presence of inorganic pigments. Three organic pigments from the benzimidazolone class were present in a number of the permanent makeup inks. One benzimidazolone reference standard, C.I. Pigment Brown 25, unexpectedly decomposed under the mild conditions of the analysis. Therefore, a secondary HPLC peak was used as a marker to indicate the presence of this pigment in several ink samples.
Conclusions: The method developed in this study found that most of the inks contained mixtures of organic pigments from several chemical classes, including benzimidazolones. The complexity of the ink mixtures complicates the correlation of adverse events with the presence of specific organic pigments.
Isolation and Quantification of Sudan I in Worcestershire Sauce
B. R. Petigara, A. L. Scher, CFSAN, FDA, College Park, MD
Background: The E.U. has recently reported that Sudan I was found in a batch of Worcestershire sauce and in other food products. More than 400 foods marketed in Europe were thought to be contaminated and were recalled. Sudan I is an orange dye that is used to color solvents, oils, waxes, petrol, and shoe and floor polishes. It has been reported to be a carcinogen, and may not be used as a food additive in the EU nor as a color additive in the United States. The FDA District Laboratories need methods for the identification of Sudan I in foods.
Methods: A method was developed using Extrelut solid phase adsorption columns to isolate Sudan I from Worcestershire sauce. The isolated color was then analyzed using reversed-phase high performance liquid chromatography (HPLC) with photodiode-array detection.
Results: Method validation was performed by spiking Worcestershire sauce with Sudan I at levels from 0.2 to 5 ppm. The detection limit is ca 0.2 ppm. Calibration was linear giving a R value of 0.9995.
Conclusions: A quantitative method was developed to determine Sudan I in Worcestershire sauce. The method can be expanded to other food products for use by the FDA District laboratories.
A New Technique for Protein Purification - Centrifugal Precipitation Chromatography
L. Qi1 , Y. Ito2 , 1CDER, FDA, Rockville, MD, 2Center for Biochemistry and Biophysics, NHLBI, NIH, Bethesda, MD
For decades proteins were fractionated with ammonium sulfate by stepwise precipitation. Even now 80% of protein purification protocols include at least one step of ammonium sulfate precipitation. A new technique, Centrifugal Precipitation Chromatography (CPC), was used to replace the tedious and inefficient manual operation.
The separation column consists of a pair of disks with mirror-imaged spiral grooves and a dialysis membrane sandwiched between the disks to form two identical channels. The disk assembly is mounted on the seal-less continuous-flow centrifuge. The solvent system contains ammonium sulfate and phosphate buffer.
The method is optimized using human serum albumin and globulin. The effects of the sample sizes were investigated on lysozyme. Hyaluronidase and monoclonal antibodies were purified using standard CPC procedure. Recombinant ketosteroid isomerase was purified by affinity CPC using estradiol-PEG conjugate as an affinity ligand.
CPC is simple and useful with the following advantages: 1) the native conformation and bioactivities of the protein are preserved in ammonium sulfate and phosphate buffer system; 2) high recovery is ensured due to the absence of a solid support; 3) protein fractions are concentrated, separated, and collected without stepwise centrifugation; 4) affinity CPC gives highly purified proteins; 5) can be used to determine the critical precipitation point for large scale protein precipitation; and 6) can be used to purify other biopolymers, such as DNA, RNA, and polysaccharides.
Idiosyncrasies of a Porous Graphitic Carbon Stationary Phase During Analysis of Conjugated Estrogens by Liquid Chromatography-Mass Spectrometry
J. C. Reepmeyer, H. Ye, FDA Division of Pharmaceutical Analysis, St. Louis, MO
Background: While developing a method for the differentiation of more than 100 steroidal components in equine conjugated estrogens through a combination of chromatographic separation on a porous graphitic carbon (PGC) stationary phase and mass selectivity using LC-MS, unexpected changes occurred in the chromatography. The purpose of this work was to determine the cause for such changes and establish procedures to maintain reproducible chromatography on PGC.
Methods: Gradients using aqueous organic solvents containing unbuffered amine additives initially gave excellent chromatography and MS response, but were subject to retention time drift and chromatographic degradation. In attempts to regenerate a deteriorated PGC column, it was washed with solutions of acids (trifluoroacetic acid or acetic acid) and bases (NaOH, triethylamine, or ammonia) and with various organic solvents. The effects of an aging mobile solvent and the addition of acids to the mobile phase were evaluated.
Results: For our application, washing the PGC stationary phase periodically with acetone followed by 95% methanol was a reasonably effective means of column regeneration. Aging amine-containing mobile solvents and the addition of acids promoted longer retention times. Long retention times, retention time drifting and peak distortion occurred when the column was pre-washed with polar solvents.
Conclusions: PGC columns can degrade which may cause changes in retention time and gross peak distortion. Regenerating the column periodically, conditioning the column prior to analysis, and using fresh mobile solvents can minimize these effects.
Analysis of the nitrogen mustard mechlorethamine in compounded topical pharmaceutical preparations by high performance liquid chromatography
J.C. Reepmeyer, Division of Pharmaceutical Analysis, St. Louis, MO
Background: Mechlorethamine hydrochloride, an anticancer agent used to treat lymphoma of the skin, is unstable, and therefore, must be freshly compounded as a topical pharmaceutical formulation by a pharmacist. The purpose of this study was to develop a sensitive HPLC-UV assay procedure specific for mechlorethamine in ointment formulations by trapping the highly reactive compound as a stable derivative with a UV chromophore.
Methods: Mechlorethamine was derivatized with benzenethiol and analyzed by normal phase HPLC on silica gel using dibutyl phthalate as an internal standard. The derivatization reaction, purification, and isolation were conveniently performed in a single test tube. The method was applied to different types of ointment formulations containing 0.02% mechlorethamine and evaluated for its precision and recovery.
Results: Mechlorethamine reacts with benzenethiol to give a high yield of the stable UV-sensitive disubstitution product bis(2-phenylthioethyl)methylamine. Recovery from ointment formulations ranged from 98.4-100.4%. Precision for the assay of mechlorethamine HCl standard and mechlorethamine HCl at 0.02% in ointment formulations was 0.08-0.52% RSD (n=6). There was no chromatographic interference from ointment excipients.
Conclusions: A precise, accurate, and sensitive liquid chromatographic method was developed for the assay of mechlorethamine hydrochloride in topical pharmaceutical formulations.
Drug Substance Stability in Liposomal Drug Products (LDPs) under Various Stress Conditions
L. K. Revelle, T. G. Lipe, W. H. Doub, FDA/CDER/OPS/OTR/DPA
Introduction: Methods have been developed toto monitor the stability of Doxorubicin HCl (Doxil®, pegylated LDP) and Daunorubicin citrate (DaunoXome®) under thermal, oxidative and various pH stress conditions.
Method: Fresh Doxil® and DaunoXome® aliquots were stressed for 6 days at 50° C, 0.3% hydrogen peroxide, pH 8.5, and pH 3.0 conditions. Liposomes were disrupted and the drug substance was analyzed by an isocratic HPLC-UV method. A Supelcosil C18, 50 x 4.6 mm, 5 µm column was used with a mobile phase consisting of 20 mM (pH 4.0) ammonium acetate buffer/methanol (composition is drug substance dependent) with detection at 254 nm. The percent degradation of the drug substance was determined by comparing the concentration of the stressed drug to the concentration of the unstressed drug for each liposomal drug product at each set of stress conditions.
Results: Under thermal and under oxidative stress, doxorubicin showed considerably greater stability than daunorubicin. On the other hand, under basic pH stress, daunorubicin appeared more stable than doxorubicin. Under acid pH stress, doxorubicin and daunorubicin appear to be equally stable.
Conclusion: Doxorubicin shows equal or better stability than daunorubicin under all stress conditions other than base stress where the opposite trend is observed.
Monitoring Lipid degradation in Liposomal Drug Products (LDPs) using HPLC with Evaporative Light Scattering Detection (ELS)
L. K. Revelle, T. G. Lipe, W. H. Doub, FDA/CDER/OPS/OTR/DPA
Introduction: Methods have been developed to monitor the degradation of lipid components (MPEG-DSPE, DSPC, LysoPC, cholesterol, & HSPC) in LDPs. We comparecompare degradation for a pegylated LDP (Doxil®)vs. a "normal" LDP (DaunoXome®).
Method: LDPs Doxil® and DaunoXome® were stressed for six days at 50° C, 1% H2O2, acid pH, and base pH. The change in lipid composition was monitored by HPLC-ELS. The HPLC separation was carried out on a Chromegabond PEP 5µmm 250 x 4.0mm id. column at 40° C with an ethanol:water gradient at a flow rate of 1.0 mL/min. ELS detection was achieved using a nebulizer temp of 35° C and evaporatorevaporator temperature of 55° C. The change in lipid composition was followed by comparing the peak areas for unstressed solutions of Doxil® and DaunoXome® with those for stressed solutions.
Results: All lipid components were verified by spiking experiments with standards. Hydrolysis of HSPC and DSPC into LysoPC and hydrolysis of MPEG are observed. Upon comparison of lipid component ratios, the most dramatic change was observed for acid stress conditions. Doxil® and DaunoXome® samples showed significant increases in the ratios of LPC/HSPC (3920%) and the LPC/DSPC (268%). Under basic stress, Doxil® and DaunoXome® samples showed more moderate increases in the ratios of LPC/HSPC (534%) and LPC/DSPC (68%). Under oxidation and thermal stress, relatively little change in lipid composition ratios was observed.
Conclusion: The above LC-ELS method is an effective, simple, and time efficient way of monitoring the lipid decomposition in stressed Doxil® and DaunoXome®.
Assessment of Encapsulation Efficiency Stability of Liposomal Drug Products (LDPs) under Various Stress Conditions
L. K. Revelle, T. G. Lipe, K. D. Story, W. H. Doub, FDA/CDER/OPS/OTR/DPA
Introduction: Methods have been developed to monitor the change in encapsulation efficiency (EE) of Doxil® (pegylated) and DaunoXome® (normal) LDPs under conditions thermal, oxidative, acid pH, basic pH, and detergent stress.
Method: Fresh LDP aliquots were stressed for 6 days under 50° C, 0.3% hydrogen peroxide, pH 3.0, and pH 8.5. Stressed and unstressed Doxil® samples were analyzed by UV (480nm) while stressed and unstressed DaunoXome® samples were analyzed by fluorescence ( λEx = 476 nm, λEm = 588 nm) to obtain EE values for each set of stress conditions. Fresh LDP aliquots were also stressed by addition of a non-ionic detergent and analyzed immediately by the fluorescence method to determine EE. The change in EE was determined by comparing the EE of the fresh liposomal drug product to the EE of the stressed drug for each set of conditions.
Results: Under thermal stress, DaunoXome® showed a significant decrease in EE while Doxil® showed a slight increase. Under oxidative stress, both LDPsLDPs showed more moderate decrease in EE. Under basic pH stress, neither LDPLDP showed much change in EE. Under acidic pH stress, DaunoXome®showed a moderate decrease in EE while Doxil® showed a slight increase. Under detergent stress, both LDPsLDPs showed significant but similar loss of EE.
Conclusion: Under thermal, oxidative, acid pH, and detergent stress conditions, DaunoXome® showed expected decreases in EE, while Doxil® showed paradoxical increases in EE under thermal and acidic pH stress conditions. Neither drug showed much change in EE under moderate basic pH stress.
Multi-Laboratory Evaluation of the QuEChERS Method for Pesticide Residue Analysis
F. J. Schenck1 , J. Wong2 , A. N. Brown3 , L. V. Podhorniak4 , A. N. Parker1 , M. K. Hennessy2 , M. Reliford3 , A. J. Krynitsky2 , 1ORA, FDA, Atlanta, GA, 2CFSAN, FDA, College Park, MD, 3Florida Dept. of Agriculture & Consumer Services, Tallahassee, FL, 4OPP, EPA, Fort Meade, MD
Current methods used for the analysis of pesticide residues in foods are labor intensive and consume large volumes of solvent. QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) a procedure which entails extracting the pesticides by vortexing with a 1% acetic acid/acetonitrile mix, followed by salting out with sodium acetate and magnesium sulfate, has been reported. Initially a dispersive SPE cleanup (vortexing with primary-secondary amine [PSA] SPE sorbent and MgSO4) was used. We modified the method in order to obtain a better cleanup. Two cleanups were evaluated: 1) dispersive SPE with both PSA and graphitized carbon black (GCB) sorbents, and 2) SPE column cleanup using PSA, aminopropyl, or GCB/PSA SPE columns. Recovery data was obtained for >330 pesticide residues, using gas chromatography and/or liquid chromatography with mass spectrometric and/or element specific detection. Recoveries were >80% for most of the compounds tested. When compounds were analyzed by two or more of the laboratories, recoveries were similar, even when different cleanups and detection systems were used. Using the QuEChERS method resulted in a 66-92% reduction in solvent usage and a 56-87% reduction in cost, compared to methods currently being used in our laboratories.
Oligonucleotide microarray based detection of bacterial pathogens relevant to food and reused medical devices
N. V. Sergeev1 , A. D. Nandanie1 , D. Volokhov2 , A. Jacobson3 , D. Ranamukhaarachchi1 , 1FDA/CDRH/OSEL/DB, Silver Spring, MD 20903, 2FDA/CBER/OVRR/DVP, Kensington, MD 20895, 3FDA/CFSAN/OPDF/DMS, College Park, MD 20740
Background: Microbial pathogens relevant to food and reused medical devices impact public health and, accordingly, have significant relevance to FDA regulatory affairs. Currently used biochemical methods for bacterial detection are time consuming. Rapid and simultaneous detection of multiple pathogens will improve the FDA's ability to evaluate safety of foodand reused medical devices, and respond to bioterrorism outbreaks.
Methods: We developed DNA microarray methods for the rapid and simultaneous detection of bacterial pathogens relevant to food (Listeria monocytogenes, Staphylococcus aureus) and reused medical devices (Staphylococcus epidermidis, Streptococcus pneumonia, Streptococcus agalactiae, Streptococcus pyogenes). Fluorescently-labeled single stranded PCR amplicons were generated in one step by asymmetric PCR using hyper-variable regions of topA gene sequences. Oligonucleotide probes (19-25 nt) specific for Listeria, Staphylococcus and Streptococcus genera were developed for unambiguous detection and identification of specific pathogens.
Results: Based on our microarray method, bacterial detection limits were improved with the ability to detect approximately 10 microbial genomes. The assay was sensitive and specific for all species tested. Reproducibility of the assay was assessed by the analysis of more than 70 ATCC strains and clinical isolates collected from different sources. Success rate of bacterial species identification was 100%. Protocols were optimized to reduce the entire assay time to 4 hours.
Conclusions: Compared to other methods of identification using DNA sequence variation (e.g. RFLP or DNA sequencing), our microarray method was far more suitable for the analysis of mixtures of microbes, as it allows simultaneous detection of multiple pathogens present in one sample.
Comparison of whole genome amplification strategies for microarray-based analysis of bacterial pathogens
N. V. Sergeev, A. Rasooly, FDA/CDRH/OSEL/DB, Silver Spring, MD 20903
Background: Detection, identification and characterization of microbial pathogens are important tasks in evaluating the safety of food, biologics and reused medical devices. DNA-microarray technology has revolutionized microbial characterization by allowing the detection of thousands of genetic markers simultaneously. The main shortcoming of the technology is the necessity to use PCR amplification when the starting amount of DNA is limited. When the pathogen is unknown or if many genes have to be analyzed, PCR is impractical.
Methods: We evaluated the utility of several whole genome amplification strategies for microarray-based analysis of microbial pathogens. These approaches include Phi29 DNA polymerase-based, isothermal Klenow polymerase-based and one-primer amplification of randomly generated genomic library. The performance was assessed by hybridizing amplicons to microarrays consisting of 20-25-mer oligoprobes specific to 16 virulent factors and characteristic genes of pathogens belonging to Bacillus genus. Sensitivity limits were determined for different substrates inoculated by specified number of colony forming units and purified DNA samples.
Results: Random amplification strategies appeared to be less sensitive than PCR-based approaches, but provide more uniform amplification of the whole bacterial genome. Phi29 DNA polymerase-based amplification was the only technique capable of generating long products (>10 kb), while other approaches tend to produce relatively short amplicons. PCR-based amplification of a genomic library appears to be a very promising technique for rapid identification of multiple genetic markers when the source of starting material is limited.
Conclusions: Various whole genome amplification strategies have a considerable potential use in microarray-based detection and analysis of bacterial pathogens.
Development of a Simple Stability Indicating HPLC Method for Ranitidine Hydrochloride
R. B. Shah, H. R. Prasanna, M. Tawakul, M. A. Khan, DPQR, OPS, CDER, FDA,, Silver spring, MD
Purpose. The purpose of the present study was to develop a simple high performance liquid chromatography (HPLC) method for the determination of ranitidine HCl from its syrup. The current USP method was found to interfere with some of the excipients. Therefore a modification was required in our laboratory.
Background. The current method is based on the gradient elution method that was outlined in Pharmeuropa. With appropriate modification of the column, buffer, and operating conditions, a new isocratic elution method has been developed to assay ranitidine HCl and its related compounds.
Methods. The samples were analyzed on a Phenomenex Luna (2) reversed phase column (250 X 4.6 mm, 5 µ, 26 0C) with a mobile phase of phosphate buffer (10 mM, pH = 7.1) - acetonitrile (80:20, v/v) and a flow rate of 1 ml/min. Detection was at 230 nm.
Results. Ranitidine related compound A, ranitidine related compound C, and ranitidine HCl eluted at 2.07±0.001, 3.19±0.003, and 6.46±0.007min, respectively, free from any other peaks. Linear relationship (r > 0.99) was observed between the ranitidine HCl peak area and the concentrations within the range of 1-112 µg/mL. The total time for analysis was 10 minutes. Ranitidine HCl from the syrup also eluted at 6.47±0.001min with the concentration consistent with the labeled amount.
Conclusions. A simple and selective RP stability-indicating HPLC method for ranitidine HCl was developed that efficiently separated it from its related compounds. This novel analytical technique will be useful in drug product quality characterization.
Determination of albendazole and its metabolites in catfish after oral administration
B. Shaikh, R. Reimschuessel, N. Rummel, C. Gieseker, FDA/CVM/OR, Laurel, MD
Residue depletion of albendazole and its potential metabolites was studied in the muscle tissue of channel catfish. Albendazole, at the dose level of 10 mg/kg, was administered to channel catfish via stomach tube with manual restraint. Necropsy of catfish was performed at 8, 16, 24, 48, 72, 96, and 120 hour, post dose. Catfish were de-skinned and muscle fillets were collected. Six fish were used at each post dose period and the muscle tissues collected were homogenized and powdered in dry ice and stored at - 80 °C, until used. One gram of powdered tissue sample was carried through extraction and cleanup procedures followed by high performance liquid chromatography for quantification of albendazole and its metabolites. The results indicate that parent albendazole (ABZ) and its two metabolites, albendazole sulfoxide (ABZ-SO) and albendazole aminosulfone (ABZ-NH2-SO2) were depleted rapidly (<16 hour). However, the third metabolite albendazole sulfone (ABZ-SO2) was present until 48 hour post dose, albeit in low concentrations (~ 1 ppb).
HPTLC analysis of blue cohosh for alkaloids
S. Satchithanandam, E. Grundel, K. D. White, J. I. Rader, CFSAN, FDA, College Park, MD
Background: Blue Cohosh (Caulophyllum thalictroides) has been actively promoted as an alternative to estrogen therapies, since it is believed to have estrogen-like benefits without the unpleasant or harmful side effects. However, recent studies indicate that some of the alkaloids present in Blue Cohosh are toxic to humans. The purpose of this work is to identify alkaloids in Blue Cohosh.
Methods: The rhizomes of authenticated Blue Cohosh were obtained from Botanical Liaisons, Boulder, CO. They were powdered in a blender and sieved through a # 60 sieve. Ten grams of powder were extracted overnight with 50 mL methanol. The extract was centrifuged and the supernatant was analyzed by High Performance Thin Layer Chromatography (HPTLC) using chloroform-methanol-ammonium hydroxide (200:20:1) as the mobile phase.
Results: Five bands were observed when stained with Dragendorff's reagent. On the basis of HPLC-MS data, the molecular weights of compounds in three of the bands were consistent with those of baptifoline, N-methylcytisine and anagyrine.
Conclusions: Three alkaloids have been tentatively identified. We are in the process of identifying the compounds in the other two bands.
Differences in the constituents of two subspecies of Teucrium, Teucrium chamaedrys L. and T.canadense determined by analytical reversed-phase method.
P. R. Sundaresan1 , S. A. Slavoff2 , E. Grundel1 , K. D. White1 , J. I. Rader1 , 1CFSAN,FDA,College Park,MD, 2JIFSAN,FDA,College Park,MD
Neoclerodane diterpenoids are accepted as markers of the Teucrium species which are commonly known as germander. Preparations from germander have been used in traditional folk medicine as stimulants, tonics, diuretics, diaporetics and treatments for asthma and gout. Germander powder was marketed in France and Germany as an adjuvant to weight control. However, in 1992 these preparations were prohibited from sale in France and Germany because of severe hepatotoxicity resulting from their use. Germander has also been identified as an adulterant in several skull cap (Scutellaria lateriflora L.) herbal preparations. In the present report, we have demonstrated by analytical HPLC that teucrin A and teuflin (TC-5) are present in the aerial parts of T.chamaedrys L. in mg/g concentrations. However, teucrin A is absent in the subspecies T. canadense, while TC-5 and teucvidin (TC-6) are present at much higher concentrations. These results lead to the conclusion that teucrin A cannot be used as a marker compound for the genus as a whole. Determination of teucrin A and TC-5, both of which are relatively abundant and at least one of which is present in both Teucrium species, is the preferred method of identifying the presence of germander in herbal preparations.
Determination of Mycotoxins in Botanical Roots
M. W. Trucksess1 , C. M. Weaver1 , C. J. Oles1 , B. A. Cohen2 , J. I. Rader1 , 1CFSAN, FDA, College Park, MD, 2Vicam, Watertown, MA
The public health concerns related to both acute and chronic effects of mycotoxins in animals have prompted more than 100 countries to establish regulatory limits for some of the well-known mycotoxins. Our research focused on method development for three of these toxins: aflatoxins, ochratoxin A and fumonisins in botanical roots, specifically on ginseng and other selected roots. Methods using an immunoaffinity column cleanup, liquid chromatographic separation, and fluorescence detection were modified and evaluated. Three derivatization techniques to enhance the fluorescence of the aflatoxins were compared: pre-column trifluoroacetic acid, post-column bromination and post-column UV irradiation. Pre-column derivatization was used for fumonisins and no derivatization was needed for ochratoxin A. Results for aflaotoxins were all comparable for ginseng and for other roots such as ginger, licorice, and kava-kava. Recoveries of added total aflatoxins for ginseng at levels ranging from 2 ng/g to 16 ng/g were from 77 to 90 %. Recoveries of added toxins for ginger, licorice and kava-kava were from 50 to 70%. Recoveries of added fumonisins and ochratoxin A for ginseng at various levels and preliminary results of using commercially available ELISA kits for the three mycotoxins will also be presented.
Confirmation of Diminazene Diaceturate (Berenil) in Bovine Milk by LC-MS
S. B. Turnipseed, J. E. Robyal, S. B. Clark, C. M. Karbiwnyk, W. C. Andersen, ADRC, ORA, FDA
Background: Diminazene diaceturate (BerenilTM) is an antiprotozoal drug that could be used to treat diseases such as bebesiosis and trypanosomiasis in cattle in tropical regions. Unintended exposure to residues of this drug may cause possible human health concerns. While LC-UV methods are available for the quantitation of diminazene diaceturate in bovine milk and plasma, there are no published MS confirmatory procedures.
Methods: An LC-MSn method using electrospray ionization coupled with an ion trap mass spectrometer was developed for the confirmation of diminazene diaceturate in bovine milk. Samples were prepared using the methods described for LC-UV analysis with some modifications to obtain final extracts that would be compatible with LC-MSn. Chromatographic separation from matrix components was obtained using ammonium formate/acetonitrile with a C18 analytical column. Characteristic ions from the full MS spectra (m/z 282, MH+; 141.6), as well as MS2 (m/z 254) and MS3 (m/z 237, 220) product ions were monitored.
Results: Specific confirmation criteria for retention time as well as relative abundances of the ions monitored were developed. Using these criteria, diminazene diaceturate could be confirmed in raw and whole milk samples fortified at 1, 5, 10, and 25 ppb. Earlier dosing studies showed that the drug may be present in milk at levels below 10 ppb. The drug residue was not confirmed in reagent blanks or control milk. The application of this method to bovine plasma extracts is also being investigated.
Conclusion: Diminazene diaceturate can be confirmed at low ppb levels in bovine milk using LC-MSn.
Analysis of Protective Antigen in Final Product of Human Anthrax Vaccine
H. Wang, J. C. May, CBER, FDA, Rockville, MD
There is no assay to determine the amount of protective antigen (PA) and other protein components in the final product of US anthrax vaccine --- Anthrax Vaccine Adsorbed (AVA). The aim of this study is to develop a proper method to quantitatively analysis the various proteins in the vaccine and monitor lot-to lot change of the final product due to the nature of the fermentation procedure. There are two main steps for the analysis. The first one is to effectively dissolve PA and other proteins from the adjuvant --- Alhydrogel. The developed dissolving method could detect equivalent amount of PA content based on the protein analysis with the aliquots of the fermentation solution from manufacturer. The second step is to find a suitable method to determine the amounts of PA and other proteins and their molecular weights. SDS-PAGE, Bioanalyzer (a micro-electrophoresis method) and HPLC methods are used for the purpose. The results and limitation of each method are discussed.
Determination of Niacin and Biotin Using Microwell Microbiological Assays
G. M. Ware, Y. J.. Morris, M. D.. Hill, M. Haynes, SRL
A microwell microbiological assay method using 96-well format was developed for the determination of niacin and biotin in infant formula. The method is based on the assay principles of the AOAC official microbiological method [AOAC, 17ed, 45.2.01 (960.46)]. The microwell assay was optimized with respect to inoclum size, time of incubation, and dilution ratio of standards and samples. Samples were digested with 1.0 N H2SO4, pH adjusted to 4.5, filtered to remove precipitated protein, and pH adjusted to 6.8. Samples and standards were serially diluted with water. Each microwell was inoculated with Lactobacillus plantarum (ATTC) cells. The microplate was incubated at 37°C until maximum (constant) turbidity was obtained. Concentration-response curves for niacin and biotin were prepared with ranges 20.0 to 0.31 and 0.2 to 0.0031 ng/mL, respectively. The average correlation coefficient (R-value) for niacin and biotin were 0.9976±0.002 and 0.9984±0.001 (n=4), respectively. Using the microwell method, we analyzed 16 replicates of NITS's SRM-1846 for niacin and biotin and found 62.9±9.3 and 0.421±0.022 mg/kg, respectively. These results compare favorably with NIST certified value 63.3 ±7.6 and 0.411±0.066 for niacin and biotin respectively.
The Use of Electrodialysis to Prepare Aqueous Bread Extracts for Bromate Determination by Chemiluminescence
K. Himata1 , C. R. Warner2 , D. Currie3 , Q. Graves4 , G. W. Diachenko2 , 1Yamazaki Baking Co., 2OFAS/DCRER, 3Univ. of Maryland/Physics, 4OO/OSAS/DM
Background: Potassium bromate (PB) has been used by the baking industry as
a dough conditioner since 1914 when a patent was issued by the United States
Patent Office. Long-term toxicological studies based on drinking water for rats have established PB as a renal carcinogen. Quantitative risk analysis indicates that residues in the finished bread above 20 ppb in baked goods would lead to a potentially significant level of risk. Electrophoresis and chemiluminescence were studied as fundamental principles for a quick test for bromate at 20 ppb in bread.
Methods: A cleanup procedure based upon electrodialysis with three chambers separated by semi-permeable membranes is used to prepare the bread extracts for chemiluminescence. The relative merits of reverse osmosis (RO), ultrafiltration and nanofiltration membranes were evaluated. After electrophoretic separation with a RO membrane the bromate concentration in the collection chamber was typically 2 to 3 times greater than the concentration in the bread extract.
The chemiluminescent reaction of bromate with sulfite with hydrocortisone as the enhancer was selected for detection of bromate. The emission, with a wavelength maximum at 575 nm, was found to "glow" rather than "flash" after the reagents were mixed; therefore, it was possible to optimize the light collection period.
The method was validated with a variety of commercial bread products. White bread, hot dog buns, hamburger rolls and a multigrain bread from seven different manufacturers were studied.
Conclusions: The combination of electrodialysis and chemiluminescence provides a quick test for bromate in baked goods at levels of 20 ppb and above.
High-Performance Liquid Chromatographic Method for the Determination of Triiodoresorcinol in the Color Additive FD&C Red No. 3 (Erythrosine)
H. Mai, D. L. Brodie, M. B. Meyers, A. L. Baldo, Z. Krantz, A. Weisz, Office of Cosmetics and Colors, CFSAN, College Park, MD 20740
Background: FD&C Red No. 3 (R3, Colour Index No. 45430, mainly the disodium salt of 2',4',5',7'-tetraiodofluorescein) is a color additive listed in the U.S. Code of Federal Regulations (CFR) for use in foods and ingested drugs. The manufacturing process involves several steps: condensing phthalic anhydride with resorcinol; partially purifying and iodinating the resulting fluorescein; and finally hydrolyzing the product with sodium hydroxide. During manufacture, various impurities may be produced. R3 is batch-certified by the FDA to ensure compliance with certain limiting specifications, as described in the CFR. The specification limit for triiodoresorcinol (I3R) in R3 is 0.2%. Currently, the intermediates of R3 (including I3R) are analyzed by a multi-step procedure that includes gravity-column chromatography followed by spectrophotometric quantification. While this method is reproducible, it is labor-intensive. A more efficient, automated method was needed for the analysis of I3R in R3.
Methods: I3R was synthesized and purified for use as a reference standard. The solubility of I3R and its stability in the presence of R3 was determined using selected solvent systems. A high-performance liquid chromatographic (HPLC) method was developed to determine I3R in R3.
Results: For the quantification of I3R in R3, a five-point calibration curve was prepared by analyzing samples of R3 spiked with varying amounts of I3R. The calibration curve was linear over the needed range (0.1% to 0.3% w/w). Eight certified samples representing four manufacturers were analyzed by the developed HPLC method. No I3R was detected in the samples, which agrees with the results obtained using the gravity-column chromatography method.
Conclusions: The present study reports the development of an automated, reliable high-performance liquid chromatographic (HPLC) method for the determination of I3R in R3 that could replace the currently used procedure. The HPLC method is applicable for use in routine batch-certification analyses and will be extended to other CFR-specified intermediates of R3.
Simultaneous Determination of Protein Aggregation and Stability as well as Absolute Molecular Weight by SEC-MALLS
H. Ye, DPA, CDER, FDA, St. Louis, MO
Background. Increasingly, recombinant proteins and protein related products are being formulated as biopharmaceuticals. In this study the aggregation of glycosylated and non-glycosylated proteins, as well as antibodies are investigated using size exclusion chromatography (SEC) with on-line UV, refractive index, and multi-angle laser-light scattering (MALLS) detectors.
Methods. Proteins were dissolved in appropriate buffers with or without Tween 80. Separation of protein and its aggregates was performed on TSK-GEL G3000SWXL or G2000SWXL (7.8mm x 30cm). The eluent, 0.1M Na2SO4 and 0.05% NaN3 in 0.1 M phosphate buffer at pH 6.7, was pumped at 0.8 mL/min. Detection was carried out using HP G1362A refractive index, HP G1314A UV, and Wyatt Technology miniDAWN light scattering detectors. Quantitative and molecular weight determination was performed by the Astra software.
Results. Monomer and aggregates are well resolved although different proteins require different columns for separation. Aggregation and stability were monitored under various conditions and quantitative results were obtained. Observations on bovine serum albumin, choriogonadotropin, Herceptin and ReoPro are reported. Stability of these proteins is varied under conditions such as addition of detergent, pH adjustment, variation of protein concentration and temperature.
Conclusions. This method can simultaneously determine the quantities and molecular weights of macromolecules from a single injection. The determination of molecular weight is absolute which avoids the sometimes misleading effects caused by molecular shape or interactions with the column matrix. This is not only valuable for determination of aggregates, but also for effective monitoring of their stability as molecular degradation may be evidenced by molecular weight reduction.
Patulin Analysis Using A MycoSep Column
M. B. Young, V. A. Vega, FDA
The analysis of patulin in apple juice and apple juice products is a routine analysis performed in the mycotoxin group at the Food and Drug Administration. Patulin is an important mycotoxin because children are exposed to patulin through their consumption of apple products. This research focuses on a patulin method being developed that uses a MycoSep column and high performance liquid chromatography - diode array detector (HPLC-DAD) instrument. The goal of this method is to significantly reduce the number of clean-up steps and the analysis time for patulin. This research also includes recovery studies for precision and repetitive HPLC runs for accuracy.
Synthesis and prophylactic antimalarial activities of 2-guanidinylimidazolidinedione derivatives
Q. Zhang1 , J. Guan2 , G. Montip3 , E. William2 , A. Ager4 , W. K. Milhlus2 , D. R. Skillman2 , A. J. Lin2 , 1OGD, CDER, FDA/WRAIR, 2Div. of ET, WRAIR, 3AFRIMS, 4Univ. of Miami
The reported synthetic procedure of WR182393, a 2-guanidinoimidazolidinedione derivative with high prophylactic antimalarial activity, was found to be a mixture of 3 closely related products. Poor solubility of
WR182393 in both water and organic solvents and its impractical synthetic method have made the purification and structure identification of the reaction mixture a highly challenging task. The problems were circumvented by pro-drug approach involving carbamate formation of the mixture, which enhances the solubility of the mixture in common organic solvents. Structure activity relationship studies (SAR) resulted in discovery of a number of new compounds with 100% oral protection activity against P. yoelii sporozoites challenged mice at a dose as low as 2.5 mg/kg. Two new carbamates 3b and 4d were found to possess higher intramuscular (im) efficacy than the parent compound WR182393 againstPlasmodium cynomolgi in Rhesus monkey. The novel synthetic procedure and the SAR studies of the new carbamates will be presented.
HPLC Determination of Novobiocin in Dosage Forms
Louis P. Lue1, Susan T. Hadman1 and Ales Vancura2
1NRL, ORA, FDA, 158-15 Liberty Avenue, Jamaica, New York 11433
2St. John's University, Jamaica, NY 11439
AlbadryPlus® and Biodry® are two dosage forms of novobiocin (NOV) used to treat mastitis in dairy cattle. In addition to NOV, AlbadryPlus® also contains penicillin (PNC). We developed an HPLC method for simultaneous determination of NOV and PNC based on isocratic elution (0.85 mL/min) of µBondapak C18 column (300 mm X 3.9 mm) with mobile phase consisting of acetonitrile : 10 mM potassium phosphate buffer (35:65, v/v). The injection volume was 5 µL, and UV detection set at 254 nm for NOV and at 220 nm for PNC. NOV, penicillin, and procaine had retention times 4.56, 2.93, and 7.99 min, respectively. In contrast to the official USP microbial assay that does not allow simultaneous determination of both antibiotics, the developed HPLC method is suitable for determination of NOV and PNC in the same sample. In addition this HPLC method is more selective, sensitive and offers positive identification.
Development of Method for the Measurement of Tissue Steroids in Pregnant Rats.
P. P. Sapienza, I. A. Ross, W. D. Johnson, R. L. Sprando, S. C. Sahu, T. J. Flynn, P. L. Wiesenfeld, T. F. Collins, C. S. Kim, FDA
Background: An alternative method to existing radioimmunoassays (RIA) for measurement of plasma and tissue steroids was needed in order to chemically analyze for androstenedione and its steroid metabolites in pregnant rats dosed orally with androstenedione.
Method: Iced tissue homogenates (1:4) were prepared in tris-buffer. An internal standard (cortexolone) was added and the homogenates extracted twice with 3 mL ethanol/acetone (1:1) followed by centrifugation. Supernatants were concentrated under nitrogen at 37° C. Concentrates were delipidated in 3 mL of 70% methanol and stored at -20° C overnight and then centrifuged. The methanol was evaporated under nitrogen at 37° C. The residue was extracted twice with 3 mL ether supernatants dried with anhydrous sodium sulfate. The ether was evaporated and the dried extract was reconstituted in 70% methanol, centrifuged and the supernatant injected for HPLC analysis. The initial mobile phase was water:methanol:acetonitrile:isopropanol (62:28:5:5); changing to a linear gradient of 3.3%/min of water:methanol:butanol (35:45:20) using a C18 column. Flow rate was 0.5 mL/min and wavelengths were 254 nm and 280 nm (for β-estradiol ). An internal standard calibration method was used for quantification based on the molar concentrations calculated from a calibration curve.
Results and Conclusion: A method for the extraction and chemical analysis (HPLC) of steroids from plasma, liver, and brain has been developed for androstenedione, testosterone, β-estradiol, and progesterone. This method has an advantage over previous methods in that steroids can be determined chemically without using radioisotopes for steroid levels in various target organs and it is less costly than other RIA procedures.
A Cost Saving Approach to the HPLC-Postcolumn Derivatization Analysis of 35 N-Methylcarbamate Pesticide Residues in Fruits and Vegetables
J. A. Casanova, F. J. Schenck, ORA, FDA, Atlanta, GA
N-methylcarbamate pesticides (carbamates) are widely used on fruits and vegetable crops. Carbamate residues were found in 10% of >800 fruit and vegetable samples analyzed in our laboratory in the past year. An economical liquid chromatographic method using postcolumn derivatization for the LC-fluorescence determination of carbamates is presented. Luke extracts of fruit and vegetable samples were subjected to a single primary secondary amine (PSA) solid phase extraction (SPE) column cleanup and then split for use on both GC and HPLC systems. Solvent and reagent consumption and hazardous waste production for the HPLC analysis were reduced by >60% by using narrowbore (2.0 mm and 3.0 mm id) HPLC columns with a commercial postcolumn reaction system that had been designed for use with 4.6 mm id columns. All 35 carbamate residues were recovered from spiked tomato samples. The recoveries for 34 of the residues tested ranged from 91-107%. Some loss of chromatographic resolution for the early eluting polar compounds was noticed with the 2.0 mm id column. Excellent chromatography for all the compounds was obtained using the 3.0 mm id columns.
Melatonin Analysis in Dietary Supplements by HPLC using UV-VIS detector
J. O. Vega, J. Moreno, J. Bloom, FDA
A simple method for the determination and quantitation of melatonin in dietary supplements using HPLC with UV/VIS (DAD) detection was developed. Several matrices (capsules, tablets, capsules with herb) from different manufacturers were analyzed. A simple extraction method for all the samples was also developed using sonication and centrifugation and methanol as the extraction solvent. Results obtained for the assay of the samples were between 86 - 105% of label claim. Spike recovery results (95.1 - 101.2%) showed that components in the matrices do not interfere with the analysis or the precision of the method. The linearity range determined was from 0.040 ug/mL to 25.0 ug/mL, with an LOD at 40 ng/mL and an LOQ of 80 ng/mL. The calibration curves range used during analysis were between 1.60 - 25.00 ug/mL as recommended by USP for an assay of a drug product. We observed that the analyte chromatographic peak for melatonin at 4.4 minutes is attributable to only one peak due to the use of a DAD detector. In addition, the chromatographic patterns among the samples were very similar.
Identification of Metals and Other Elements in Tattoo Inks by X-Ray Fluorescence Spectrometry
N.M. Hepp, OCAC, FDA, College Park, MD
Background:. Reports of adverse events from permanent makeup/tattoos and from the effects of tattoo removal have recently increased. Color additives permitted in cosmetics are listed in the CFR, but these approvals do not include injection into the skin. Historically, certain metals (Hg and Ni) have been implicated in skin reactions. Also, some forms of titanium dioxide are photoactive. The Agency has initiated studies on the identification and safety of inorganic and organic pigments used in permanent makeup/tattoo inks. This work describes the x-ray fluorescence (XRF) determined elemental composition of selected commercial inks.
Methods: XRF was used to identify elements with atomic number 11 (Na) and above in several permanent makeup/tattoo inks. A standardless software program UniQuant (ODS) was used to estimate quantities of the elemental components. Only elements above trace (ppm) levels were reported.
Results: The permanent makeup/tattoo inks examined included various shades of red, yellow, black, brown, white, blue, and green. High levels of iron and titanium were found in many of the inks. Several other elements were also seen at levels higher than trace amounts. No mercury- or nickel-based pigments were found. The detection of non-metallic elements indicates the additional presence of organic pigments.
Conclusion: This study identified elements present in the selected inks. The results indicate that mixtures of pigments are present in many of the inks. The complexity of the mixtures complicates the correlation of adverse events with inorganic pigment identity.
Immunogenicity of West Nile Virus-like Particles in Mice
J. E. Adamo, C. A. Meseda, J. Soto, J. M. Muller, J. P. Weir, CBER, FDA, Bethesda, MD
West Nile Virus (WNV) is an emerging pathogen for which there is currently no human vaccine available. We have utilized a Modified Vaccinia Ankara virus to express two WNV structural proteins, pre-Membrane (prM) and Envelope (E). When co-expressed in cells, prM and E self-assemble into virus-like particles (VLPs) which are devoid of viral RNA, but retain immunogenicity similar to the complete virion. The immune response to these purified particles was evaluated by immunization of mice and was compared to the response elicited by DNA vectors expressing prM and E. We compared a needle-free injection device, Biojector 2000, and standard intramuscular injection for the VLPs and the DNA vectors. Since the immune response to West Nile infection is thought to be primarily humoral, we developed an ELISA to measure the IgG response. After immunization there was a detectable WNV-specific IgG response to both the VLPs and the DNA vaccine. One limitation to evaluating the efficacy of current WNV vaccine candidates is the required use of BioSafety Level 3 facilities for virus challenge. We are generating a replicating but non-infectious WNV cDNA which will be used to quantitate the amount of neutralizing antibodies elicited post-immunization. This replicon lacks the structural genes of WNV and contains a fluorescent reporter gene. When used in tandem with the VLPs, it will allow for analysis of neutralization and vaccine potency.
Molecular Identification of Yersinia enterocolitica Using DNA Microarray Chip Hybridization
K. M. Myeres1 , J. Gaba2 , S. F. Al-khaldi3 , 1JIFSAN University Of Maryland, 2JIFSNA University of Maryland, 3FDA College Park
A DNA microarray chip of four Yersinia enterocolitica virulence genes and non-specific 16S RNA gene as positive control was developed and evaluated using 22 Y. enterocolitica isolates and 10 different non-Yersinia bacteria. Eight different oligonucleotide probes (oligoprobes), complementary to the unique sequences of each gene, were designed and immobilized on the surface of chemically modified slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all genes and single-stranded DNA (ssDNA) samples for microarray analysis were prepared by using a primer extension of amplicons in the presence of one primer. Fluorescent labeling using Cy3 was incorporated into the ssDNA by chemical modification of guanine bases. The presence of genes in Y. enterocolitica was established by hybridization of the fluorescently labeled ssDNA representing different samples of the microarray gene-specific oligoprobes and confirmed by PCR. Results of the study showed sensitivity and specificity of genotyping Y. enterocolitica using multiple microarray-based assays.
Lowering the Detection Limits of HIV-1 Viral Load Using Real-time Immuno-PCR (IPCR) for HIV-1 p24 Antigen
J. Barletta, Ph.D.1 , D. C. Edelman, M.S.2 , N. T. Constantine, Ph.D.2 , 1CDER, FDA, Rockville, MD, 2University of Maryland Baltimore, Baltimore, MD
Purpose: Currently, nucleic acid tests can detect HIV-1 infection 12 days post infection, and can identify 50 copies HIV-1 RNA/mL. Our objective was to develop a real-time IPCR method able to detect lower levels of viremia than testing methods presently available based on the premise that there are 75,000 HIV-1 p24 moecules available for detection (i.e., 3,000 molecules of p24 antigen) per HIV-1 virion.
Methods: The IPCR is a technique whereby exponential amplification ability of the PCR is coupled to detection of proteins in an ELISA format. To assess performance of the IPCR, 14-37 replicates from HIV-1 antibody positive patients with known HIV-1 RNA viral loads were diluted within three log groups (between 1.68 - 6514 viral RNA copies) for analysis. 52 samples from HIV-1 infected persons (antibody positive) who had viral loads <50 RNA copies/mL were also tested.
Results: IPCR detected 42 % of patient samples which were not detected by RT-PCR (i.e., <50 RNA copies/mL). The limit of detection for the IPCR was equivalent to 20 viral RNA copies/mL (0.66 viral RNA copies per reaction or 40 attograms/reaction at approximately 1000 p24 antigen molecules).
Conclusions: IPCR for HIV-1 p24 antigen detection was shown to have higher analytical sensitivity for the detection of viremia than approved nucleic acid tests. It has the potential to confirm the diagnosis of HIV-1 at an earlier time than current methods, and to monitor the response to anti-retroviral treatment when HIV-1 RNA copy number is below 50.
(Experimental work was performed at the University of Maryland Baltimore)
Barletta J.M., Edelman D.C., Constantine N.T., Lowering the detection limits of HIV-1 viral load using real-time immuno-PCR for HIV-1 p24 antigen. Amer J Clin Pathol. 122(1): 20-27, 2004.
Immune Complex Repression of Interferon-gamma Signaling by a Novel Fc-gamma-Receptor-1 Function
G. H. Boekhoudt, M. R. Frazier-Jessen, G. M. Feldman, DMA, OBP, OPS, CDER, FDA, Bethesda, MD 20892
Background: Immune complexes (IC) have been implicated in the initiation and the progression of inflammatory human diseases. The mechanisms by which the IC and its ligand the Fc-receptor accomplish these effects are still unknown. In an effort to better understand the ability of IC to inhibit IFNγ-induced activation we concentrated on dissecting out the point where the signaling pathways of the Fc-receptor and the IFNγ-receptor cross.
Method: Human peripheral blood monocytes that were pretreated with or without IC followed by stimulation with or without IFNγ were used in numerous in vitro assays including RT-PCR, EMSA, immunoprecipitation and pull-down. In addition, knockout mice for the FcRγ chain and SHP-1 genes were also studied.
Results: IC inhibits IFNγ-induced genes IP-10 and CD64. Data from the FcR gamma chain knockout mice demonstrates that the gamma chain is critical to this inhibition. Blocking the FcγR1 with specific Fab2 fragments mimic the results from the knockout model. Cross-linking FcγR1/Fab2 complexes reproduced the inhibition of IFNγ induction by IC. The src kinase inhibitor PP2 indicate its requirement in the function of IC while the data from Motheaten mouse showed a role for SHP-1 in the repressive function of IC.
Conclusion: The initiation of FcγR1 signaling by IC activates downstream signaling molecules such as src kinase and SHP-1, which are required for the inhibitory function of IC. These findings further elucidate the mechanism by which IC regulates IFNγ-signaling, while adding a level of complexity to the signaling crosstalk between Fc receptors and by which IFNγ is regulated.
Development of a Quantitative Multiplex RT-PCR Assay for the Detection of Noro- and Enteroviruses
W. Burkhardt III, J. Woods, M. Vickery, FDA, GCSL, Dauphin Island, AL
Noroviruses and enteroviruses have been detected in municipal wastewater and polluted receiving waters. Bivalve molluscs can become vectors of these agents if the water in which they feed is contaminated by human fecal material. Noroviruses have been implicated as the causative agents responsible for the majority of non-bacterial gastroenteritis in shellfish consumers. While certain enteroviruses can be cultured by conventional tissue culture techniques, the use of RT-PCR bases assays has increased detection sensitivity and reduced analysis time. The predominant human noroviruses, which are placed into two genogroups, GI and GII , are non-culturable, so their detection is based primarily upon non-quantitative RT- PCR assays. Advancements in real-time quantitative RT-PCR (qRT-PCR) technology have allowed the recent development of several qRT-PCR assays for the rapid detection and enumeration of enteroviruses and Norovirus; however, the Taqman style assay developed for Norovirus requires separate simplex reactions to distinguish the GI and GII genogroups, and the SYBR Green based assay is unable to reliable distinguish these two genogroups. To date, no single assay has been capable of simultaneous detection and enumeration of enteroviruses, and noroviruses GI and GII. In the present study, using nucleotide sequences based upon previously published oligonucleotide primers and Taqman-style probes, we developed a multiplex qRT-PCR assay on the Cepheid SmartCycler® system for the simultaneous the detection and enumeration of enteroviruses and norovirus genogroups I and II. We also incorporated a novel quantitative internal control to prevent the reporting of false negatives due to inhibition or failure of the qRT-PCR. The development of this methodology should allow the rapid semi-quantitative determination of Norovirus and enterovirus levels in nucleic acids extracted from clinical and environmental matrices.
Identification of a new drug target for treatment of human leishmaniasis
N. Lee, S. Gannavaram, A. Selvapandiyan, H. L. Nakhasi, A. Debrabant, CBER, FDA, Bethesda, MD
The protozoan parasite Leishmania donovani is the causative agent of human visceral leishmaniasis that is responsible for hundred of thousands of deaths per year worldwide. In addition, U.S. Military personnel and travelers to endemic areas are at risk for leishmaniasis and are also deferred for blood donations. In that regard, in the last two years, over 900 cases of leishmaniasis have been diagnosed among the troops in Iraq and Afghanistan. To date, there is no effective vaccine against Leishmania and parasites show increasing resistance to current anti-leishmanial drugs. Programmed cell death (PCD) is an essential part of cell biology and is thought to have evolved not only to regulate growth and development in multicellular organisms but also to have a functional role in the biology of unicellular organisms. In higher eukaryotes caspases are key effector molecules of PCD. There is no caspase gene is the Leishmania genome, however, we identified and characterized a caspase-like gene in L. donovani and showed that this unique enzyme (metacaspase) is probably involved in the Leishmania PCD pathway. Since metacaspases are not present in the human host, the L. donovani metacaspase represents a new target that could be exploited for the development of novel anti-leishmanial drugs that would trigger the parasite PCD pathway. This study contributes to our better understanding of the Leishmania parasite biology and pathogenesis which is essential for making informed regulatory decision with regard to this infectious agent and other related blood borne pathogens.
The Effects of Agricultural Pesticide and Antibiotic Applications on Apples and Apple Juice Microflora.
B. S. Eblen1 , A. R. Ottensen2 , E. C. Hawkins3 , C. S. Walsh2 , A. J. Miller1 , 1CFSAN, FDA College Park, MD, 2NSRL Department, University of Maryland, College Park, MD, 3JIFSAN, Student Intern, University of Maryland, College Park, MD
The impact of pesticide and antibiotic sprays, on microflora levels in apple and expressed juice was investigated. Three plots with randomized repetitions Enterprise and Goldrush trees were designed to receive three distinct treatments. Plot 1 received no pesticide applications of any kind for 2 years. Plot 2 received a spray regime mimicking garden type applications, with cessation of sprays after first cover for the same 2 year period. Plot 3 was treated with a full spray regime for two consecutive years. Apples were sampled at different time-points throughout the season in each plot and analyzed for total aerobic (TA), yeast and mold(Y&M), total coliform (TC) and Gram negative (GN) counts. Apples from each plot were harvested at the end of the growing season, juiced then analyzed. Results indicated that there were no consistently, significant differences found between plots at all time-points in the Y&M and TA counts. TC counts were not significantly different between plots in early season samples. A significant difference between Plot 1 and Plot 3 was observed during late season samples (p<0.001). A consistent, significant difference in GN counts was observed at all time-points after first cover between Plots 1 and 3 (p<0.009). In expressed juice significant differences were noted for all microbial enumerations between Plot 1 and 3 (p<0.0005) and between Plot 2 and 3 (<0.002). The observations demonstrate the impact of different agricultural applications on apples and juice microflora. Any effect on apple or juice microflora levels is of relevance to food safety and quality.
Steroid Hormones are Substrates for the Major RND- and MFS-type Tripartite Multiple Drug Efflux Pumps of Escherichia coli
C. A. Elkins, L. B. Mullis, NCTR, FDA, Jefferson, AR
Gastrointestinal tract (GI) bile acids are steroid molecules that exert antibacterial effects due to their detergent nature; they also serve as the potential "natural substrates" for several multiple drug efflux (MDE) pumps in gram-negative bacteria. We assessed whether other steroid molecules, such as hormones, are also recognized as substrates of certain MDE pumps although they are not antibacterial at physiological concentrations. Uptake of several tritium-labeled steroids was measured using E. coli AG102 (marR1) cells and its isogenic mutant derivatives containing deficiencies in two major MDE pumps: AG102MB (acrB::kan), CE1 (emrAB::cat), and HNCE4 (acrB::kan, emrAB::cat). Interestingly, uptake of [2,4-3H(N)]cholic acid, [1,2-3H(N)]progesterone, [6,7-3H(N)]estradiol, and [1,2-3H]hydrocortisone increased with AG102MB cells approximately 2-, 6-, 10-, and 15-fold, respectively, over wild-type AG102 cells, suggesting that the primary resistance nodulation division (RND)-type tripartite efflux pump (AcrAB-TolC) recognizes and expels these steroid molecules. In addition, uptake of progesterone, cholic acid, and estradiol in HNCE4 cells increased approximately 2-, 3-, and 4-fold, respectively, over that of AG102MB cells (11-, 7-, and 38-fold over AG102 cells, respectively) further suggesting that the primary major facilitator superfamily (MFS)-type tripartite system (EmrAB-TolC) also recognizes at least two steroid hormones in addition to the bile acid, cholic acid, but not hydrocortisone. With CE1 cells, uptake profiles were similar (expectedly so) to wild-type AG102 cells presumably because of the overriding influence of the AcrAB-TolC system. In comparison, uptake levels of [1,2-3H(N)]cholesterol were indistinguishable between AG102, AG102MB, and CE1 cells but were also approximately 2-fold lower than in HNCE4 cells. When these strains were exposed to the ionophore and protonmotive force uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), at 0.1 mM, uptake of estradiol and progesterone increased to levels that were generally similar in all four strains and approximately 2-fold higher than with the respective non-CCCP-treated HNCE4 cells which indicate either (i.) the presence of additional steroid efflux mechanism(s) or (ii.) a non-specific protective effect of the protonmotive force on steroid uptake. In substrate competition experiments, estradiol uptake was unaffected in wild-type AG102 cells at increasing concentrations of the conjugated bile acid, taurocholic acid (pKa ~1.4), supplied exogenously in the assay medium, whereas a small inhibitory effect was observed when using the deconjugated bile acid, cholic acid (a weak neutral lipophile, pKa ~6.4). Conversely, cholic acid uptake in AG102 cells increased consistently at successively higher concentrations of exogenous estradiol. Taken in aggregate, these data suggest that steroid hormones are "good" substrates for at least two major efflux pumps in E. coli and that this activity may be important for survival in and transit through the GI tract.
Assessment of Dermal Absorption, Metabolism, and Irritation Potential of Arachidonic Acid & Glyceryl Arachidonate Using In Vitro Diffusion Cell Techniques
A. R. Eppler1 , M. E.K. Kraeling1 , R. R. Wickett2 , R. L. Bronaugh1 , 1Office of Cosmetics and Colors, US FDA, Laurel, MD, 2College of Pharmacy, University of Cincinnati, Cincinnati, OH
Arachidonic acid (AA), which is known to be metabolized to pro-inflammatory mediators, and its glycerin ester, glyceryl arachidonate (GA), have been used as ingredients in cosmetic products. Therefore, in vitro diffusion cell studies were conducted to evaluate skin absorption and metabolism of AA & GA and skin irritation by AA. To simulate normal consumer use conditions, [14C]AA and [3H]GA were topically applied in an oil in water emulsion (2mg/cm2). AA penetration in viable rat skin was found to be 50.3% of the applied dose while penetration in viable human skin was found to be 18.4%. GA penetration in viable human, human cadaver, and Epiderm cultured skin was found to be 21.4%, 14.8%, and 55.8% respectively. Approximately 3% of the absorbed dose of GA in Epiderm was metabolized to AA, based on HPLC analysis. Skin irritation by model compounds and AA was assessed on rat skin and Epiderm assembled in flow-through diffusion cells. Irritation was determined after 24hr exposure by evaluating barrier integrity changes using TEWL measurements and skin cell viability changes using the MTT assay. TEWL measurements taken immediately prior to the skin surface wash were inaccurate due to the occlusion of the emulsion vehicle, while measurements taken immediately following the wash were elevated due to evaporation of the wash and rinse solutions. For the emulsion-vehicle treatments (Tween 80, sodium lauryl sulfate, and AA applied in an oil in water emulsion at 10 mg/cm2), TEWL measurements obtained 40-60 minutes after the wash had returned to baseline values taken prior to dosing. Therefore, no significant irritation could be observed with TEWL analysis of these treatments. However, aqueous 5% sodium lauryl sulfate (78 µl/cm2) on rat skin resulted in a significant increase in TEWL. Irritation assessment with the MTT assay showed greater sensitivity when using Epiderm than when using rat skin. In conclusion, AA and GA dermal penetration was measured and Epiderm was determined to be potentially useful in metabolism studies. Additionally, irritation assessment in flow-through diffusion cells may be possible by means of the MTT and TEWL assays.
Synergistic inhibition of rat liver cytochrome P450 (CYP) activities by co-exposure to a bacterial (lipopolysaccharide) and mold (deoxynivalenol) toxin
T. J. Flynn, P. P. Sapienza, P. W. Wiesenfeld, S. C. Sahu, C. S. Kim, M. E. Lorenzo, L. H. Garthoff, FDA, CFSAN, OARSA, Laurel, MD
Background: Bacterial and mold toxins could occur in foods together either from natural co-contamination or intentional tampering. Little is known about the toxicological effects of such co-exposures.
Methods: Young adult male rats were injected i.p. with 50-100 µg/kg E. coli lipopolysaccharide (LPS) and/or 5-10 mg/kg deoxynivalenol (DON). Animals were euthanized at 3, 24 and 72 hours after injection. Livers were collected, and liver microsomes were incubated with 200 µM testosterone. Hydroxylated metabolites of testosterone (a measure of multiple CYP activities) were identified and quantitated by HPLC.
Results: LPS alone caused a significant drop in metabolite formation by 24 hours which persisted through 72 hours post injection. DON alone caused a marginally significant drop in metabolite formation at 24 hours that recovered to control levels by 72 hours. Combined LPS and DON treatment caused a drop in metabolite formation that, at 24 hours, was significantly different from control but not from LPS alone. At 72 hours, metabolite formation in the combined treatment group had decreased further and was significantly lower than that in all other treatment groups at all other time points. Evaluation of the proportions of individual hydroxylated metabolites at 72 hours after combined treatment showed that male-specific metabolites (16α and 2α) decreased while the relative proportion of the female-specific metabolite (7α) increased.
Conclusions: The effects of combined exposure to LPS and DON on inhibition of rat liver CYP were more severe than an additive effect of LPS and DON alone, and sexually dimorphic CYP isoforms were affected differently.
Mechanistic studies on the photocytotoxicity of 2,3-diaminophenazine
P. K. Fu1 , J. J. Yin1 , C. Turro2 , 1CFSAN, FDA, College Park, MD, 2The Ohio State University, Columbus, OH
Background: 2,3-diaminophenazine (DAP), a major contaminant in commercial hair dyes and an agricultural pesticide, exhibits profound effects on human skin fibroblasts and plasmid DNA upon irradiation.
Methods: The photobiological properties of DAP toward human skin cells and plasmid DNA was evaluated using a cytotoxicity test and a DNA nicking assay. The photophysical and metal chelating characteristics were investigated using laser flash photolysis, and electronic absorption, emission, and EPR spectroscopies.
Results: DAP exhibits weak intercalative binding to double stranded DNA (Kb = 2.8 x 103 M-1), and is able to nick plasmid DNA in the presence of oxygen upon irradiation with visible light (400 - 700 nm, 30 min, 5 J/cm2). The concentration of DAP which resulted in 50% cell death (LC50) was 172 ± 9 µM in the dark and 13 ± 1 µM upon irradiation, a 13-fold increase in toxicity. The mechanism behind DNA photocleavage and the phototoxicity was further investigated using EPR spectroscopy. Preliminary data showed the generation of reactive oxygen species. Bathochromic and hypsochromic shifts in UV/Vis absorption and emission spectra were observed, which indicates the possibility of chelating to heavy metal cations such as Fe (III), Zn (II), Cu (II), Co (II) and Cr (III).
Conclusion: These results suggest reactive oxygen species may be involved in the photocytotoxicity of DAP. The role of metal chelation must be further investigated.
In Vitro Dermal Absorption and Metabolism of D&C Red No. 17.
C. T.. Haynes, R. L. Bronaugh, J. J. Yourick, CFSAN, FDA, Laurel, MD
D&C Red No. 17 is approved for use in externally applied drug and cosmetic applications, in amounts consistent with good manufacturing practice. Concerns about the safety of this color additive (1-[4-phenylazophenylazo]-2-naphthol (PAN) is the primary color constituent) have been raised due to potential metabolic cleavage of PAN to yield 4-aminoazobenzene. [14C]-PAN was applied in a sunscreen vehicle containing D&C Red 17 to viable porcine and non-viable human skin in flow-through diffusion cells. At the end of 24 hours, unabsorbed material was removed from the skin and some cells were allowed to continue for an additional 48 hours. In human skin 0.02% and 0.07% of the applied dose was absorbed into the receptor fluid after 24 and 72 hours, respectively. At the end of 24 hours 12% of the applied dose remained in the skin and this amount had not decreased at 72 hours. Similar results were found when PAN was applied to porcine skin, with 0.02% of applied dose absorbed and 12% remaining in the skin. Results examining PAN metabolism by HPLC found no metabolites in viable porcine skin. Skin absorption of PAN was low from this sunscreen product.
The evaluation of the role of A27L protein in vaccinia induced protective immunity
Y. He, J. Manischewitz, C. Meseada, R. Vassell, H. Goolding, C. D. Weiss, FDA
Vaccinia immune globulins (VIG) is currently the only licensed treatment for severe adverse reactions to the smallpox vaccine (Dryvax). However VIG was never tested in randomized clinical trials, and its immune correlates to protection are unknown. Therefore, we undertook studies to dissect the role of the A27L protein, a well known neutralizing target of vaccinia virus(VV), in vaccine-induced protective immunity in order to gain information for evaluating and designing new smallpox vaccines and therapeutics. To map the neutralizing epitope in A27L protein, we made a recombinant A27L protein (rA27L). Rabbits were immunized with rA27L and peptides corresponding to different regions of A27L. Our results showed that only antisera to rA27L, not the individual peptides, had potent neutralization activity, suggesting that neutralizing determinants may be conformational. To dissect the antibody components in VIG that contribute to protection, we analyzed VIG by rA27L affinity chromatography. Our results showed that only very small of VIG is composed of A27L antibodies. Finally we tested the in vivo protection by immune compromised animal model. The results show the first time that therapeutic administration of polyclonal antibody specific to A27L protein alone can be an effective means of control of VV replication in the nude mice. Animals receiving both A27L antibody +VIG survived longer than the groups that received A27L antibody or VIG alone. The results of VIG fractionations, along with the in vivo studies indicate that Dryvax vaccination dose not generate a robust A27L antibody response. Thus it appears that A27L antibodies play a minor role in Dryvax induced protection and therapeutic activity of VIG. These findings suggests that ways to augment existing therapeutics and vaccines.
Multiplex Real-Time PCR to Simultaneously Detect Listeria spp. and L. monocytogenes from a Variety of Food Enrichments
A. D. Hitchins1 , K. C. Jinneman2 , K. J. Yoshitomi2 , G. M. Blackstone3 , K. Thammasouk2 , J. M. Johnson2 , M. D. Feist2 , 1CFSAN, College Park MD, 2ORA, Bothell, WA, 3CFSAN, Dauphin Is., AL
Background: Faster methods of detecting the foodborne pathogen Listeria monocytogenes are needed.
Methods: We developed a multiplex real-time PCR 5' nuclease based assay targeting the Listeria invasion -associated protein gene. Its conserved and variable regions were targeted with generic or specific primer-probe sets to identify all Listeria or just L. monocytogenes. A preliminary enrichment step was used to cope with low target organism concentration and possible polymerase inhibitors in food. An internal control system was included. Fifteen foods were spiked at about 8 CFU L. monocytogenes /g and enriched for 48 h at 30°C. Enriched cells from 0.5 ml aliquots were washed with saline, and boiled (10 min) in water to extract target templates.
Results: The genus and specific gene targets were detected in most of the food enrichment templates. Only one was reliably detected with lettuce salad mix or brie while none with sour cream. L. monocytogenes culture dilutions were added to 48 h food enrichments in duplicate and templates prepared. Genus and specific gene targets were detected at about 2 x 105 CFU/ml in shrimp, smoked salmon jerky, cold smoked salmon, mozzarella cheese and clam dip enrichments; at 2 x 106 in roe, crab, sour cream, hot dog, deli turkey and salami enrichments; and 2 x 107 in salad mix and milk. Results were variable with brie and roast beef spread. Mean PCR efficiencies were 1.93 (genus) and 1.58(species) out of a possible 2.0.
Conclusion: Additional procedures may help improve real-time PCR screening of the refractory food matrices observed in this study.
Atypical Non-hemolytic Listeria seeligeri Isolated in the USA
D. V. Volokhov1 , V. E. Chizhikov1 , R. E. Duvall2 , A. D. Hitchins2 , 1CBER, FDA, Rockville, MD, 2CFSAN, FDA, College Park, MD
Background: Regulation of food pathogens, like Listeria monocytogenes, requires accurate identification of them and their related species. This may be confounded when aberrant variants are isolated as is exemplified by this study of non-hemolytic L. seeligeri isolates presenting as L. welshimeri.
Methods: Identities of 7extant isolates of the Rha- biotype of L. welshimeri were re-determined phenotypically. Their hybridization patterns with general iap- and hly-specific probes were determined with a validated species identification oligonucleotide microarray. Their reactivity with L. seeligeri hly-specific oligonucleotide probes was determined. The hly region of the isolates' genome was compared with that of L. seeligeri, ATCC 35967, the type strain, using PCR and nucleotide sequencing. Housekeeping genes were sequenced and phylogenetically analyzed.
Results: The 7 isolates were confirmed as the Rha- biotype of L. welshimeri, not its Rha+ biotype, by standard tests. In contrast, they identified as L. seeligeri based on their hybridization patterns in the species identification oligonucleotide microarray. The isolates did not hybridize with special L. seeligeri hly-specific probes. PCR and sequencing showed that the PrfA regulated virulence-gene cluster was altered in the variants by the loss of 13 genes: orfD-prfA-orfE-plcA-hly-orfK-mpl-actA-dplcB-plcB-orfH-orfX-orfI. Thus the isolates' cluster is: prs-orfA2-[13-gene deletion]-orfP-orfB-orfA-ldh. The isolates' housekeeping genes (iap, 16S rRNA gene, the inter-16S-23S rRNA gene region, prs, comK, groEL, cat, sigB, recA, hsp60, and ldh) were L. seeligeri specific.
Conclusions: Atypical variants of Listeria species, like Hly- L. seeligeri, can sometimes only be recognized genotypically. Phenotypic tests may be insufficient.
Bile Salts Tolerance of Clinical and Food Isolates of Listeria monocytogenes
D. M. Winkler1 , A. D. Hitchins2 , 1JIFSAN, College Park, MD, 2CFSAN, College Park, MD
Background: Two strains of Listeria monocytogenes (Lmo), a foodborne pathogen, are reported to able to exist and grow in the murine gall bladder system, tolerating bile salts (BS). The universality of the tolerance was tested in vitro with 100 strains from clinical and food sources.
Methods: Listeria strains (N=30) were tested in Trypticase Soy Broth with yeast extract (TSBye) plus ox gall (0.625-25%w/v). Growth rates on Trypticase Soy Agar-yeast extract (TSAye) plus 4%w/v ox-gall were estimated from colony diameters measurements. Cells were stained and observed by light microscopy. Bile salt hydrolase activity (BSH) was detected anaerobically by production of opacity zones and/or chalk-white growth of stabs in deMan-Rogosa-Sharpe agar (MRS) with (0.5 %w/v) Na taurodeoxycholate (TDCNa) or Na glycodeoxycholate (GDCNa). All incubations were at 37°C. The GDCNa responses of 40 clinical and 60 food isolates were compared.
Results: Lmo strains were able to tolerate at least 18.75%w/v bile in TSBye. Lmo grew half as fast on 5%-oxbile-TSAye as on TSAye tending to form filaments. Lmo formed distinctive chalk-white colonies on TDCNa- and GDCNa-MRS agar. An opacity zone was seen sometimes especially with GDCNa. No BSH activity was observed with the ox-gall or bile salts # 3. GDCNa-opacity-zones occurred more often with clinical than with food isolates. This may help in screening the pathogenic potential of Lmo strains.
Conclusions: BS concentrations higher than found in the human biliary system are tolerated by Lmo. Conceivably, at least, a human carrier might disseminate Lmo just like the cook "Typhoid Mary" spread Salmonella typhi.
A simplified and efficient method for full-length cDNA Preparation
Y. Hu1 , I. Hirshfield2 , 1U.S. Food and Drug Administration, Northeast Regional Laboratory, Jamaica, NY 11433, 2St. John's University, Jamaica, NY 11439
PCR amplification of long DNA fragments have been advanced significantly in the past few years, but the synthesis of full-length cDNA from long mRNA transcripts still remains a challenge. Traditional reverse transcription of mRNA can produce only partial cDNAs. There is a need to develop an efficient method for creating full-length cDNAs in high quantity that contain the entire mRNA sequence for subtractive hybridization, DNA microarray analysis, cDNA library generation or for viral sample preparation and other regulatory applications. We developed a useful method to generate full-length cDNA probes for subtractive hybridization that requires two major steps. Briefly, after mRNA preparation, the 1st strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase with a modified (dT) primer. When RT reaches the 5' end of the mRNA, the enzyme's terminal transferase activity added a few additional nucleotides to the 3' end of the cDNA to create an extended template. The 3' cDNA sequence and the 5' poly T sequence serve as universal priming sites. The 1st strand cDNA was used as a template for direct PCR amplification. The double-stranded cDNA was then synthesized by universal long-PCR. Unlike most traditional cDNA preparation methods which are not well suited for long mRNA transcripts, our method is able to efficiently synthesize a full-length cDNA. We have successfully used this method to generate a full-length cDNA probe for subtractive hybridization and also synthesis of a potential full-length HCV cDNA up to 9.6 kb. This method can be used routinely for regulatory purposes in our FDA field laboratory for gene discovery and viral cDNA preparation.
Antibiotic Resistance of Salmonellae Isolated from Various Products, 2003-2004
C. R. Kiessling1 , M. H. Loftis1 , W. M. Kiessling1 , M. B. Buen1 , E. W. Laster1 , J. N. Sofos2 , 1FDA, 2Colorado State University
Foodborne salmonellosis continues to be a problem within the Unites States. FDA routinely analyzes food products and animal feeds for presence of Salmonella and subsequently determines antibiotic resistance/sensitivity patterns of isolates. Currently, Salmonella isolates are subjected to minimum inhibitory concentration (MIC) testing to determine resistance patterns; previously, resistance patterns were obtained utilizing disk assays. This report presents MIC data for isolates collected from August 2003 through December 2004, and compares the results with data of previous years collected with disk assays. A total of 494 isolates were submitted for analysis (391 from foods and 103 from feeds/miscellaneous sources). The most frequently isolated serotypes (23.8% of all isolates) from foods continued to be, in decreasing order, Weltevreden, Newport, Saintpaul, and Senftenberg. The frequency of isolates exhibiting resistance to one or more antibiotics was 16.4% as compared with previous findings (1999-2003) of 45.9%. Serotypes Enteritidis, Montevideo, Mbandaka, Senftenberg and Cerro constitute 40.8% of the isolates from animal feeds, dog treats and environmental swabs which is consistent with previous findings; however, only 5.8 % of these isolates exhibited resistance to one or more antibiotics (down from 63.0 % for 1999-2003). All isolates continued to show the highest resistance to sulfamethoxazole and tetracycline, with isolates originating from foods also showing resistance to streptomycin. It will be interesting to compare these results with future data in order to confirm whether there is a real downward trend in resistance or whether variation in methodologies and sources of isolates may also be involved in the decline.
Absorption of Lawsone through Human Skin.
M. E.K. Kraeling1 , C. T. Jung2 , R. L. Bronaugh1 , 1Office of Cosmetics and Colors, CFSAN, USFDA, Laurel, MD, 2Office of Pharmaceutical Science, CDER, USFDA, Rockville, MD
Lawsone (2- hydroxy-1, 4-naphthoquinone) is the principal color ingredient in henna, a color additive approved with limitations for coloring hair by the Food and Drug Administration under 21 CFR 73.2190. The safety of lawsone as a coloring agent in hair dye products has recently been evaluated by the Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) of the EU. The SCCNFP concluded that lawsone was mutagenic and not suitable for use as a hair coloring agent. Studies were conducted to measure the extent of lawsone absorption through human skin. Lawsone skin absorption was determined from two hair coloring products and two shampoo products, all containing henna. [14C]-Lawsone (Sp. Ac. 22.9 mCi/mmol) was added to each commercial product and applied to excised, non-viable human skin (approx. 240 µm thick) mounted in flow-through diffusion cells perfused with a physiological buffer (HEPES-buffered Hanks' balanced salt solution, pH 7.4). Products remained on the skin for 5 min (shampoos) and 1 h (hair color paste). For the henna hair paste products, 0.29 and 1.4% of the applied dose was absorbed into the receptor fluid in 24 h while 2.2 and 3.7% remained in the skin. For the henna shampoo products, 0.32 and 0.34% of the applied dose was absorbed into the receptor fluid at 24 h while 3.6 and 6.8% remained in the skin. For all products, most of the lawsone applied was washed from the surface of the skin (83 - 102%) at the end of the exposure period. Extended absorption studies were conducted for 72 h to determine if skin levels of lawsone in the 24 h studies might eventually be percutaneously absorbed. These studies determined that the majority of the lawsone remained in the skin with little diffusing out into the receptor fluid. For example, the 72 h receptor fluid values following administration of henna paste products were only 0.48 and 1.61%. Therefore, it appears that the 24 h receptor fluid values would be a good estimate of lawsone absorption in a 24 h exposure estimate.
Methyleugenol Skin Absorption in Human and Fuzzy Rat Skin.
J. J. Yourick, R. L. Bronaugh, Office of Cosmetics and Colors, CFSAN, USFDA, Laurel, MD
Methyleugenol (ME) is used as a fragrance ingredient in perfumes, soaps, detergents, creams, and lotions. Methyleugenol is an allylbenzene that is structurally related to safrole, isosafrole and estragole. Concerns about ME's safety have been raised by National Toxicology Program carcinogenicity testing in rodents. Therefore, we initiated studies to measure ME dermal absorption in human (in vitro) and fuzzy rat (in vitro and in vivo) skin. In vitro [14C]ME (approx. 0.5 ìCi/cell) skin absorption was measured using either ethanol or an emulsion dosing vehicle. In vitro absorption was measured for 24 h by using flow-through diffusion cells (0.64 cm2) with a receptor fluid consisting of HHBSS (pH 7.4). In vivo ME rat skin absorption was determined from an ethanol vehicle applied to skin for 24 h. In human skin (n=3), the percentage of applied dose absorbed (%ADA) from an ethanol vehicle over 24 h was 9.3 ± 1.82 (mean ± SEM) with approximately 0.9% remaining in skin. In rat skin in vitro (n=3), the %ADA from an ethanol vehicle over 24 h was 34.2 ± 2.82 with approximately 1.3% remaining in skin. Absorption of ME from an emulsion vehicle resulted in similar receptor fluid and skin levels when compared to levels measured in human or rat skin with an ethanol vehicle. Occlusion of the diffusion cells resulted in significantly higher in vitro absorption of ME with receptor fluid %ADA values of 49.7 ± 16.9 and 77.3 ± 6.0 for human and rat skin, respectively. ME rapidly penetrated both human and rat skin in vitro with approximately 7 and 30%, respectively, of the absorbed dose found in the receptor fluid within 6 hr. Use of a charcoal trap attached to the top of the diffusion cell improved the recovery of the volatile ME. In vivo absorption of ME in the rat resulted in systemic absorption of 19.8 ± 0.1 %ADA with 0.7 %ADA remaining in skin. These studies indicate there is considerable ME absorption in skin. Systemic ME absorption should be expected after dermal application of ME-containing consumer products.
Study of the Effect of Different Vaccination Schedules on Excretion and Reversion of Vaccine Poliovirus
M. Laassri1 , K. Lottenbach2 , R. Belshe2 , S. Plotkin3 , K. Chumakov1 , 1CBER, FDA, Rockville, MD, 2Saint Louis University, Saint Louis, MO, 3University of Pennsylvania, Philadelphia, PA
To prevent vaccine-associated paralytic poliomyelitis, the advisory committee on immunization practices (ACIP) recommended in 1996 to begin immunizations with two doses of inactivated polio vaccine (IPV) flowed by two doses of oral polio vaccine (OPV). There were reports that prior immunization with IPV may increase the rate neurovirulent reversions. Recently the ACIP recommended the use IPV exclusively. However, it is still unclear whether IPV could be used to stop a potential future epidemic since it is believed to induce inadequate intestinal immunity that does not prevent virus shedding. In this work we studied shedding of vaccine poliovirus as a measure of intestinal immunity in vaccine recipients, and also genetic stability of attenuated strains after different immunization regimens.
The analysis of OPV shedding and reversion in stool specimens collected from 281 infants who were previously immunized with either inactivated or live vaccine was performed by direct FL-PCR and quantitative real-time PCR of viral cDNA. It demonstrated that three weeks after vaccination there was a clear decrease of OPV shedding in infant groups that received three doses of OPV and two doses of IPV followed by one OPV dose. This decrease of OPV shedding was significant (5%) three weeks after three doses of OPV than in what observed in infant group that received two doses of IPV followed by one OPV dose (37%). Three weeks after vaccination the quantity of shedded virus decreased significantly in infants that received two IPV doses followed by one OPV dose compared to infants that received only one OPV dose. Three weeks after three doses of OPV shedding of Sabin 1 and Sabin 3 was dramatically reduced, and shedding of Sabin 2 stopped completely. Quantitative analysis of mutations in the 5'-noncoding region performed by microarray hybridization showed that Sabin types 2 and 3 strains reverted easier than type 1. The highest percent of reversion was observed in Sabin 2. The results demonstrate that even though IPV is less efficient stimulator of intestinal immunity, it primes the immune system of vaccine recipients so that viral shedding is substantially reduced.
Residual total protein levels on reprocessed gastrointestinal (GI) biopsy forceps
S. K. Lappalainen1 , H. S. Baskar2 , V. M. Hitchins1 , 1FDA, Rockville, MD, 2University of Maryland, College Park, MD
Background: Many single use devices (SUDs) are reprocessed between patient uses. In the past, a visual determination was accepted as the endpoint of "clean." However, it is not possible to visually determine "clean" on opaque, lumened devices.
Methods: To explore a quantitative endpoint for clean, we measured residual total protein levels on GI biopsy forceps before and after reprocessing with commercially available cleaners. We simulated clinical use by inoculating the devices with a 3-protein test soil. Soiled devices were left to dry to simulate worst-case transport conditions. Total proteins were extracted from each device before and after cleaning. This inoculation and cleaning procedure was repeated 6 times to simulate up to 7 clinical uses. Each device was extracted separately and tested for total protein using 2 different commercially available Bradford's reagents and employing the more sensitive micro-protein protocols.
Results: A total of 30 GI forceps were used in the study. Pre-cleaned devices had an average total protein of 48 ìg/mL. Post-cleaned devices had average total protein of 3.8 ìg/mL or 0.47 ug/cm2. All of the post-cleaned devices were found visually clean.
Conclusions: Our residual protein results remaining on cleaned SUDs are comparable to that found by others after cleaning reusable medical devices. Visual detection alone underestimates residual bio-soil remaining on medical devices after reprocessing. Our results provide a quantitative protein acceptance criterion for cleaned medical devices.
Cytotoxicity of residual cleaning agents used in reprocessing medical devices
H. S. Baskar1 , V. M. Hitchins2 , 1University of Maryland, College Park, MD, 2CDRH, FDA, Rockville, MD
Cytotoxicity of residual cleaning agents used in reprocessing medical devices.
H.S. Baskar1, S.K. Lappalainen2 and V.M. Hitchins2. 1 University of Maryland, College Park, MD 20742 and 2CDRH, FDA, Rockville, MD. 20850.
Background: Many medical devices used on patients are reprocessed prior to use on the next patient. One reprocessing step involves the use of detergents to remove patient materials and microorganisms. There is a potential for cytotoxic effects from detergent residues remaining on reprocessed medical devices.
Methods: We explored this potential for cytotoxic effects by testing two commercially available detergents on two established mammalian cell lines. One agent contained proteolytic enzymes and the other did not. Different detergent concentrations were tested against murine macrophages (RAW 264.7) and murine fibroblast (L929) cell lines. The cells were treated for 1 or 24 hours with the detergents. Cytotoxicity was estimated by counting the viable cells using Trypan blue dye exclusion and a hemocytometer. Relative cell survival was calculated with respect to the control cells. Data are presented as the mean of 3 experiments performed in duplicate.
Results: The enzyme free detergent exhibited more cytotoxic effects than the detergent with enzymes after both 1or 24 hour exposures. The macrophage cells were more sensitive to these detergents than the fibroblast cells. One hour exposure to either detergent in both cell lines was not very cytotoxic even at high concentrations. With 24-hour exposure to detergents, very low concentrations of detergents killed all cells.
Conclusions: It is important that reprocessed medical devices have very low levels of detergent residuals to avoid cytotoxic effects. A one hour exposure time of cleaning agents with two different cell lines is not adequate to determine the potentially cytotoxic effects of these cleaning agents.
TRANSPLACENTAL AND POSTNATAL EXPOSURE TO AIDS DRUGS ZIDOVUDINE (AZT) AND LAMIVUDINE (3TC) IN C3B6F1trp53(+/-) TRANSGENIC MICE.
F. W. Lee, S. M. Lewis, C. Crawford, W. T. Allaben, J. E. Leakey, OSC, NCTR
AZT/3TC antiviral drug combinations are given during pregnancy to reduce maternal-fetal HIV transmission. AZT is genotoxic in fetal mice and monkeys and carcinogenic in mice. We assessed a new C3B6F1trp53(+/-) p53 haplodeficient transgenic mouse model to be used for cancer bioassays. These mice, produced by mating Taconic C57Bl6(N12)trp53(-/-) males and C3H females, possibly have similar tumor profiles to B6C3F1 mice. Haplodeficient C3B6F1trp53(+/-) and wild-type C3B6F1trp53(+/+) mice were dosed with 0, 40, 80, 160 mg/kg AZT or 160 mg/kg AZT combined with100 mg/kg 3TC, by gavage in aqueous methylcellulose/polysorbate 80 (2/0.1%), transplacentally from GD12 to GD18 then postnatally from PND1 to PND 28. P53 deficient mice are susceptible to fetal and neonatal mortality particularly when exposed to genotoxic compounds in utero. In a previous study using perinatal C57Bl6(N5)trp53(+/-) mice dosed with 200 mg/kg AZT, we obtained relatively low pup survival in both control and treated groups (75 & 17% respectively at PND-28). In contrast, C3B6F1trp53(+/-) were more robust. Survival in these pups was >95% and >85% for the control and dosed groups respectively and was greater for haplodeficient than for wild type mice. The AZT and AZT/3TC treatment produced only small (<10%) reductions in body weight gain, but mice from the AZT/3TC dose groups showed increased hprt mutation frequency when evaluated at PND 28. In adult humans hepatic AZT glucuronidation by UDP-glucuronosyltransferase (UGT) is a major detoxication pathway. In contrast, liver from C3B6F1trp53(+/-) and C3H mice expressed very low levels UGT activity towards AZT (0.2 - 1.5 pmol/min/mg microsomal protein; human and rhesus monkey liver activities ranged between 1.5 - 2.5 nmol/min/mg), and only low levels of AZT-glucuronide were detected in serum from AZT-treated mice. Since human neonates also express low levels of hepatic UGT activity, the C3B6F1trp53(+/-) mouse may be a good model for evaluating risk of perinatal AZT exposure.
Characterization of amino acid substitutions associated with the neuroattenuation of a mumps virus.
C. L. Wolbert, T. H. Malik, S. Rubin, K. Carbone, CBER, FDA, Rockville, MD
Mumps virus, a member of the Paramyxoviridae family, is a non-segmented, negative-stranded, enveloped RNA virus. This virus is highly neurotropic and often neurovirulent, though the genetic basis of neurovirulence is unknown. In order to understand the molecular basis for neurovirulence, a neurovirulent wild-type clinical isolate of mumps virus, 88-1961, was neuroattenuated (as confirmed in an animal model) by serial passage in chicken embryo fibroblast (CEF) cells. The sequence of the attenuated 88-1961 was compared with wild-type 88-1961. Amino acid substitutions were identified within the F (91, Pro/Thr→Thr), HN (466, Ser→Asn) and L (736, Ile→Val) proteins. Through the use of several in vitro functional assays, our data indicate that amino acid substitutions in both the F and L are associated with neuroattenuation of the virus by affecting fusogenicity and replication/transcription, respectively. The amino acid change in the HN protein does not appear to be involved. The possible relevance of these amino acid substitutions to neurovirulence will be discussed.
Caspase-12 expression increases prior to induction of apoptosis during HAV 18f infection in FrhK4 cells
M. Kulka, D. Ngo, T. A. Cebula, CFSAN, FDA, Laurel, MD
Background: Hepatitis A virus (HAV) is the principal agent responsible for foodborne outbreaks of infectious hepatitis. Identifying the molecular events responsible for the noncytopathic versus cytopathic growth characteristics observed among different culture-adapted HAV strains is a prerequisite for development of rapid culture methods for the detection of infectious virus in contaminated foods.
Methods: FrhK4 (monkey) cells were HAV 18f (cytopathic) infected in the presence or absence of caspase inhibitors. Clone 1 cells are HAV (noncytopathic) persistently infected FrHK4 cells. Apoptosis, caspase and stress marker expression/cleavage, and rRNA degradation were analyzed either by microscopy (cultures), immunoblotting (lysates) or agarose gel electrophoresis. Caspase-12 amino acid sequence was derived from RT-PCR generated cDNA.
Results: RNaseL-like activity, apoptosis and caspase-3, but not caspase-8 or 9, cleavage was detected following 18f infection, but not in clone1 cells; specific caspase inhibitors had no effect on apoptosis. Caspase-12 and endoplasmic reticulum (ER) stress have been implicated in some viral induced apoptotic responses. Caspase-12 expression was elevated in both 18f infected and clone 1 cells; GRP78 (an ER stress marker) expression increased only in 18f infected cells. FrhK4 caspase-12 cDNA encodes a protein having 57 and 85% identity to murine and human caspase-12, respectively. FrhK4 caspase-12 is similar in size to murine but larger than human caspase-12. FrhK4 caspase-12 contains a wild-type catalytic site as reported for murine, but not human, caspase-12.
Conclusions: Induction of apoptosis in 18f infected cells may involve activation of additional cellular events such as ER stress response and/or RNaseL-like activity.
Comparison of Viral Extraction Protocols by qRT-PCR for Enumeration of Calicivirus in Shellfish
W. Burkhardt III, J. L. Nordstrom, FDA
A majority of shellfish-associated viral gastroenteritis illnesses in the U.S. are attributed to Noroviruses of the family Caliciviridae. Noroviruses are currently not culturable by conventional cell culture techniques so epidemiological investigations rely primarily on molecular detection methods such as RT-PCR. These molecular techniques, however, are unable to distinguish infectious from non-infectious agents and are unable to effectively quantify the virus. These drawbacks have thwarted investigations to characterize the environmental stability of these viruses, and to determine the effectiveness of various protocols to liberate and quantitate these viruses from illness-implicated foods, including shellfish. In studies which viral infectivity must be determined, surrogates for Noroviruses have been utilized. One such surrogate, San-Miguel Sealion virus-17 (SMSV-17), is genetically and morphologically similar to the human Calicivirus and is culturable. Numerous protocols have been established for the extraction of Noroviruses from shellfish tissue but each has been ineffective in determining the precise levels of recovery. We developed a quantitative reverse transcriptase PCR (qRT-PCR) assay using Cepheid's SmartCycler® for the Norovirus surrogate SMSV-17. This quantitative assay was used to establish the efficiency of five viral extraction protocols for recovery of SMSV-17 from seeded oyster tissue. The qRT-PCR assay developed has a sensitivity of <1 PFU/ rxn and <5 PFU/ rxn in 10% newborn calf serum and oyster tissue (0.25 g shellfish tissue equivalent), respectively. Of the five extraction protocols examined, the protocol developed by Shieh et al. (2001) was the most successful in our laboratory for the extraction of SMSV-17 from shellfish tissue, with a mean recovery of 34 %. This combined use of methodologies demonstrates the effectiveness of qRT-PCR for quantification of viral particles.
A conventional multiplex PCR application for the detection and speciation of Microsporidia in clinical specimens
P. A. Orlandi1 , L. Carter1 , G. S.. Visvesvara2 , 1CFSAN, FDA, Laurel, MD, 2CDC, Atlanta, GA
Background: Microsporidia are obligate intracellular, spore forming parasites recognized as important emerging pathogens in immunocompromised and immunosuppressed patients. Infections in immunocompetent individuals are also steadily rising. Species of Enterocytozoon and Encephalitozoon are most commonly associated with infection in humans, particularly Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem and Vittaforma corneae. As the number of reported cases in AIDS patients, organ transplant patients, and the immunocompetent increase, so too does the need for a rapid, sensitive, and accurate method for clinical diagnosis to the species level. Here we report the design and development of a PCR detection protocol for microsporidia that combines the sensitivity and timeliness of FTA filter PCR technology with the specificity of a novel multiplex PCR assay.
Methods: Fresh and formalin-fixed fecal, urine, and mucosal aspirates were applied onto FTA filters to serve as DNA templates. Primary PCR assays amplified all species of the Microsporidia 18S rRNA gene. A nested, multiplex reaction based on a single, common forward primer and five novel genera and species-specific reverse primers then followed. This design exploited limited regions of microheterogeneity within the gene target to generate a series of size-specific amplicons for easy identification of human pathogenic microsporidia.
Results: This assay detected and identified single or mixed infections of E. bieneusi, E. intestinalis, E. cuniculi, E. hellem, and V. corneae and was able to accurately identify infections in a number of archival samples from HIV-infected patients and artificial mixtures.
Conclusions: The novel design of genera and species-specific PCR primers and the unique properties of FTA-PCR provided an effective means of rapidly and reliably detecting pathogenic Microsporidia spores in a diverse number of clinical specimens.
Immunological Response to MaPE of Mycobacterium avium 104
M. Parra1 , N. Cadieux1 , V. Dheenadahyalan1 , T. Pickett1 , G. Delogu2 , G. Fadda2 , M. J. Brennan1 , 1CBER, FDA, NIH, 2Catholic University of the Scred Heart, Rome, Italy
One subgroup of the unique PE (Pro-Glu) multi-gene family of Mycobacterium tuberculosis (Mtb) H37Rv contains ~37 members, encoding proteins that average 110 amino acids in length. Genomic analyses suggests that a few PE genes are also present in Mycobacterium avium. In our studies, we have focused on one M. avium PE gene, (MaPE), which is homologous to PE 18 and PE 19 of Mtb H37Rv. Recombinant his-tagged MaPE protein (rMaPE) and a MaPE DNA vaccine (MaPE-DNA) were constructed using DNA from M avium strain 104 and were characterized in immunological studies. To analyze the immune response to MaPE following infection or immunization, C57Bl/6 female mice were infected I.P. with 200 ul of 5 x 105 M.avium 104 or immunized intramuscularly three times at three week intervals with 200 ug of MaPE-DNA or with vector only. When the rMaPE protein was used as a recall antigen to stimulate cytokine production from lymph node cells or splenocytes from infected or immunized animals, significant levels of INF-γ were detected by cytokine ELISA. T cell enrichment demonstrated that the INF-γ response was due mostly to CD4 positive T cells. Moreover, MaPE-DNA immunized mice were significantly protected (~0.5 log reduction compared with vector control) when aerogenically exposed to a low challenge dose of M. tuberculosis Erdman. Sera collected at different timepoints post infection or post immunization and used in Western blots containing purified rMaPE protein, contained no antibodies reactive with the rMaPE antigen. Spleen cells isolated from mice infected with M. bovis BCG Pasteur strain also produced INF-γ when pulsed with the rMaPE protein suggesting that M. bovis BCG expresses a cross-reactive PE gene. These results are the first to show that M.avium 104 has an immunologically functional PE protein that may have therapeutic value. These data also have implications for the influence of exposure to M. avium expressing PE antigens on BCG vaccination and on infections caused by M. tuberculosis that contain similar PE genes.
Characterizing the mutator phenotype by global gene expression profiling of MMR-defective Salmonella enterica and Escherichia coli O157: H7
I. R. Patel, S. A. Jackson, J. E. LeClerc, T. A. Cebula, OARSA,DMB,Laurel,MD
Background: The CDC estimates between six and 33 million cases of bacterial food poisoning occur annually in the United States, and Salmonella and Escherichia coli pathogens are common causative agents. The ability of these pathogens to rapidly adapt to novel environments may be explained, in part, by the prevalence of mutators found among natural populations of these pathogens. Mutators typically have defects in the mutS gene, an essential part of the methyl-directed mismatch repair (MMR) system that maintains genome fidelity. MutS may play additional roles that have previously gone unrecognized, explaining some of the unusual phenotypes of mutator strains that we and others have observed.
Methods: To further characterize the mutator phenotype, we have used DNA microarrays to analyze the global gene expression profiles of mutator strains of Salmonella Typhimurium, Salmonella Enteritidis, and E. coli O157:H7. The microarrays represent ~98% of the genes found in five Salmonella serovars and all of the genes found in E. coli O157:H7 strain EDL933.
Results: Microarray profiling demonstrated that stress response genes, genes involved in transcriptional control of virulence, and various biosynthetic genes are strongly up-regulated in Salmonella mutator strains. Similar global differences were observed in mutator strains of E. coli O157:H7. Real-time PCR analysis confirmed that many of these genes are differentially expressed.
Conclusions: These data suggest that MutS acts as a global regulator and may help to explain the prevalence of mutS phenotypes among Salmonella and E. coli pathogens in nature.
CpG ODN enhance survival of pregnant mice to infection with Herpes Simplex type 2 virus during late-stage-pregnancy
J. A. Pedras-Vasconcelos, D. Goucher, D. Verthelyi, DTP, CDER, FDA, Bethesda, MD
Synthetic oligodeoxynucleotides encoding unmethylated CpG motifs (CpG ODN) trigger an immune cascade that results in improved antigen uptake/presentation by APC, and the secretion of polyreactive Ig, chemokines and cytokines by B cells, NK cells, DCs, and monocytes. These effects limit the early spread of pathogens while promoting the development of type 1 immunity. Studies in murine and primate models indicate that CpG ODN facilitate host clearance of infectious pathogens such as Leishmania, Listeria and Herpes Simplex type 2 (HSV2). In this study we assess the immunoprotective effects of CpG ODN during pregnancy, a state that is associated with suppressed cellular immune responses and increased susceptibility to intracellular infections.
Primary HSV2 infection during gestation is associated with a 30-50% neonatal infection rate (2000 cases /year in USA). Neonatal HSV2 infections usually occur at the end of pregnancy or during birth, and are associated with pre-term labor and low-birth-weight infants. In spite of the availability of cesarean section and antiviral therapy for neonatal HSV2, the outcome remains poor, particularly for neonates with disseminated multi-organ infection or central nervous system disease.
Using a murine model of HSV2, we assessed whether CpG ODN could protect pregnant mice from viral infection. Multiparous females challenged with 104 PFU of HSV2 during the 3rd week (day 16-20) of pregnancy have significantly reduced litter sizes (mean of 3 pups/litter compared with 8 pups/litter in uninfected controls, p=0.002) and high mortality (89% of mothers die within 2 weeks of infection). Treatment with CpG ODN at the time of infection resulted in improved maternal survival (64%, p=0.037 by Fischer's Exact test), larger litter sizes (mean litter size was 5, p=0.096 by T test) and fewer teratogenic sequelae such as blindness, low birth weight and delayed growth. Importantly, administration of CpG ODN to uninfected pregnant females did not impact litter size. Splenocytes harvested two weeks after infection secreted high levels of IFN-a, but low IL-12 and IFN-g upon stimulation with HSV2 antigens. In contrast, those from CpG ODN-treated mice secreted higher levels of IL-12 and IFN-g, but lower IFN-a.
The immune response to HSV in the offspring was assessed in litters born to mothers infected with 103 CFU. The offspring from CpG-treated mothers had increased HSV2-specific IgG antibodies compared to controls, but their levels were lower than those from mice born to untreated infected mothers. Furthermore, their splenocytes failed to secrete high levels of IFN-a and IFN-g in response to HSV antigens in vitro, suggesting that CpG ODN treatment prevented vertical transmission of the virus. After weaning the offspring were challenged with HSV2. Offspring from mothers that survived an infection with HSV2 during gestation are less susceptible to HSV2 infection later in life (40% survival). However, those born to HSV-infected mothers treated with CpG ODN had lower survival rates (9%, p=0.027), confirming that these mice had been exposed to little or no virus. In summary, CpG ODN treatment improved the outcome of the HSV2 infection for the pregnant mouse model by enhancing an early maternal anti-viral type 1 response, which decreased morbidity and mortality in both mother and offspring.
Ingestion of Staphylococcal Enterotoxin B (SEB) in the Diabetic Mouse
R. Jones, Jr1 , R. Michelson2 , J. Stone2 , M. G. Robl3 , M. A. Principato3 , 1CFSAN,FDA, Laurel, MD, 2JIFSAN, Univ MD, 3CFSAN,FDA,Laurel, MD
Ingestion of Staphylococcal Enterotoxin B (SEB) in the Diabetic Mouse
Robert Jones1, Russel Michelson2, Jeffrey Stone2, Martin G. Robl1, MaryAnn Principato1 1 CFSAN/FDA Laurel, MD 20708 2 JIFSAN,FDA/UMD Laurel, MD 20708
Background: The Staphylococcal enterotoxins are known to be etiologic agents of food poisoning in man and are also recognized for their ability to exert profound effects upon the immune system due to their superantigenic characteristics. Significantly, superantigens have been implicated in the pathogenesis of certain autoimmune diseases. This project sought to assess the effect of superantigen ingestion upon the progression of autoimmune diabetes using the NOD mouse model of human Type I Insulin-dependent diabetes mellitus (IDDM).
Method: The development and onset of diabetic disease was monitored in NOD mice via weight changes and blood glucose monitoring. High-dose SEB was fed to animals at a time when it was determined that disease onset was imminent or in its very early stages.
Results: The progression of disease was monitored in untreated NOD and compared to those treated with SEB. Preliminary results demonstrate that SEB-treated animals demonstrated an averaged 1.5-fold increase in blood glucose 12 days following ingestion. This increase was apparent 36 days after treatment. SEB-treated NOD lived a lifespan comparable to the untreated NOD. Histologic examination of treated and untreated NOD at the time of death demonstrated significant splenic lymphoproliferation, an overall decrease in the total number of pancreatic islets, and marked lymphocytic infiltration in the pancreatic islets, consistent with the development of autoimmune disease.
| Sigma Xi Outstanding Poster Award - 2005 FDA Science Forum |
Genetic Variability in West Nile Virus (WNV) Isolates From Blood Donors Specimens From 2002, 2003 and 2004 USA Epidemics.
A. Grinev, S. Daniel, I. K. Hewlett, M. Rios, CBER, FDA, Rockville, MD
BACKGROUND: The objective of this study was to investigate potential genetic variation of WNV over time during the 2002, 2003 and 2004 epidemics in the US. Variation in the viral genome may affect pathogenesis, possible treatment and performance of screening and diagnostic assays. WNV first appeared in the US in 1999 and since then epidemics have reoccurred for 6 consecutive years. The US epidemics of 2002 and 2003 led to the largest meningoencephalitis outbreaks in the Western hemisphere and the largest WNV outbreaks ever reported. Preliminary molecular phylogenetic studies divide isolates of WNV into two lineages. Linage II strains are mostly found in Africa. Lineage I strains are more widely distributed and have been responsible for all the recent large outbreaks. Immeasurable efforts have been directed to the study of WNV, and information about its potential for genomic variation is desirable and important for an understanding viral of evolution.
METHODS: Plasma samples from 25 blood donors, who were identified as WNV-NAT-positive during 2002 (n=7), 2003 (n=10) and 2004 (n=8) epidemics, were used for virus isolation in Vero cells. RNA was extracted from the isolates, reverse transcribed and the cDNA used for sequence analysis. Two isolates (FDA-Hu2002 and isolate Hu10-02) were completely sequenced while 23 other isolates had all viral structural regions (5'NTR, Cap, preM, M and Env) sequenced. Sequence results were analyzed using Vector NTI software and compared to genetic sequences from the prototype WN-NY99 isolate.
RESULTS: The isolate FDA-Hu2002 had 20 nucleotide (nt) mutations and one nt insertion (T at position 10497); Five of the 20 mutations were associated with amino acid (aa) substitutions (M22T; A52V; V449A; N684S; S2301G). The isolate Hu10-02 had 22nt mutations of which 3 resulted in aa substitution (V449A; V2213A; E2826G). The structural region from 22 of the 23 other isolates had 2 common mutations (1442T>C [V449A] and 2466C>T [silent]) in the envelope gene. One isolate from 2002 had two silent mutations (1487T>C and 1773C>T) in the envelope gene that were not found in the other 22 isolates. The mutation 660C>T in the prM region was present 18/25 isolates (all 8 from 2004, 7/10 from 2003 and 3/7 from 2002). Isolates from 2004 shared 2 silent mutations in the envelope gene (1320A>G in all 8 and 1974C>T in 7/8) not observed in the 17 isolates from previous years.
CONCLUSION: The small number of nucleotide mutations in the envelope gene and the single conservative amino acid substitution in the envelope variable region suggest the absence of strong selective pressure and limited evolution of West Nile virus from 1999 to 2004. However, some mutations resulted in aa substitutions that could affect viral pathogenesis; and changes in nt and aa sequences that could potentially affect the sensitivity of screening and diagnostic assays.
Monocytes/Macrophages are a Potential Target in Human Infection with West Nile Virus (WNV) through Blood Transfusion.
M. Rios, M. J. Zhang, K. Srinivasan, S. Daniel, O. Wood, I. K. Hewlett, A. I. Dayton, CBER, FDA, Rockville, MD
BACKGROUND: WNV transmission by transfusion was documented in 2002. About 80% of WNV the infections are asymptomatic and 1% develop severe neurological illness with a high mortality rate. In animals, Langerhans dendritic cells support initial viral replication, followed by replication in lymphoid tissues, dissemination to organs and possibly to the nervous system. The cellular tropism of WNV infection following transfusion and the particular human blood cells that sustain viral replication remain largely unknown. We investigated whether primary monocyte-derived-macrophages (MDM) support WNV infection/replication and produce infectious virions, using an in vitro system.
METHODS: Elutriated monocytes from blood suitable for transfusion were cultured in the presence of M-CSF, infected with WNV-NY99 strain at different time points, washed and cultivated for 47 days. Supernatants were tested for WNV replication by TaqMan RT-PCR using primers for the envelope and/or 3'NC region. Infectivity assays in Vero cells were performed to investigate the presence of functional virions.
RESULTS: RT-PCR-TaqMan assay of supernatants demonstrated productive infection. Viral load increments started 24 hours after infection, reached 2 to 5 logs above baseline in 3 to 6 days then declined, with low viral replication persisting for up to 47 days. Infected MDM cultures showed no cytopathic changes. Supernatants with TaqMan positive results infected Vero cells in culture and produced cytopathic effects within 3 to 5 days of culture.
CONCLUSION: The susceptibility of monocyte/ macrophages to productive infection with WNV in vitro is compatible with their potential role in initial WNV replication and propagation following transmission by transfusion.
Acute Histopathology and Total Blood Parameters in Young Male Rats Given Bacterial (Lipopolysaccharide-LPS) or Fungal (Deoxynivalenol-DON)Toxins
M. G. Robl, L. H. Garthoff, D. M. Hinton, OARSA, CFSAN, FDA, Laurel, MD
Background: Histopathology and total blood parameter changes in rats exposed to LPS and DON have not been reported.
Methods: In a range finding study (RFS), rats were injected intraperitoneally (i.p.) with bacterial toxin (LPS at 10, 100, and 300 ug/kg) or fungal toxin (DON @ 5, 10, and 25 mg/kg) or saline. In the 2nd study, LPS at 83 ug/kg or DON at 5 or 10 mg/kg were given alone or co-administered (LPS then DON 1 hour later). Saline served as control. Rats were euthanized in both studies after 3, 24 & 72 hours (hrs) and blood samples were taken and analyzed. Light microscopic evaluation of selected tissues was conducted in the 2nd study.
Results: In the RFS, LPS and DON alone affected platelets, white and red blood cells. Platelets, lymphocytes, and neutrophils were affected most at 3 and 24 hrs. Some changes returned to near normal levels or persisted at 72 hrs. In the 2nd study, mid-dose toxin levels were used alone or together. Blood results mimicked those in the RFS when given alone; when co-administered, some changes were more severe.
Microscopic evaluation of tissues revealed necrosis of bone marrow cells, hepatocytes, and lymphocytes with LPS, and necrosis of hepatocytes, lymphocytes, and pancreatic acinar cells with DON at 3 and 24 hrs. Changes persisted or appeared near normal at 72 hrs. With co-administration, some lesions were more severe.
Conclusions: Cellular necrosis in tissue sections and blood component changes occurred in rats given LPS or DON alone or together. Some effects were more severe when toxins were co-administered.
In Vivo Studies on the Toxicity of Adenovirus Vectors for Kupffer Cells
E. Manickan, J. S. Smith, J. Tian, A. P. Byrnes, FDA, Bethesda, MD
When adenovirus type 5 vectors (AdVs) are injected into the bloodstream, the liver macrophages known as Kupffer cells (KCs) take up the vast majority of the virions. In the current study, we provide evidence that replication-incompetent AdVs are rapidly cytotoxic for KCs in vivo.
Using two independent in vivo assays we detected signs of plasma membrane permeability as early as 10 min after injection of AdVs into the tail vein of mice. In the first assay, the membrane-impermeable dye propidium iodide was injected intravenously and could be found staining KC nuclei, exclusively in mice which had received AdV. In the second assay, we detected a transient AdV-induced elevation of serum lactate dehydrogenase which peaked at 30 min, indicative of cytoplasmic leakage from dying cells. Within 4-6 h, the number of KCs in the liver was substantially reduced. In additional experiments, when fluorescent AdVs were injected intravenously, they could be found taken up by KCs in the liver within 10 min, as expected. Unexpectedly, however, lungs also had macrophages which contained fluorescent AdV, and these increased in number with time. Close examination of the liver after AdV injection revealed that small numbers of KCs could be seen in the central venules, which carry blood away from the liver.
We interpret these data as showing that AdVs cause rapid cytotoxicity in KCs, and these cells are subsequently flushed from the liver and become lodged in the pulmonary vasculature.
Development of Selective Media for Detection Enterobacter sakazakii by Using Ferrioxamine E and alpha-glucosidase Substrates
K. Y. Song1 , K. H. Seo2 , G. Thammasuvimol2 , R. Brackett2 , 1Joint Institute for Food Safety & Applied Nutrition (JIFSAN), University of Maryland, College Park, MD, 2CFSAN, FDA, College Park, MD
Enterobacter sakazakii causes meningitis, necrotizing enterocolitis, sepsis, and bacteremia in neonates and children with high mortality rate. To detect E. sakazakii rapidly, various differential selective media have been developed by use of alpha-glucosidase substrates, 5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside (BCIG) or 4-methyl-umbellifery-alpha-D-glucoside (alpha-MUG), since only E. sakazakii among genus Enterobacter exhibits alpha-glucosidase activity. However, Escherichia vulneris in the family Enterobacteriaceae also utilizes alpha-glucosidase substrates resulting in false positives. Ferrioxamine E (FE) is considered as a selective iron source because it is utilized by E. sakazakii, but not by E. coli. This study was performed to develop a selective agar medium for E. sakazakii detection by using FE and alpha-glucosidase substrates. Three previously developed media (TPD), Oxoid, OK, and VRBG, and one developed in this study, SFC, were evaluated using 58 E. sakazakii and 6 non-E. sakazakii strains. Fifty four E. sakazakii strains appeared as fluorescent or chromogenic colonies on all of the media tested. Two strains were negative on TPD media while showing positive on SFC medium. Interestingly, the other two strains were observed vice versa showing positive on TPD media, but negative on SFC medium. None of the non-E. sakazakii strains showed fluorescent or chromogenic colonies on all of the media tested except E. vulneris, showing positive on TPD media and negative on SFC medium. This study demonstrates the newly developed medium (SFC) enabled not only detect equivalent number of positive colonies but also exclude false positives such as E. vulneris when compared with currently available media.
A universal DNA microarray for detection of gene sequence changes---Applications to the detection of Mycobacterium tuberculosis (MTB) mutant genes associated with antibiotic drug-resistance and of pathogens contaminating medical devices
X. Tang1 , L. Baeva1 , S. Morris2 , J. Langone1 , L. Bockstahler1 , 1CDRH, FDA, White Oak, MD, 2CBER, FDA, NIH, MD
DNA microarray technology has been widely used in the detection of gene sequence changes associated with genetic diseases, genotyping, and drug-resistance of pathogens. Multiple targets can be screened or examined simultaneously on one slide by hybridization to complementary probes spotted on the slide. We are developing a universal DNA microarray for the detection of point mutations in MTB genes and pathogens contaminating medical devices. Currently, ten point mutations in three MTB genes have been successfully identified. In addition, using the array Staphylococcus aureus and Staphylococcus epidermidis can also be accurately distinguished based on differences in the gene sequences in the 16S rRNA fragments of these two pathogens. Compared with the conventional detection methods, e.g. bacterial culture and drug-sensitivity test, microarray technique is more rapid, accurate, efficient, and sensitive. The hands-on experience obtained from our research will enable us to optimize this methodology and validate and standardize the microarray procedure. This knowledge will facilitate evaluation of future regulatory submissions to FDA.
Optimization of Ferrioxamine E Concentration as Effective Supplementation for Selective Isolation of Salmonella Enteritidis in Egg White
G. Thammasuvimol1 , K. H. Seo1 , K. Y. Song2 , 1U.S. Food and Drug Administration, CFSAN/OPDF, 5100 Paint Branch Parkway, College Park, Maryland 20740, 2Joint Institute for Food Safety & Applied Nutrition (JIFSAN), University of Maryland, College Park, Maryland 20742
Studies show that utilization of ferrioxamine E (FE) as a sole source of iron distinguishes Salmonellae from a number of related species, including Escherichia coli (E. coli). Ferrioxamine E is not able to feed E. coli or the Proteus-Providencia-Morganella-group. This confers a selective advantage on Salmonella Enteritidis in egg white supplemented with FE. The optimum concentration of FE for selective growth of SE in egg white was determined in this study. Four supplementation concentrations were evaluated (500 ug/ml, 200 ug/ml, 50 ug/ml, and 25 ug/ml) in egg white artificially inoculated with proportionally mixed cultures of a Rifampicin-resistant strain of Salmonella Enteritidis (0.1 ml of 102 CFU/ml) and E. coli K12 (0.1 ml of 108 - 101 CFU/ml). After 24 h incubation at 37 C, Salmonella and E. coli populations were enumerated. At higher concentrations of FE (> 50 ug/ml), both Salmonella and E.coli were able to utilize the iron supplement (log 1 - 8.5 and log 1.8 - 8 CFU/ml, respectively), however lower FE concentrations (<50 ug/ml) exclusively promoted Salmonella growth. Salmonella was unrecoverable without supplementation. This study indicates that optimum levels of FE supplementation in egg can improve selective detection for Salmonella Enteritidis against other competitive organisms.
Moulds, yeasts and aerobic plate counts in ginseng supplements
V. H.. Tournas1 , E. Katsoudas2 , E. Miracco3 , 1CFSAN, FDA, College Park, MD 20740, 2Northeast Regional Laboratory, Jamaica, NY 11433, 3JIFSAN, University of Maryland, College Park, MD 20740
Forty six ginseng supplement samples including Siberian ginseng root, Chinese ginseng herb and root, and American ginseng root and extract were purchased from retail in the Washington, DC area and from Penn Herb Co. (Philadelphia, PA) and tested for mould and yeast (MY) contamination and the presence of aerobic mesophilic bacteria (APC). Results indicated that 100% of the Siberian ginseng samples were contaminated with fungi and bacteria. MY counts ranged from 8.0 x 102 to 1.4 x 103cfu/g whereas the APCs were between 2.3 x 104 and 1.0 x 106 cfu/g. Most common fungi encountered in this commodity were Penicillium spp., Eurotium rubrum, E. chevalieri and Rhizopus spp.
Seventy-eight per cent of the Chinese ginseng herb samples were contaminated with fungi and 89% with bacteria at levels ranging between <100 and 6.0 x 104 and <100 and 1.2 x 106 cfu/g, respectively. Moulds commonly isolated were Alternaria alternata, Aspergillus niger, Aspergillus spp., Cladosporium spp., E. chevalieri, Penicillium spp. and Rhizopus spp. Fifty six percent of the Chinese ginseng root samples tested contained fungi (A. niger, Rhizopus spp. and yeasts), and 100% contained bacteria. Fungal counts ranged between <100 and 1.4 x 103 cfu/g and APCs were between 3.0 x 102 and 6.8 x 105 cfu/g. Forty-eight per cent of the American ginseng root samples contained moulds and 30% showed bacterial contamination. MY counts were between <100 and 4.3 x 105 cfu/g whereas APCs were between <100 and 4.5 x 104 cfu/g. A. flavus was isolate from 9% and Penicillium spp. were recovered from 39% of the tested samples. This is the first report of A. flavus contamination in ginseng supplements. No moulds or yeasts were found in ginseng extract, but 50% of these samples contained bacteria at levels ranging between <100 and 1.0 x 103 cfu/g.
USEFULNESS OF STUDIES ON THE MOLECULAR MECHANISM OF ACTION OF HERBALS/BOTANICALS: THE CASE OF ST. JOHN'S WORT
S. Choudhuri, L. G. Valerio, Jr., CFSAN, FDA, College Park, MD
The use of herbals/botanicals has been gaining wide popularity in recent years in the United States as well as in other parts of the world. The mechanism of action of most of these herbals/botanicals has not been subjected to thorough scientific investigations. St. John's Wort (Hypericum perforatum) represents a useful case study in this sense. Traditionally, it is used as a natural treatment for depression; however, in recent years its molecular mechanism of action has been elucidated by a number of laboratories across the world. Such studies have helped understand potential interactions of St. John's Wort with drugs and other xenobiotics. St. John's Wort activates a nuclear receptor called pregnane X receptor (PXR). PXR is a ligand-activated transcription factor that induces a number of xenobiotic-metabolizing enzymes and transporters including cytochrome P4503A4 (CYP3A4) in humans. Because CYP3A4 alone metabolizes about 60% of all clinically relevant drugs, induction of CYP3A4 results in the rapid elimination of these drugs and a consequent reduction in drug efficacy. Ironically, such enzyme-inducing effects may not produce any immediate adverse symptomatology in the person taking St. John's Wort. Therefore, the case of St. John's Wort should serve as a good example of the usefulness and importance of studies on the mechanism of action of the herbals/botanicals, particularly those with widespread use. Scientists, physicians and other health professionals can make use of the knowledge from such studies as an additional risk management tool.
EVALUATION OF FLOW CTYOMETRIC ENDPOINTS FOR THE LYMPH NODE PROLIFERATION ASSAY (LNPA).
J. L. Weaver, D. D. Broud, DAPR, OTR, Silver Spring, MD
The LNPA is being developed for use in detection of the potential for systemically administered drugs to cause clinical hypersensitivity reactions. This method, modified from the local lymph node assay, uses in vivo uptake of 3H-thymidine in the superficial cervical lymph node as the endpoint. The research reported here was performed to determine whether changes in lymphocyte subpopulation numbers or cell surface marker expression could be used as a non-radioactive endpoint in the LNPA. Groups of BALB/c mice were injected s.c. with saline or test drugs for three days, rested for two days, and sacrificed. The drugs tested were: assay control: streptozotocin; negative - phenobarbital, metformin; positive - nevirapine, abacavir, lamotrigine, ofloxacin, zomepirac, clonidine, procainamide, and sulfamethoxazole. Leukocytes from draining lymph nodes and peripheral blood were analyzed for changes in cell surface proteins using flow cytometry. These markers were initially measured in lymph node cells: sIgE, Ia, CD45R/B220, CD4, CD8, CD25, TcR-beta, CD62L, CD44, CD71, CD54, CD86. In the lymph nodes, changes were seen in cell numbers only with streptozotocin, lamotrigine, abacavir and nevirapine. Changes in the percent of B220+ cells were seen with nevirapine, and lamotrigine. Alterations among T-cell populations were observed with nevirapine, lamotrigine, and abacavir with changes in proportions of CD4, CD8 and CD62L populations. Significant changes in absolute numbers among T-cell populations were seen only with streptozotocin and nevirapine, due to the larger variability in lymph node cell counts. No changes in CD25, CD44, CD71, CD54, or CD86 were seen with any drug. Changes in percentages of cells in peripheral blood were also seen following treatment with abacavir, lamotrigine, nevirapine, and ofloxacin. The expression of CD62L was increased only with nevirapine treatment. The presence of the alterations in expression of cell surface proteins and in the proportions of cells in both lymph nodes and peripheral blood are in general agreement with the results of the 3H-thymidine-uptake results.
Mutagenicity of chromium picolinate and its components in Salmonella typhimurium and L5178Y mouse lymphoma cells
P. Whittaker1 , R. H.C.. San2 , J. J.. Clarke2 , H. E.. Seifried3 , V. C.. Dunkel1 , 1CFSAN, FDA, College Park, MD, 2BioReliance Corporation, Rockville, MD, 3NCI, NIH, Bethesda, MD
Chromium picolinate is one of the most commonly used chromium dietary supplements available in the United States, and it has been marketed to consumers for use in weight loss, increasing muscle mass, and lowering serum cholesterol. Chromium picolinate is a synthetic compound that provides a bioavailable form of Cr(III) that is absorbed better than dietary chromium. However, there are several reports that it can have adverse effects. In order to study the mechanism of observed cellular toxicity and mutagenicity, chromium picolinate and its component compounds, chromium (III) chloride and picolinic acid, were evaluated in Salmonella typhimurium and L5178Y mouse lymphoma cells. Neither chromium picolinate nor chromium chloride induced a mutagenic response in S. typhimurium. However, in the L5178Y mouse lymphoma mutation assay chromium picolinate induced mutagenic responses without and with the addition of S9.
Evaluating the Risk of Transmission of Porcine Endogenous Retroviruses in Xenotransplantation Products.
C. A. Wilson, T. Argaw, W. Colon-Moran, M. Gemeniano, O. Mpanju, CBER, FDA, Bethesda, MD
Carolyn A. Wilson, Takele Argaw, Winston Colon-Moran, Malou Gemeniano, and Onesmo Mpanju, CBER/FDA, Bethesda, MD 20892.
The use of pig cells to treat human disease, either by direct transplantation or by ex vivo exposure, is being explored in clinical trials. Clinical investigations using this class of products, termed xenotransplantation products, are under regulation by the FDA's Center for Biologics Evaluation and Research. Xenotransplantation carries the long-term potential to alleviate the shortage of human organs to treat a broad spectrum of fatal diseases. However, this potential positive outcome is counterbalanced by the possible risk of transmitting infectious disease to the recipients. Indeed, recent zoonotic outbreaks, such as the SARS virus, underscore the infectious disease risks associated with exposures to animals. Xenotransplantation procedures exacerbate these risks by breaking down the usual barriers to transmission of infectious disease. Our laboratory was the first to demonstrate that primary cultures of porcine peripheral blood mononuclear cells are capable of transmitting porcine endogenous retrovirus (PERV) to human cell during in vitro coculture assays (Wilson, C., et al., Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells. Journal of Virology, 1998. 72(4): p. 3082-3087). More recently, we have been investigating the viral and cellular determinants that affect the tropism and replication properties of PERV in order to enhance our understanding of the potential risk of transmission to xenotransplantation recipients. In addition, the results of our studies may elucidate means to block or treat infections, should they occur. The details of these studies will be presented.
Bioeffects of ultrasound and ultrasound microcontrast agent on three mammalian cell lines.
S. C. Wood, R. P. Brown, J. Chen, E. A. Gordon, G. R. Harris, V. M. Hitchins, S. Maruvada, A. R. Mtungwa, M. E. Stratmeyer, CDRH, FDA, Rockville, MD
Background: Ultrasound (US) imaging is enhanced with the use of microbubble contrast agents to assess cardiac function. One agent, Optison® (OPT), is a suspension of microspheres of human albumin with perflutren. Pre-market testing did not reveal overt toxicity. However, these studies did not examine if US+OPT altered vascular function. Our studies focused on cells that would be subjected to the high energy cavitation of the microbubbles induced by US: fibroblasts, macrophages, and endothelial cells.
Methods: US exposures were performed using a clinical imaging system operating at an ultrasonic frequency of 2 MHz and a mechanical index of 1.8. All cell types were grown to confluence in Opticells and divided into 4 groups: untreated, OPT, US, and OPT + US. Endpoints included the generation of tumor necrosis factor-alpha (TNF-á), nitric oxide (NO) and apoptosis. At selected time points after treatment, macrophage lysates were assayed for TNF-á and NO. Apoptosis was assessed by flow cytometry.
Results: Apoptosis was seen as early as 1 hour after addition of OPT to macrophages. The combination of US+OPT or pre-treatment with lipopolysaccharide (LPS) did not enhance apoptosis. In contrast, both fibroblast and endothelial cells were resistant to both OPT and US. TNF- á was not produced by macrophages by the addition of OPT alone or OPT+LPS.
Conclusions: While these results indicate that OPT does not stimulate inflammation directly, the induction of apoptosis in macrophages is significant. Apoptotic cells and microparticles are highly thrombogenic and pro-inflammatory and may potentially accelerate pathogenesis of vascular disease.
Mechanistic insights into stent-mediated thrombosis and adverse events by stent-delivered drugs sirolimus and taxol.
S. C. Wood1 , G. Bushar1 , B. Tesfamariam2 , 1CDRH, FDA, Rockville, MD, 2CDER, FDA, Rockville, MD
Background: Drug-eluting stents (DES) combine a drug delivery system with a mechanical scaffold to maintain vascular patency in atherosclerotic lesions. The two approved DES elute either sirolimus or taxol from a polymer matrix. Significantly, the clinical trials for sirolimus did not reveal any stent associated thrombosis (SAT). However, upon approval, numerous SAT were reported. Additionally, late SAT (>6 months) has been noted when anti-platelet therapy has been suspended. The detailed mechanism of action of these agents on platelet/vascular function is unknown. Given the high concentrations of anti-restenotic drugs from DES, activation of platelets, endothelial damage and alterations in endothelial function may increase the risk to SAT.
Methods: Platelets were obtained from donors via plateletphoresis. Platelets were pre- treated with either sirolimus or taxol, stimulated with agonists and platelet activation was followed by flow cytometry. Endothelial cells were exposed to sirolimus or taxol and apoptosis was followed by flow cytometry and fluorescence microscopy.
Results: Interestingly, platelet activation was inhibited by sirolimus but not by taxol. Currently, we are examining the effects of sirolimus and taxol upon platelet degranulation. Endothelial cells were quite sensitive to taxol and apoptosis was readily induced. Sirolimus did not induce apoptosis. Future experiments will focus on the effects of sirolimus and taxol on the extrinsic coagulation cascade.
Conclusions: The preliminary data suggest that the mechanism of SAT may be due primarily to changes in the endothelium. Anti-platelet therapy may be crucial in reducing both the initial and long term risks of SAT.
Characterization of Salmonella Typhimurium of Animal Origin Obtained from the National Antimicrobial Resistance Monitoring System (NARMS)
S. Zhao1 , P. J.. Fedorka-Cray2 , S. Friedman1 , P. F. McDermott1 , R. D. Walker1 , S. Qaiyumi1 , S. L.. Foley1 , S. K. Hubert1 , S. L. Ayers1 , L. L. English1 , D. A.. Dargatz3 , B. Salamone4 , D. G. White1 , 1CVM, FDA, Laurel, MD, 2USDA, ARS, Athens, GA, 3USDA, Fort Collins, CO, 4USDA, FSIS, Washington, DC
ABSTRACT
Background: Salmonella Typhimurium remains one of the most common causes of salmonellosis in animals and humans in the United States. The emergence of multi-drug resistant Salmonella reduces the therapeutic options in cases of invasive infections, and has been shown to be associated with an increased burden of illness.
Methods: In this study, 588 S. Typhimurium (including var. Copenhagen) isolates obtained from either animal diagnostic specimens (n=199) or food animals after slaughter/processing (n=389) were examined for antimicrobial susceptibility, presence of class-1 integrons, and characterized using pulsed-field gel electrophoresis and phage typing.
Results: Seventy-six percent (448/588) of isolates were resistant to at least one antimicrobial. Salmonella isolates displayed resistance most often to streptomycin (63%), tetracycline (61%), ampicillin (61%), and to a lesser extent, chloramphenicol (36%), ceftiofur (15%), gentamicin (9%), and nalidixic acid (4%), with more resistance observed among diagnostic isolates. Salmonella of turkey origin (n=38) exhibited the highest rates of resistance, with 92% of isolates resistant to least one antimicrobial, and 60% resistant to > 10 antimicrobials. Class 1 integrons were present in 51% of all isolates. Five integron associated resistance genes (aadA, aadB, pse-1, oxa-2 and dhfr) were identified. A total of 311 PFGE patterns were generated using XbaI, indicating a genetically diverse population. The largest PFGE cluster contained 146 isolates, including DT104 isolates obtained from all seven animal species.
Conclusions: Results demonstrated a varied spectrum of antimicrobial resistance, including several multidrug resistant clonal groups, among S. Typhimurium and S. Typhimurium var. Copenhagen isolates recovered from both diagnostic and slaughter/processing samples.
Rubella Virus and Birth Defects: Molecular Insights into the Viral Teratogenesis at the Cellular Level
C. D. Atreya1 , K. VK. Mohan2 , S. Kulkarni2 , 1CBER, FDA, Rockville, MD, 2CBER, FDA, Rockville Pike, MD
Background: In utero Rubella virus (RV) infection of a fetus can result in birth defects often collectively referred to as congenital rubella syndrome, or CRS. In extreme cases fetal death can occur. In spite of the availability of a safe and effective vaccine against rubella, recent worldwide estimates are that more than 100,000 infants are born with CRS annually.
Recent progress: Recently several significant findings in the field of cell biology as well as in the RV replication and virus-cell interactions, originated from the authors laboratory and others have provided insights into RV teratogenesis. It has been shown that, a) a RV protein induces cell cycle arrest by generating a subpopulation of 4N cells, perhaps representative of tetraploid state following S phase in cell cycle, due to its interaction with Citron-K kinase, b) RV infection induces apoptosis in cell culture and, c) Citron-K kinase functional perturbations leads to tetraploidy, followed by apoptosis, in specific cell types.
Conclusions: Based on several similarities between known rubella virus-associated fetal and cellular manifestations and CK deficiency-associated phenotypes, it is reasonable to postulate that P90-CK interaction in RV infected cells interferes with CK function and induces cell cycle arrest following S phase in a subpopulation, perhaps representative of tetraploid stage, which could lead to subsequent apoptosis in RV infection. Taking all these observations to the fetal organogenesis level, it is plausible that P90-CK interaction could perhaps be one of the initial steps in RV infection-induced apoptosis-associated fetal birth defects in utero.
UVA1-Mediated Receptor and Cytokine Changes of Transformed Lymphocytes.
D. E. Godar1 , A. D. Lucas2 , 1OSEL, CDRH, FDA, Silver Spring, MD, 2OSEL, CDRH. FDA, Silver Spring, MD
UVA1 radiation (340-400 nm) causes singlet-oxygen damage that depolarizes mitochondrial membranes triggering immediate apoptosis (T¡Ü4 h), while it also causes oxidative damage to DNA inducing delayed apoptosis (T¡Ý24 h). In this study, we examined some potential therapeutic endpoints associated with UVA1-mediated immediate and delayed apoptosis, such as receptor and cytokine changes. We quantified the number of membrane-bound CD3 receptors on transformed T lymphocytes (Jurkat) and the number of membrane-bound CD19 receptors on transformed B lymphocytes (Daudi) using flow cytometry. We also quantified the release of the cytokines IFN-γ and IL-2 using enzyme-linked immunosorbent assays. Out of the entire population of cells, only the apoptotic Daudi cells immediately decreased CD19 expression via capping, while only the apoptotic Jurkat cells increased CD3 receptor expression 24 h post exposure. Both receptor changes occurred in a UVA1 dose-dependent manner. We also examined other T-cell receptors, such as CD4, CD25 and CD69, but they did not change for up to 24 h following exposure. During UVA1-triggered immediate apoptosis of Jurkat T cells, IFN-γ levels increased in a dose-dependent manner at 4 h, but returned to base-line levels at 24 h post exposure; whereas, there was no significant change in IL-2 at 4 or 24 h. Thus, UVA1-triggered immediate apoptosis causes a rapid decrease in the number of CD19 receptors on Daudi B cells and release of IFN-γ from Jurkat T cells at 4 h, and UVA1-mediated delayed apoptosis causes an increase in the number of CD3 receptors on Jurkat T cells.
Comparative Kinetics of Invasion into Human Intestinal INT407 Cells by Different Intestinal Bacterial Pathogens
L. Hu, T. T. Wai, D. J. Kopecko, CBER, FDA, Bethesda, MD
Background: Salmonella, Shigella, EHEC, and Yersinia cause invasive diarrheal illnesses, but the kinetics and severity of illness differ with each organism. The study purpose was to determine if in vitro invasion assays might reveal invasion differences that reflect natural disease severity/kinetics. In addition, this knowledge is helpful in evaluating safety of live attenuated bacterial vaccines.
Methods: A standard tissue culture invasion assay was employed. Mid-log phase bacteria at different multiplicities of infection (MOI of 0.04, 0.4, 4, 40, 400, 4000) were added onto cultured INT407 cells. Infected monolayers were incubated two hrs. at 37°C to allow for invasion. Then the monolayer was washed, and incubated another two hrs. in culture media containing 100 µg/ml gentamicin to kill remaining extracellular bacteria. Intracellular bacteria were released by monolayer lysis and enumerated by colony count on LB agar.
Results: These pathogens displayed markedly different kinetics of invasion over the MOI range tested. Salmonella Typhi showed a discrete invasion optimum at MOI=40, possibly indicative that natural infection requires a higher dose of organisms. In contrast, Shigella flexneri invaded at a constant efficiency over all MOI's, and this organism is known to cause disease at very low infectious doses (<10 bacteria). EHEC showed a broad invasion optimum at MOI=0.4 to 4.0. Yersinia invasion efficiency was constant up to MOI=40 and then decreased sharply, reflecting saturation of host integrin receptors at higher MOI's.
Conclusion: In vitro kinetic analyses of bacterial invasion can be used in a targeted manner to understand the kinetics of disease pathogenesis.
Rapid DNA Preparation for Real-Time PCR Detection
D. K. Lau, J. K. Yee, FDA, ORA, San Francisco District
Most of the current rapid detection methods for pathogens involving gene probes, conventional PCR, and even real-time PCR have shown that a detection level of < 10 and even 1 cfu can be achieved from a cell suspension in broth. Once a sample matrix, such as food or environmental components, enters the picture, inhibitors or the sample itself can affect the detection limit of these molecular methods. A robust sample preparation method is a necessity in a successful detection of pathogens if DNA is the main sample for the molecular method. The simplest DNA preparation step is the boil prep method which involves the boiling of the sample to release the DNA from the cells. There are also complicated DNA purification kits involving extraction columns and special reagents that can take up to 4 hours for sample preparation. Here, we demonstrate a rapid DNA isolation method involving the FTA technology in comparison to the boil prep method and a more extensive extraction method to purify DNA from several food matrices. The findings showed that the GenSpin FTA based genomic DNA purification kit can provide rapid DNA template preparation to be used for a real time PCR detection method with a detection limit of < 3 cfu from spiked food matrices.
Detection of Salmonella Enteritidis in Incubated Pools of Shell Eggs Supplemented with Ferrioxamine E by Lateral Flow Test Kit
K. H. Seo1 , G. Thammasuvimol1 , K. Y. Song2 , 1CFSAN, FDA, College Park, MD, 2JIFSAN, UMD, College Park, MD
Shell eggs and egg products have been associated with many of food borne outbreaks caused by Salmonella Enteritidis (SE). A rapid and simple method for detecting SE from poultry samples is critical for the effective implementation of such testing strategies. However, rapid methods have been reportedly able to consistently detect SE in egg pools only at levels of >107 CFU/ml. Therefore, sample preparation for the rapid methods is critical to detect low numbers of SE cells in eggs. In the present study, an optimum incubation method was determined for the promotion of the multiplication of small initial numbers of SE to permit efficient detection of SE using a rapid lateral flow test kit. A lateral flow device for the detection of SE utilized in this study was manufactured by Neogen, Lansing, MI. A series of iron supplementation using ferrioxamine E and ferric sulfate were conducted to optimize the test procedure for raw eggs in combination with different incubation temperatures (25, 37, and 42 degrees C). Detection of SE was 100% only in FE-supplemented raw egg pools inoculated with 10 SE cells per pool of 10 eggs when combined with an enrichment step at a 1:10 ratio in mTSB for 24 h at 37 or 42 degrees C. The lateral flow test kit could provide a simple, rapid, and inexpensive method for egg producers and processors to test specifically for Salmonella group D1 serovars, such as SE, in egg samples.
The Evaluation of Ferrioxamine E as a Selective Iron Source for Enterobacter sakazakii in iron Limited Nutrient Media and Egg White
K. Y. Song1 , K. H. Seo2 , G. Thammasuvimol2 , R. Brackett2 , 1Joint Institute for Food Safety & Applied Nutrition (JIFSAN), University of Maryland, College Park, MD, 2CFSAN, FDA, College Park, MD
This study was conducted to find the optimal concentration of iron sources to detect Enterbacter sakazakii and the effectiveness among Ferric Ammonium Citrate (FAC), Ferrous Sulfate (FS), and Ferrioxamine E as an iron supplement. FE has been known as selective iron sources for certain groups of bacteria such as Salmonella and Enterobacter. In recent years, Enterobacter sakazakii has been associated with necrotizing enterocolitis, bacteremia, and infant meningitis through the ingestion of contaminated powdered infant formula milk. The iron-free medium was composed of NaCl-glucose and the amount of iron source was added to give a final concentration from 200 ng/ml to 5 mg/ml inoculated with 0.1 ml of 102 CFU/ml of Enterobacter sakazakii. After 24 h of incubation at 37 C, the Enterobacter sakazakii populations recovered from iron-free media supplemented with FAC, FS, and FE were log 6.2, 6.3 and 6.4 CFU/ml at the concentration of 200 ng/ml, respectively. However, only log 1.8 CFU/ml Enterobacter sakazakii were isolated from iron-free media without supplementation. Also, in egg white known as perfect iron-free media, the same procedure was performed with the Enterobacter sakazakii populations in the egg white media supplemented with FAC were log 8.7 CFU/ml in 5 mg/ml, log 8.6 CFU/ml in 2 mg/ml, and log 5.0 CFU/ml in 200 ug/ml; FS were log 6.0 CFU/ml in 2 mg/ml and log 4.2 CFU/ml in 200 ug/ml; and FE were log 6.5 CFU/ml in 200 ug/ml, log 6.4 CFU/ml in 20 ug/ml, and 3.0 CFU/ml in 2 ug/ml. No Enterobacter sakazakii was detected from the media without supplementation in egg white. FE could be used as the stimulator to detect Enterobacter sakazakii in selective media by accelerating the growth of the bacteria that is usually present in low number in infant formula or dairy food.
The Rapid Detection of Salmonella Enteritidis by Quantum Dot Biolabeling Coupled with Immunomagnetic Separation
K. Y. Song1 , K. H. Seo2 , G. Thammasuvimol2 , R. Brackett2 , 1Joint Institute for Food Safety & Applied Nutrition (JIFSAN), University of Maryland, College Park, MD, 2CFSAN, FDA, College Park, MD
Colloidal semiconductor CdSe-ZnS core-shell nanocrystal quantum dots (Qdot) are luminescent inorganic fluorophores that have the potential to overcome some of the functional limitations encountered by organic dyes in fluorescence labeling application. Salmonella Enteritidis emerged as a major cause of human salmonellosis worldwide from the 1980s through 1990s and remains an important serovar. A rapid, specific, and sensitive method for the detection of Salmonella Enteritidis was developed using Qdot as a fluorescence marker coupled with immunomagnetic separation. Magnetic beads coated with anti-Salmonella Enteritidis antibodies were employed to selectively capture the target bacteria and biotin-conjugated anti-Salmonella antibodies were added to form sandwich immuno complexes. After magnetic separation, the immuno complexes were labeled with Qdot via biotin-streptavidin conjugation. This was followed by a fluorescence measurement using a RF-5301 PC fluorometer. The detection limit in Qdot method was 103 CFU/ml for the cell concentration of Salmonella Enteritidis, while the FITC-based method required over 105 CFU/ml. The total detection time was within 2 h. In addition to the nanotechnology developments, the results could play a role in the new rapid detection of various pathogenic bacteria.
Persistence of Yersinia pestis in Bottled Water
S. D.. Torosian1 , P. Regan1 , R. Zsigray2 , 1FDA, 2UNH
Historical anecdotal evidence, existence of pharyngeal plague combined with recent studies using cats and mice suggest that Yersinia pestis, the causative agent of bubonic plague, can be transmitted via ingestion. This means of transmission might be utilized for bio-terror purposes. It is therefore of value for the FDA to know the persistence capabilities of Yersinia pestis in various food matrices.
We selected sixteen strains of Y. pestis of varying genetic make-up and prior genetic manipulation for examination of persistence in bottled water. Strains were grown in Heart Infusion Broth and enumerated via plate count. Sixteen separate , filter sterilized reservoirs of one liter of bottled water (purchased at random from a local grocer) were placed in clean, sterilized screw cap bottles. Each bottle received an inoculum of 2 x 106 cells of one of the sixteen strains. This number was chosen to allow accurate plate counts using 0.1 ml samples initially. The final concentration of 2 x 103 cells per ml was visually undetectable. All reservoirs were kept at 26oC to approximate shelf storage of bottled waters.
Of the sixteen strains four reached persistence endpoints between 100 and 120 days post-inoculation. Four more strains persisted between 160 to 190 days post-inoculation. The remaining eight strains have not reached the endpoint as of this abstract submission, 210 days post-inoculation. The endpoint samples taken of each strain were verified as Y. pestis by multiplex PCR utilizing published primers
Detection of Yersinia pestis in Selective Enrichment of Certain Foods by Multiplex PCR.
S. D.. Torosian1 , P. Regan1 , R. M.. Zsigray2 , 1FDA, 2UNH
Sixteen strains of Yersinia pestis of varied genetic make-up were examined in this study for detectability in a variety of matrices. The matrices employed were lettuce, cucumbers, tomatoes, peppers, salad, pepperoni pizza, and mashed potatoes. Each food was either rinsed in or seeded with a portion of an overnight culture of Y. pestis. Twenty five gram samples of Y. pestis contaminated foods were stomached for five minutes in 100 ml of selective enrichment broth (1X Heart Infusion Broth, 12ug/ml Sodium Azide, 0.08 ug.ml Irgasan). and allowed to incubate at 26oC. Samples were taken at 0, 24 and 48 hour time points for assessment by multiplex PCR. Ten microliters of selectively enriched culture were utilized in a 50 microliter PCR reaction. Published primers were used for this study. All primer sets were chosen for amplicon sizes amenable to probe or beacon utilization. PCR products for this study were analyzed using agarose gel electrophoresis. Samples of Lettuce and tomato have been processed and verified as of abstract submission. Other samples are being processed.
Analysis of time 0 samples by PCR showed anplification of six of the sixteen strains for both lettuce and tomato.. For the 24 hour timepoint all sixteen samples amplified for both lettuce and tomato. For the 48 hour samples fourteen of the sixteen samples amplified for lettuce and twelve of sixteen for tomato.
This data indicates that with this selective enrichment media and these samples contaminated with a high number of Y. pestis, detection is best after 24 hours of enrichment. It is probable that the reduced sensitivity at 48 hours is due to interference by outgrowth of other flora. Further studies using specific and smaller inocula are warranted.
The biological effects of nanoparticles may be different than those of larger sized particles, therefore, it is crucial to develop appropriate toxicity testing approaches for the safety assessment of nanoparticles. To develop methodologies for a comprehensive immunotoxicological evaluation, we are using a combined in vivo and in vitro approach for studying the effects of nanoparticles on immune responses that are frequently undetected at the time of acute exposure. Our previous data with micron-size particles indicate that smaller particles induce a stronger inflammatory response than larger particles, suggesting that the response to nanoparticles may be different from that produced by micron-sized particles. Changes in the balance among various types of immune responses may have significant long term impact on health. The in vivo part of our study evaluates the significance of the chemical and physical characteristics of the materials, the size of the particles, and the routes of exposure. Histological and functional analyses of in vivo particle-affected tissues will determine the further in vitro evaluation of immune cell functions. Immune effects of in vivo exposure will be correlated with the in vitro exposure effects on macrophages, spleen and lymph node cells. The goal of this approach is to identify in vitro indicators of the effects observed in vivo. The immunological studies compared here are part of a multi-pronged approach involving numerous toxicological endpoints.
Elimination of alpha-Gal Alone From Source Pigs is Not Sufficient to Avoid NK Cell-Mediated Destruction of Porcine Endothelial Cells
J. A. Arcidiacono, C. M. Porter, E. T. Bloom, CBER, FDA, Bethesda, MD
Xenotransplantation of pig organs into humans provides a potential approach to alleviating the shortage of human organs. Primates reject pig organs in a hyperacute fashion due to complement and natural antibody to the Galα(1,3)-Gal (αGal) epitope. However, evidence for the role of αGal in the NK cell-mediated xenoresponse is contradictory. The identification of endo-β-galactosidase C, a novel enzyme that cleaves αGal, and creation of α1,3-galactosyltransferase knockout pigs provide two novel systems for investigating this issue. We found that αGal removal by endo-β-galactosidase C variably reduces the susceptibility of these cells to lysis by fresh human NK cells. We then examined αGal-null porcine endothelial cells (EC) using lines from α1,3-galactosyltransferase gene null pigs. The two αGal null lines showed no consistent difference in expression of adhesion molecules CD106 (VCAM-1), CD31 (PECAM), CD62E (E-selectin) or CD62P (P-selectin) compared to αGal positive cells. Nevertheless, immortalized EC from αGal-/- pigs were significantly less susceptible to lysis by naïve human NK cells compared to immortalized αGal-expressing cells (p<0.05). However, nonimmortalized wild type and knockout cells were equivalently sensitive. All αGal-null EC failed to display reduced susceptibility to killing by activated human NK cells cultured for 5 days in the presence or absence of IL-2. Since NK cells are likely to be activated in a transplantation setting, these findings suggest that elimination of αGal alone from source pigs will be insufficient to circumvent NK cell mediated destruction of porcine xenografts.
Characterization of differentiated stem cells on the basis of their gene expression profile
B. Bhattacharya1 , Y. Luo2 , T. Miura2 , J. Cai2 , J. Mejido1 , T. C. Schultz3 , X. Zheng2 , S. N. Brimble3 , R. K. Puri1 , 1CBER, FDA, Bethesda, MD, 2NIA, Baltimore, MD, 3Bresagen Inc. Athens, GA
Background: Embryonic stem cell lines available from NIH have generated huge scientific and public interest. These cells have the ability to differentiate into a variety of cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. However, little is known about the mechanism of differentiation and factors regulating cell development. Understanding the molecular identity of these cells will help understand the pathway these cells will adopt.
Method: We have produced high quality microarrays containing 16,659 seventy base pair oligonucleotides to examine gene expression profile in three stem cell differentiated cell lines.
Results: Ninety-two genes identified as 'stemness' genes showed down modulation in differentiated cells while new subset of 194 genes showed over expression of greater than 3 fold. These included 37 novel and 157 known genes. Gene expression was validated by a variety of techniques including RT-PCR, focused cDNA microarrays, massively parallel signature sequencing analysis and immunocytochemisty. Several novel differentiation specific ESTs were mapped to the human genome database and their expression profile characterized. A hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among ES cells and differentiated cells and confirmed that microarray could readily distinguish between undifferentiated and differentiated cells.
Conclusion: These results represent a detailed characterization of a unique set of differentiation specific genes, which can be used to assess ES cell differentiation.
Use of Methacrylated Beta-Cyclodextrin in Dental Restorative Fillings
L.A. Hussain, CDER, FDA, Rockville, MD
Background: Methacrylated ß-cyclodextrin (MCD) is a novel monomer that can be used to overcome drawbacks such as shrinkage that causes secondary dental caries and inadequate wear resistance of polymer-based dental restorative fillings.
Methods: MCD was synthesized by dissolving beta-cyclodextrin in pyridine, followed by the addition of an excess of methacrylic anhydride. The average number of double bonds (DB's) per MCD molecule was established using a bromination technique. The MCD was mixed at a mass ratio of 1: 1 with each of the methacrylate monomers: 1) hydroxyethyl methacrylate; 2) triethyleneglycol dimethacrylate; 3) 1,6-hexanediol dimethacrylate; 4) 1,10-decamethylenediol dimethacrylate; 5) 1,12-dodecanediol dimethacrylate. The mixtures were activated with 4.8 % camphorquinone and 7.8 % ethyl p-dimethylaminobenzoate (mol/mol resin mixture). Composites were prepared from each resin mixture and silanated BaO-containing glass (1 : 3; w/w), light cured for 2 min per side, stored in water (37 oC) for 24 h, and Flexural Strength(FS) was measured. Volumetric Shrinkage(VS) was determined by a computer-controlled mercury dilatometer (n = 3), and Degree of Conversion(DC) with near-IR spectrometry (n = 3).
Results: The DB's per MCD were 16.6 ± 2.0 (n = 3). The FS values (MPa ± standard deviation) for the composite formulations 1) through 5) were (62±11A), (117±16B), (71±10A,C), (110±7B), and (81±6C). The % VS were (3.6±0.2a), (2.9±0.2b), (3.1±0.1b), (2.8±0.3b), and (3.1±0.2a,b). The corresponding DC (%) were (73.5±0.5a), (60.6±1.1b), (57.3±1.0c), (52.0±0.8d), and (54.1±0.2d). Different superscript letters indicate significant differences; ANOVA; Tukey test; p < 0.05. In comparison, the FS, VS, and DC of similarly filled 50 % Bis-GMA, 50 % triethyleneglycol dimethacrylate (w/w) were (125±13) MPa, (3.0±0.1) %, and (79.6±0.5) % respectively.
Conclusions: The properties of the MCD-based formulations 2) and 4) show excellent properties approaching those of the Bis-GMA/triethyleneglycol dimethacrylate control.
Development of a Focused Microarray to Assess Stem Cell Differentiation
A. X. Yang1 , J. Mejido1 , Y. Luo2 , X. Zheng3 , C. Schwartz2 , T. Wu4 , R. S. Thies5 , B. Bhattacharya1 , J. Han1 , B. Freed3 , M. Rao2 , R. K. Puri1 , 1CBER, FDA, Bethesda, MD, 2NIA, NIH, Baltimore, MD, 3NIDA, NIH, Baltimore, MD, 4NIDCR, NIH, Bethesda, MD, 5Geron Corporation, Menlo Park, CA
Background: Embryonic stem (hESC) cells have great therapeutic potential but also have the potential to form teratoma tumors if transplanted as undifferentiated cells. hESC cells must be differentiated before clinical use and the extent of contamination with undifferentiated cells must be assessed.
Method: We developed a focused DNA microarray containing 729 oligonucleotides spotted in triplicate belonging to genes that may distinguish differentiated stem cell from their progenitors. The focused array was tested with DNA derived from two hESC lines, three differentiated stem cell lines, and a human testicular embryonic carcinoma cell line.
Results: Stem cells and differentiated cells showed different gene expression patterns when cDNA hybridized to the focused array. A cluster of 248 genes was identified that can readily distinguish stem cells from differentiated cells. Interestingly, the testicular embryonic carcinoma cell tested had similar characteristics with stem cells.
Conclusion: Thisfocused array is a valuable tool to rapidly identify stem cell population without major efforts. This array will be highly useful in the pre-clinical testing of stem cell identity and distinguishing ES cells from differentiated cells.
Diminished Neural Signaling in Psammomys Obesus (Fat Sand Rat): Possible Predictor for Diabetes
C. L. Zimliki1 , D. Mears2 , V. M. Chenault1 , 1CDRH, FDA, Rockville, MD, 2Uniformed Services University, Bethesda, MD
BACKGROUND: Pancreatic beta-cells are neurohormonal cells that secrete the hormone insulin in response to elevated glucose levels. Understanding the physiology of these cells is necessary for engineering glucose-sensing cells, but the neural stimulatory pathways are not fully defined. Muscarinic stimulation of pancreatic beta-cells (a neural signaling pathway) is critical for increased insulin release after meal consumption. This pathway is exaggerated in diabetic animals resulting in greater insulin secretion. The Psammomys Obesus is an animal model in which diet-induced diabetes occurs more frequently with diets containing high energy than high fiber. Questions still remain as to how these glucose and muscarinic pathways are altered in Psammomys Obesus. OBJECTIVE: The goal is to identify the glucose and muscarinic pathways of Psammomys Obesus. METHODS: Islet electrical activity and calcium imaging were used to detect the stimulus-secretion coupling pathways for glucose and muscarinic agonists. RESULTS: For the first time, we show cells in islets from non-diabetic sand rats display glucose-dependent electrical oscillations having half-maximal activity at 8mM glucose with activity occurring as low as 3mM. This glucose dose response was left-shifted when compared to that of mouse islets (half-maximal activity, 15mM). Interestingly, the muscarinic-induced activity was diminished in the non-diabetic sand rat when compared to control mice. CONCLUSIONS: These results suggest that the diminished muscarinic pathway limits the compensatory response in Psammomys Obesus making them prone to diabetes. Further work is necessary to determine if reduced muscarinic activity can be used as a predictor for diabetes in Psammomys Obesus.
Radial efficiency function in refractive surgery: Ablation losses caused by corneal curvature
B. Drum, CDRH, FDA, Rockville, MD
Background:Refractive surgical lasers reshape the cornea by ablating corneal tissue. If the wrong amount of tissue is removed, refractive errors may be over- or under-corrected, and higher order aberrations may be induced that cannot be corrected with glasses. Early ablation algorithms assumed that ablation was equally efficient everywhere on the cornea. Because the cornea is curved, however, basic physical principles dictate that the beam must ablate less tissue at the edge, where it is tilted, than at the center, where it is flat.
Theory: Factors contributing to reduced ablation on tilted surfaces include: (1) larger beam area and correspondingly lower irradiance; (2) more reflection and less absorption; (3) smaller proportion of beam area above ablation threshold, from attenuation of nonuniform beam. Failure to compensate for radial ablation efficiency loss predicts: (1) overcorrection of myopia; (2) undercorrection of hyperopia; (3) induction of spherical aberration; (4) induction of spherical error when treating astigmatism; (5) transformation from prolate to oblate corneal shape.
Results: Theoretical calculations of radial efficiency effects predict ablation depth errors of up to 25-30% on normal human corneas. Ablation depth measurements on model corneas and tilted plastic samples confirm these predictions. Refraction data in clinical trials also show the predicted refractive errors and induced aberrations.
Conclusions: Refractive surgical lasers must compensate for changes in ablation efficiency due to corneal curvature in order to accurately correct refractive errors. Compensation is even more important for correcting higher order aberrations, which typically require ablations of only a few micrometers for correction.
Nanoshells Enhance Optical Coherence Tomography Images of Tissue Phantoms
S. Huang, A. Agrawal, J. Pfefer, CDRH, FDA, Rockville, MD
Background
Optical coherence tomography (OCT) has recently emerged as a powerful biomedical imaging modality that uses reflected light - in a manner analogous to ultrasound - to achieve imaging resolutions of 20 µm or better. Nanoshells are novel, sub-micron diameter particles which can be manufactured to have specific optical scattering and absorption characteristics. We have characterized the performance of nanoshells as a contrast agent for OCT.
Methods
Solutions of polystyrene microspheres at three different concentrations (phantoms 1, 2, and 3) are used to mimic tissues with scattering coefficients of 7.5, 15 and 30 cm-1 respectively. These samples are imaged by our OCT system, at 1300 nm wavelength, with and without nanoshells. Furthermore, various concentrations of nanoshells are added to phantom 3 to study the effects of concentration on OCT image enhancement.
Results
The addition of nanoshells causes OCT signal intensities (brightness) to increase by 2-5 decibels (dB) in all three phantoms. The amount of increase depends on the original scattering properties of the phantom. The difference in signal intensities (contrast) between phantoms is also amplified by adding nanoshells; for example, the contrast between phantom 1 and 3 increases to 3.5 dB from 2.8 dB. Furthermore, increasing nanoshell concentration produces an increase in signal intensity of up to 13 dB.
Conclusions
Our results demonstrate that nanoshells can enhance OCT image brightness of individual phantoms and the image contrast between phantoms of different scattering coefficients. Nanoshell concentration must be optimized in order to achieve the greatest OCT image enhancement at the desired imaging depth.
Continuous-wave vs. ultrashort (pico- and femtosecond) pulsed laser beam propagation through turbid tissue in minimally invasive laser medical devices
I. K.. Ilev1 , R. W. Waynant1 , A. Amat1 , D. Royston1 , R. Weiblinger1 , T. Romanczyk2 , J. Anders2 , A. Gandjbakhche3 , 1CDRH, FDA, Rockville, MD, 2USUHS, Bethesda, MD, 3NIH, Bethesda, MD
Background: To optimize the effectiveness and safety of both continuous-wave (cw) and ultrashort (pico- and femtosecond) pulsed laser systems used as diagnostic and therapeutic medical devices, knowledge of basic laser parameters (such as wavelength, intensity, specific temporal and spatial beam characteristics) and optical tissue properties (including absorption and scattering characteristics) is of fundamental importance.
Methods: The primary objective of this project is to study the fundamental principles and effects of both cw and ultrashort pulsed laser beam propagation through biological tissue with various optical properties. We include highly scattering and thick tissue samples in order to improve the accuracy and specificity of photodosimetry in minimally invasive laser therapeutic procedures and devices. The study includes various independent theoretical and experimental approaches such as: 1) direct three-dimensional imaging (both optical and thermal imaging) of cw and ultrashort Gaussian laser beam profiles though tissue using a single-mode-fiber based technique; and 2) a simulation analytical method to compare cw vs. ultrashort laser beam parameters on laser beam propagation in highly scattering tissue samples with various optical features (scattering and absorption coefficient, anisotropy factor, thickness).
Results: We have experimentally and theoreticallystudied fundamental cw and ultrashort laser beam propagation parameters including intensity distribution, divergence, beam focal sizes, temporal pulse shape dispersion, possible thermal lensing and defocusing effects, for Gaussian laser beam propagation in single and multi-layered tissue samples.
Conclusions: The findings of this work are useful for understanding basic laser-tissue interaction mechanisms for cw and ultrashort pulsed laser beam propagation in highly scattered tissue media. Relevant results will be incorporated into test methods for evaluation of critical laser photodosimetry parameters and safety criteria for diagnostic and therapeutic laser devices.
Designing and building a laboratory based high-resolution, high-frame-rate imaging system with extended dynamic range for cone-beam computed tomography and dynamic imaging applications.
I. S. Kyprianou, R. J. Jennings, R. M. Gagne, N. A. Petrick, B. D. Gallas, K. J. Myers, US FDA/NIBIB Laboratory for the Assessment of medical imaging devices, Rockville, MD, 20852
The technology of medical imaging devices reviewed by the FDA advances rapidly. New digital imaging modalities such as tomosynthesis, high-resolution, cone-beam computed tomography (CB-CT), as well as multi-slice spiral CT are currently being introduced in clinical practice. Novel evaluation methodologies based on the statistical and physical properties of these multi-dimensional, high-resolution digital imaging devices are currently under development in our laboratory. A custom, flexible, multifunctional imaging system which uses the latest technological advances in flat panel x-ray imaging is being developed to provide experimental support for this effort. The imaging system design consists of a large field (40cm x 30cm) flat panel indirect detector with a columnar CsI(Tl) scintillator (600µm thick). The detector has 16-bit analog to digital conversion giving it a dynamic range from 4µR to 4R. It has a pixel size of 200µm, and it can acquire 30 frames per second. Two x-ray sources are used for image acquisition: a low output, constant potential, microfocus x-ray tube (15-40µm focal spot size) with custom shutter mechanism and liquid cooling for the high resolution requirements, and a high output pulsed x-ray tube (0.3 and 0.6mm focal spots) for fluoroscopic image acquisition. A computer-controlled, three-axis rotational stage is used to provide positioning and rotation of the sample to be imaged. Hardware and software are being developed for object positioning, and detector, shutter, and x-ray pulse synchronization and triggering. Various image reconstruction algorithms have been implemented and are used for the acquisition of the 3D data sets.
Quantum Yield Measurements of Drugs with the Potential to Interfere with Fluorescence-Based Diagnostic Devices.
L. S. Matchette, A. Agrawal, T. J. Pfefer, CDRH, FDA, Rockville
Background:
Fluorescence, both intrinsic and exogenously induced, is being used for diagnosis and treatment of abnormal tissue. The presence of optically active (absorbing, fluorescing) drugs is rarely taken into account by practitioners of fluorescence diagnosis and has the potential to yield either false positive or false negative results. Therefore, we are measuring, in vitro, the relative quantum yields of commonly prescribed drugs in an effort to identify those that require further characterization in vivo. Our aim is to quantify a drug's potential to interfere by (1) comparing the relative quantum yields of suspicious candidate drugs to those of known tissue fluorophores and (2) accounting for drug tissue concentrations and pharmacokinetics.
Methods:
Quantum yields are determined relative to a working standard of Rhodamine 6G in ethanol. The working standard is calibrated against a fluorescein standard developed by the National Institute of Standards and Technology (NIST). We are concentrating our initial efforts on (1) the fluoroquinolone antibiotics, ciprofloxacin and norfloxacin and (2) intrinsic tissue fluorophores, NADH FAD and protoporphyrin IX.
Results: When Ciprofloxacin and norfloxacin are excited at wavelengths 320 to 450 nm, emission occurs from 350-650 nm with quantum yields around 0.16. The quantum yields for intrinsic fluorophores excited at their peak absorption wavelengths were 0.02 (NADH, 340 nm), 0.035 (FAD, 450 nm) and 0.087 (protoporphyrin IX, 408 nm).
Conclusions:
From an optics perspective, these fluoroquinolones have the potential to interfere during fluorescence diagnosis techniques.
Electromagnetic Compatibility (EMC) of Active Medical Devices exposed to Wireless Personal Digital Assistants (PDAs)
G. G. Mendoza, D. M. Witters, H. I. Bassen, CDRH, FDA, Rockville, MD
Background: FDA is concerned that the emissions from some wireless PDAs may cause electromagnetic interference (EMI) in nearby active medical devices.
Methods: EMC and coexistence testing was performed for 11 active medical devices using common wireless PDA technology (802.11, Bluetooth) and a Blackberry PDA. Five glucometers, five patient monitors, and a surgical endoscopy device with a wireless operation control unit were tested inside the gigahertz transverse electromagnetic cell (GTEM) electromagnetic exposure facility with 802.11 and Bluetooth exposure signals at field strengths 3, 10, and 20 V/m. Additional EMC tests were performed using the Blackberry in proximity to the active medical devices.
Results: The link between the wireless control unit and the surgical endoscopy device lost communications when exposed to 802.11 and Bluetooth signals above 10 V/m. Also, the function of one patient monitor was disrupted by the Blackberry emissions when the Blackberry PDA was closer than 10 cm. The rest of the active medical devices did not show any interference.
Conclusions: Our findings indicate that certain active medical devices can be affected by emissions from wireless PDAs. These findings suggest that EMC and coexistence of wireless technologies with active medical devices needs to be examined further by FDA, and tested over a wide range of active medical devices. This work highlights the need to develop guidance and standards for the design, testing, and use of wireless PDAs around active medical devices.
Accidental Disconnection of Luer Locks
M. R. Schwerin1 , D. N. Busick2 , L. D. Cash3 , A. E. Morrison4 , 1CDRH, FDA, Rockville, MD, 2Formerly of CDRH, FDA, Rockville, MD, 3ORA, FDA, Winchester, MA, 4CDRH,FDA, Rockville, MD
Luer lock connections are widely used in medical settings. Over the years, FDA has received hundreds of adverse event reports involving the accidental disconnection of luer lock connectors. Even though most instances are not fatal, death may occur. The purpose of this study is to identify the common factors for disconnection and help reduce the incidence of future luer lock disconnections. Luer connectors were obtained from nine companies and a number of samples were sterilized. Samples were tested for conformance with the dimensional specifications of ISO 594-1. They also were tested in accordance to ISO 594-2 for ease of assembly, unscrewing torque, separation force, stress cracking, and effects of sterilization. Several samples were also tested to failure using a variation of the separation force test. It was discovered that not all of the luer connectors tested were ISO 594 compliant, as 7 of 23 different designs failed at least one of the tests outlined in ISO 594-1 or ISO 594-2. Lack of compliance with ISO 594 may contribute to luer disconnections. Several recommendations were also made to reduce the potential of future disconnections of luer connectors.
Development of High Speed Nano-scale PCR for Detection and Identification of FDA Relevant Pathogens
A. V. Matviyenko1 , M. Lewis1 , N. V. Sergeev2 , L. Bockstahler2 , A. Rasooly2 , K. Herold1 , 1University of Maryland, Department of Mechanical Engineering, College Park, MD 20742, 2FDA/CDRH/OSEL/DB, Silver Spring, MD 20903
The motivation for miniaturization of the PCR reaction comes from the need to speed up the reaction so as to increase its utility for use as a component in assays for microbial contamination. Minimization of the mass of the reaction allows more rapid attainment of temperatures and lower power requirements. Spin-off benefits include small sample size and reagent volumes.
We performed a series of proof-of-concept experiments. We found that reaction volumes less then 500 nl produced PCR amplicons in quantities hardly detectable by conventional agarose gel-electrophoresis. We also experimented with dyes included in the PCR reaction and this was found to be feasible. In parallel with that effort, we also built and tested three thermocycling schemes (Peltier, air-cooling and microfabricated heater) in attempts to arrive at a high speed system. Our preliminary results suggest that the microfabricated heater design is the most promising of these technologies due to the ability to get high heat flux while heating and cooling only a small region of space. Our current prototype device enables amplification at the rate 1 min/cycle with a sample volume of 1 µl and power consumption of 2 W. Future versions will be scaled down from this prototype. These initial results point toward the potential for development of a portable, battery-powered PCR instrument.
Development of miniature Point-of-Care microfluidic biosensor for simultaneous rapid detection of multiple microbial toxins
A. V. Matviyenko, N. V. Sergeev, A. Rasooly, FDA/CDRH/OSEL/DB, Silver Spring, MD 20903
Background: Detection of microbial pathogens and their toxins is crucial for human health and is important for assuring the safety of FDA-regulated products including medical devices, food, drugs, biologic and veterinary products. To improve the Agency's diverse regulatory operations, there is a need for point-of-care, portable, inexpensive multi-analyte detectors that can be operated without an external power source.
Methods: Our biosensor consists of a three-layer plastic cartridge assembled with a thermal press. Primary antibodies for capturing antigens are immobilized on a layer of nitrocellulose that is irreversibly attached to a polycarbonate substrate. Samples and reagents for the sandwich assay are delivered to the membrane through microcapillaries using a miniature built-in manual vacuum pump. All reagents are kept in special cartridge chambers within the device, allowing stand-alone operation.
Results: We developed a model immunosensor for the detection of staphylococcal TSST-1. We foundthat a nitrocellulose membrane allows a much higher density of antibody immobilization than any plastic substrate, improving the assay's sensitivity. The usage of a microfluidic network for reagent delivery, instead of a micro-titer plate format, considerably reduces reagent consumption and the time required for the assay. The device can process an individual sample or multiple samples simultaneously.
Conclusions: This prototype successfully demonstrates our concept for a stand-alone, inexpensive, point-of care, multi-channel biosensor. The device may be useful for rapid on-site diagnostics, supporting FDA regulatory needs and improving public health through early and sensitive detection of microbial toxins in a broad spectrum of FDA-regulated products at a low cost.
A global perspective of vitamin D Intake.
M. S. Calvo1 , S. J. Whiting2 , 1OARSA, CFSAN, FDA, Laurel, MD 20708, 2College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada S7N5C9
Background: High prevalence of vitamin D insufficiency and the global re-emergence of rickets combined with growing evidence linking poor vitamin D status with greater risk of chronic diseases have stimulated recommendations to increase exposure to sun as a source of vitamin D. Concern over increased risk of melanoma with unprotected sun exposure led to the alternative recommendation that sufficient vitamin D should be supplied by the food supply.
Methods: We examined the adequacy of vitamin D intake world-wide and the ability of fortification policies and supplement use practices among various countries to meet current dietary guidelines. We compared vitamin D intake estimates from > 80 studies conducted over the last 25 years which reported quantified vitamin D intakes estimated from food frequency questionnaires, 24 h recall or multiple day food record and plotted these values according to age and classification of the country's fortification practices (mandatory, optional or none).
Results: For many countries without mandatory staple food fortification, vitamin D intake is often too low to sustain healthy circulating levels of 25-hydroxyvitamin D, the main status indicator of vitamin D. Even in some countries with mandatory or optional fortification, vitamin D intakes are low due to unique dietary patterns such as vegetarianism. Dietary supplement use may contribute 6 - 47% of the average vitamin D intake in some countries.
Conclusions: Reliance on the food supply as an alternative to increased sun exposure will necessitate greater availability of food staples fortified with vitamin D and/or increased dietary supplement use.
Dietary recommendations for vitamin D: critical need for functional endpoints to establish an estimated average requirement(EAR).
S. J. Whiting1 , M. S. Calvo2 , 1College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada S7N5C9, 2OARSA, CFSAN, FDA, Laurel, MD 20708
Background: In 1997, the recommended intake level of vitamin D was set as an Adequate Intake (AI) value rather than an RDA (Recommended Daily Allowance). An RDA is of great public health importance because an AI cannot be used to estimate adequacy of a population's nutrient intake.
Methods: 25-hydroxyvitamin D is the major static indicator of vitamin D status. Measurement of its circulating concentration in a dose response study allowed estimation of mean adult intake requirement of at least 500 IU (12.5 µg). Functional indicators of vitamin D status are also needed to establish an EAR upon which an RDA can be based.
Results: We report how new understanding of the function of the active metabolite,1, 25-dihydroxyvitamin D goes beyond its traditional calciotropic functions to important non-calciotropic functions involving cell proliferation and immunity. We explain the novel view that circulating 25-hydroxyvitamin D needs to be available in sufficient substrate supply for the 1-alpha hydroxylase enzyme in non-renal tissues. We propose an important shift in paradigm away from a single renal source of 1, 25-dihydroxyvitamin D for calciotropic functions to new disease preventing endocrine, paracrine and autocrine functions of the active metabolite in a variety of tissues from which functional indicators of status may be derived.
Conclusions: Given the increasing evidence of a link between vitamin D deficiency and risk of chronic diseases including cancer, there is now enough data to consider setting an EAR for vitamin D, the first step toward establishing an RDA to assess population adequacy of vitamin D intake.
Vitamin D Fortification of Food in North America: Current Status.
M. S. Calvo1 , S. B. Harper2 , S. J. Whiting3 , 1OARSA, CFSAN, FDA, Laurel, MD 20708, 2Office of the Director, NIH, formerly OARSA, CFSAN, FDA, Laurel MD 20708, 3College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada, S7N5C9
Background: Vitamin D is a fat soluble vitamin with potential for toxicity if chronically consumed in very high doses, therefore in Canada and the US, its addition to foods is very carefully regulated.
Methods: We reviewed the regulatory basis and current vitamin D food fortification practices in the US and Canada.
Results: Both countries utilize the same dietary guidelines governing adequate intakes, upper limits of safe intake, and share similar labeling formats. Canada, however, has mandatory fortification of foods to insure that all people may benefit when a public health need is identified. Staple foods, milk and margarine, are the Canadian targets for vitamin D fortification. Vitamin D fortification is regulated in the US in accordance with 21 CFR 184.1(b) (2); its use has strict limitations on the category of foods, functional use and level of use and added vitamin D must not exceed the specified limitations. Any addition of vitamin D to foods not in compliance with each of these established limitations shall require a food additive regulation. In most cases the addition of vitamin D to an eligible food in the US is optional with the exception of "fortified" milk. Review of the local US market place shows that with the exception of milk and ready-to-eat breakfast cereals, few food categories eligible for vitamin D fortification are actually fortified.
Conclusions: The regulatory limitations imposed by the US and Canada provide a safeguard that limit over-fortification with vitamin D, however, problem may lie with the manufacturer's option not to fortify eligible US food products
Racial differences in vitamin D intake, dietary supplement use and vitamin D status among black and white men and women in the NHANES III survey.
M. S. Calvo1 , C. N. Barton2 , 1OARSA, CFSAN, FDA, Laurel, MD 20708, 2OFAS, CFSAN, FDA, College Park, MD
Background: Low circulating 25-hydroxyvitamin D [25(OH) D] is a significant risk factor for black men and women and has been implicated in their higher susceptibility to and severity of infections, auto-immune disorders, and high cancer mortality. Black men and women have impaired ability to synthesize vitamin D due to high melanin content in their skin and are more reliant upon dietary sources of vitamin D to maintain adequate circulating 25(OH) D. Current adult dietary reference intakes for vitamin D did not consider if race or skin pigmentation are associated with greater dietary vitamin D intake needs.
Methods: We assessed the adequacy of dietary intake, food sources and status of vitamin D in black men and women compared to whites using dietary intake data and serum measures of 25(OH) D from the third National Health and Nutrition Examination Survey (NHANES III, 1988-1994). We used estimates of vitamin D intake from food and supplements (24-h and usual) and serum 25(OH) D in adult (>20 y) Non-Hispanic white (n = 6456) and Non-Hispanic black (n = 4316) men and women.
Results: Significantly lower vitamin D intake from all sources and poorer nutritional status as measured by circulating levels of 25 (OH) D occurred in blacks compared to their white counterparts.
Conclusions: Blacks have higher incidence and mortality of certain aggressive cancers that should raise concern about their poor vitamin D intake given the established strong association between poor vitamin D status and increased cancer risk.
Physical Activity and Diet Predict Type II Diabetes Risk in the Diabetes Prone Sand Rat (Psammomys obesus)
A. A. Cotterell1 , C. L. Blue2 , V. M. Chenault1 , 1CDRH, FDA, Rockville, MD, 2Howard University, Washington, DC
BACKGROUND: Diabetes mellitus is a chronic disorder of glucose metabolism caused by inadequate production or use of the hormone insulin. It is a leading cause of death in the United States and is especially prevalent among African Americans. There are 18.2 million Americans afflicted with this disease. Physical activity and diet are known to reduce the risk of complications of diabetes. The sand rat is an excellent animal model for studying type II diabetes, as the complications that develop in the diabetic sand rat mirror those of humans. PURPOSE: To determine if physical activity and diet are good predictors of type II diabetes in the diabetes-prone Sand Rat.
METHODS: Twenty sand rats were singly housed and grouped (5 animals per group). Group I was fed a high energy, low fiber chow; group II, a high energy, low fiber chow and an enrichment toy in each cage to promote physical activity; group III, a low energy, high fiber diet and group IV, a low energy, high fiber diet with an enrichment toy in each cage. The animals were monitored for physical activity, exercise, eating, drinking, excreting and sleeping. Blood glucose levels, glycosylated hemoglobin (HbA1c) measurements and animal weights were obtained in order to monitor the onset of diabetes.
RESULTS: Blood glucose, HbA1c levels and weights were lowest in Group IV and steadily increased as follows: Group III< Group II< Group I.
CONCLUSIONS: Physical activity and low energy diets decrease HbA1c values, glucose levels and weights of diabetes prone sand rat, thereby decreasing the risk of the onset of Type II diabetes.
Impact of Dietary Food Label Use on Nutrient Intakes
L. Nadeau1 , A. I. Jessup2 , A. Serkaya1 , 1ERG, 2FDA
Background: Frequency of food label use is important in modeling the effect of nutrition labeling on diet quality.
Methods: We used data from the Continuing Survey of Food Intakes by Individuals (CSFII) and the Diet and Health Knowledge Survey (DHKS) to estimate the impact that food label use has had on intakes of calories, fat, and saturated fat. Subjects were divided into three label use groups ("always," "sometimes," and "rarely/never") based on their answers to DHKS questions. Our empirical model accounted for the likelihood that more health conscious consumers will use food labels more frequently in estimating the effect of label use on dietary intakes.
Results: The "always" food label users consume 52 fewer calories per day, 13.8% less total fat, and 13.2% less saturated fat than "sometimes" users and 213 fewer calories per day, 18.5% less total fat, and 27.3% less saturated fat than "rarely/never" users. Obese men in their 30s, who "rarely/never" use labels, can expect to lose close to 30 pounds from increasing their label to "always" over an entire year. Obese women in their 30s, who "rarely/never" use labels, can expect to lose close to 35 pounds from increasing their label to "always" over an entire year.
Conclusions: Food label use frequency is related to nutrient intakes, and hence an individual's weight. Programs and policies that increase use of food labels should be essential components of programs designed to address the nation's obesity epidemic.
REDUCTION OF OBESITY IS ASSOCIATED WITH INCREASED HEPATIC CYP3A EXPRESSION IN WOMEN.
D. Springer1 , T. I. Leakey1 , R. J. Feuers2 , C. Buffington3 , G. S. Cowen3 , J. E. Leakey1 , 1OSC, NCTR, 2DGRT, NCTR, 3UT Memphis, Memphis, TN
Although an increasing proportion of the American population is obese, there is only limited information on whether obesity or weight loss influence drug metabolism in humans. Cytochrome P450 (CYP) isoform expression is very variable in human liver samples that are used in toxicity testing and research. This variation is due to factors such as genetic polymorphism, environmental and pharmaceutical exposure to inducers and pathological and post mortem changes. It complicates the study of endogenous regulators of human CYP. Gastric bypass surgery is used in chronically obese patients as an aid to weight-loss as it both reduces food intake and absorption. Typically, once patients have lost weight a second surgery is performed to remove excess skin and muscle from the abdominal cavity. We obtained liver biopsy samples (with informed consent) from patients at both surgeries so that we could compare the effects of weight loss directly in the same patients. We investigated CYP isoform expression in these samples using isoform selective activities and western blotting. Microsomal preparations were assayed for a range of monooxygenases including acetanilide 4-hydroxylase (CYP1A2), chlorzoxazone 6-hydroxylase (CYP2E1) testosterone 6b-hydroxylase (CYP3A) and lauric acid 11-hydroxylase (CYP4A) activities. Of these, CYP3A-selective testosterone hydroxylase activity was consistently increased in samples from the second surgery as compared to corresponding samples from the first (obese) surgery 215 + 40%, P<0.05). Small increases in acetanilide and lauric acid hydroxylases were also observed (+52 + 28%, +35 + 12% respectively), whereas chlorzoxazone hydroxylase activity was slightly decreased ( -18 + 11%). This suggests that obesity does influence the expression of CYP3A and possibly other CYP isoforms in human liver.
Accumulation of saxitoxins in the non-toxic northern puffer fish (Sphoeroides maculatus) from a food chain source.
J. Deeds1 , S. M. Etheridge1 , C. Gieseker2 , R. Reimschuessel2 , S. Serfling3 , S. M. Conrad1 , S. Hall1 , 1CFSAN/OS/DSAT/WSL, FDA, Laurel, MD, 2CVM/OR/DAR, FDA, Laurel, MD, 3OC/OSCH, FDA, Rockville, MD
BACKGROUND
Recent investigations into several unexplained cases of puffer fish poisoning (PFP) that occurred in Florida, New Jersey, Virginia, and New York demonstrated that southern puffer fish (Sphoeroides nephalus) represent a new vector for the transfer of dinoflagellate-derived saxitoxins (STXs), responsible for paralytic shellfish poisoning (PSP), to human consumers. We are currently investigating the potential for the bioaccumulation of saxitoxins from a food chain source in Mid-Atlantic northern puffer fish (Sphoeroides maculatus), a species that historically sustained a large commercial and recreational fishery.
METHODS
Hard clams (Mercenaria mercenaria) were contaminated with a saxitoxin-producing isolate of the dinoflagellate Alexandrium sp. and were fed to wild-caught northern puffer fish. Fish tissue compartments were extracted and analyzed for toxins by high-performance liquid chromatography (HPLC).
RESULTS
Significant accumulation of saxitoxins were observed in puffer fish fed with hard clams maintained on STX producing Alexandrium sp., whereas no STX was detected in puffer fish fed hard clams maintained on non-toxic feeder algae. Rank order for STX concentration per tissue compartment was: ovary > mucous > muscle > skin > intestine > testis > liver.
CONCLUSION
The historically non-toxic northern puffer fish possess the same ability to accumulate PSP toxins from a food chain source as has been described for Florida southern puffer fish responsible for recent PFP events. While the directed fishery for this species has been greatly reduced in recent decades, significant recreational harvesting still occurs. PSP monitoring programs need to be aware of this additional reservoir for PSP toxins in marine systems.
Prior exposure to an acidic environment increases the acid resistance of Enterobacter sakazakii
S. G. Edelson-Mammel1 , S. Naik2 , R. L. Buchanan1 , 1CFSAN, College Park, MD, 2University of MD, College Park, MD
Previous studies with 12 strains of Enterobacter sakazakii showed BHI-grown strains were moderately acid resistant compared to other Enterobacteriacea such as Escherichia coli O157:H7, decreasing by greater than or equal to 5 log cycles when resuspended in TSB adjusted to pH 3.0, but generally decreasing by < 1.0 log cycle at pH 3.5. In the current study, the same 12 strains were evaluated to determine if they have an inducible pH-dependent stationary phase acid resistance that would enhance survival when cultures were resuspended in acidified TSB (pH 3.0) for 5 h at 36¢X C. The strains were grown individually in the presence (+G) or absence (-G) of 1% glucose for 18 h. The final pH of the 18-h cultures was 4.7-5.4 for the +G grown cells and 6.7 to 7.0 for the -G grown cells. After then being suspended in the acidified TSB (pH 3.0), samples were taken hourly and viable counts determined. With almost all strains, the +G grown E. sakazakii survived the pH 3.0 challenge longer. At times during the acid challenge, the differential in counts between the +G and -G samples was a much as 4 log cycles. The results suggest that acid resistance of E. sakazakii can be increased by prior growth in a moderately acidic environment and that the microorganism possesses one or more system for inducible pH-dependent stationary phase acid tolerance.
Faster Speeds and Higher Success Rates: The Impacts of Improved Record-keeping Practices on Trace-back Performance
A. J. Estrin1 , J. Guzewich1 , S. Pichette2 , C. Nardinelli1 , 1CFSAN, 2FDA
The analysis in this paper measures the effect on FDA traceback performance of the recently published regulation entitled "Establishment and Maintenance of Records Under the Public Health Security and Bioterrorism Preparedness and Response Act of 2002" (69 FR 71561, December 12, 2004). The regulation will allow FDA investigators to have access to storage and distribution records kept by most food facilities all along the supply chain within 24 hours of a request. In addition, the regulation will require food facilities to maintain these records at greater levels of detail than is likely to be the current practice. The requirements from this rule will allow FDA to investigate outbreaks more quickly, and reduce the probability of prematurely terminating an investigation because of poor records quality.
We use internal FDA information that tracks outbreak investigations for the period 2000 - 2003, and expert elicitation of FDA investigation personnel to estimate the impact on FDA traceback performance. Indicators of improved traceback performance are faster traceback speeds, and improved ability to complete investigations that previously would have been prematurely terminated due to poor records quality. Faster traceback speeds may permit faster preventive action and avert illnesses from on-going outbreaks. Improved investigation completion rates may reveal additional sources of outbreaks and avert illnesses by preventing outbreak recurrences. We estimate that the number of illnesses averted due to higher investigation completion rates is about double that for faster traceback speeds.
The receptor binding assay for tetrodotoxin and the saxitoxins: Progress toward the implementation of a sensitive, high throughput detection method for food defense and marine biotoxin management
S. Hall1 , S. M. Etheridge1 , J. Deeds1 , S. M. Conrad1 , F. Van Dolah2 , G. R. Strichartz3 , E. Moczydlowski4 , E. P. Mazzola5 , Y. F. Lam6 , 1Washington Seafood Laboratory, Division of Science and Applied Technology, Office of Seafood, CFSAN, FDA, Laurel, MD, 2Marine Biotoxins Program, NOAA, Charleston, SC, 3Anaesthesia Research, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 4Biology Department, Clarkson University, Potsdam, NY, 5Instrumentation and Biotechnology Branch, DGSS, OSAS, OO, 6Department of Chemistry, University of Maryland, College Park, MD
Tetrodotoxin (TTX) and saxitoxin (STX) are potent natural toxins that are of concern to food defense planners, in addition to their long-standing role as causes of human illness from naturally contaminated seafood. Detection methods for these toxins that are rugged, sensitive, and offer high throughput are of interest in both spheres. The toxicity of both TTX and STX is due to their reversible, high affinity to a specific site on the voltage activated sodium channel. The receptor binding assay (rba) exploits this strong, selective binding to provide a very sensitive detection method. Using a multi-well plate format developed at the NOAA Charleston lab, the rba offers very high throughput. Our current efforts are focused on refinement, validation, and field implementation of the rba to ensure that it is a rugged and reliable detection method that can fully address the needs of food safety and food defense.
Quantitative determination of cesium binding to Prussian Blue: A treatment for internal radioactive metal contamination
P. J. Faustino1 , Y. S. Yang1 , J. J. Progar2 , C. R. Brownell1 , N. Sadrieh3 , J. C. May2 , E. Leutzinger3 , D. A. Place3 , E. P. Duffy3 , F. Houn3 , S. A. Loweke3 , M. M. Nasr3 , A. S. Hussain3 , R. C. Lyon3 , 1CDER, FDA, Silver Spring, MD, 2CBER, FDA, Rockville MD, 3CDER, FDA, Rockville MD
Background: Prussian Blue (PB), ferric hexacyanoferrate is the Active Pharmaceutical Ingredient (API) of Radiogardase. This is the first approved drug product for treatment of internal contamination with radioactive cesium and thallium isotopes in the event of a radiological incident such as a "dirty bomb". The purpose of this study is to accurately determine the cesium binding capacity of PB simulating pH conditions encountered in the human gastrointestinal tract.
Methods: PB (0.1 g per 50 mL) API's and drug products were added to buffered solutions (pH 1.0 to 9.0) containing different concentrations of cesium ranging from 600 to 1500 ppm. Samples were incubated at 37oC in a water bath shaker. Following incubation, each sample was filtered. Measurements of cesium binding to PB were made between 30 min to 24 hours, to cover gastric and intestinal tract residence time using a validated Atomic Emission Spectroscopy (AES) method
Results: PB was found to have the highest binding capacity (340 mg/g) at pH 7.5 at 1500 ppm. The binding capacity decreased approximately 60% to a minimum at pH 1.0. Eighty percent of the binding capacity at all concentrations occurs in the first four hours of cesium exposure. Significant variation of cesium binding to PB from batch to batch was also observed among the APIs and drug products.
Conclusions: The pH, exposure time and concentration can significantly affect cesium binding to PB. These findings can be further utilized for product characterization to enhance efficacy and better predict drug product performance throughout the gastrointestinal tract.
The utility of immunomagnetic adsorption and FTA filter PCR for the screening of Cryptosporidium species in foods
A. Iddings, R. Shafran, P. A. Orlandi, CFSAN
The protozoan parasites, Cryptosporidium hominis and Cryptosporidium parvum are predominantly associated with human diarrheal illness. It is estimated that 80% of our population has been exposed to these microorganisms through contaminated food or drink. This highlights the need for a rapid and specific protocol to detect this pathogen in a wide variety of comestibles. This report details the utility of a method that combines the effectiveness of immunomagnetic bead adsorption (IMA) with the sensitivity of FTA filter PCR analysis for screening a wide variety of food matrices. Eighteen comestibles identified as most susceptible to Cryptosporidium contamination were artificially inoculated with 600 C. parvum oocysts. These included fresh and processed ciders, juices, milk, fresh berries, and leafy vegetables. A subset of commodities was also inoculated at lower levels to determine detection sensitivity. Beverages (10 ml aliquots) were processed directly by IMA. Fruits and vegetables (10-50 g) were rinsed in a bag filter and the resulting eluates were concentrated and processed by IMA. Captured oocysts were then passed through FTA filters to concentrate and lyse the oocysts for use directly in PCR reactions.The IMA-FTA PCR protocol reliably and reproducibly detected C.parvum oocysts in all matrices. Generally, PCR signal strength was proportional to the level of contamination in selected matrices. Reproducibility and calculated confidence levels validated the sensitivity of this protocol at the 50 oocyst level. In conclusion we found the combinatorial effects of IMA and FTA-PCR provided an effective means of rapidly and reliably detecting low levels of Cryptosporidium from diverse food matrices.
New Cytokine Markers for the Activity of Anthrax Lethal Toxin
H. Fang, R. Cordoba-Rodriguez, C. SR. Lankford, D. M. Frucht, Division of Monoclonal Antibodies
Purpose: Anthrax lethal toxin (LT), a critical virulence factor for Bacillus anthracis, has been proposed as a therapeutic target for the treatment of anthrax infection in humans. Anthrax LT specifically cleaves and inactivates mitogen activated protein kinase kinases (MAPKKs), thereby rendering target cells unable to respond to external stimuli that require these important signal transduction molecules. Although anthrax LT inhibitors have been proposed for use in human infections, the anthrax LT assays most commonly used are based on the species- and strain-specific action of anthrax LT to induce cell death in macrophage cell lines. Studies planned to determine the efficacy of anthrax therapeutics are currently limited in that they will be performed in animal models, due to the low frequency of naturally occurring human infection. Thus, it would be advantageous to identify new human cell-based assays for anthrax LT activity.
Methods: We screened a variety of parameters of cellular function in primary and human CD4+ T cells in response to treatment with anthrax LT, using MTT proliferation assays, flow cytometry analysis, and cytokine ELISAs. In addition, we confirmed by flow cytometry the binding of anthrax LT to target cells, and confirmed by Western blotting the effects of anthrax LT on MAPKK signal transduction pathways.
Results: Primary human CD4+ T cells, along with the model human CD4+ T cell line, Jurkat, are cellular targets for anthrax LT. Anthrax LT binds CD4+ T cells, where it acts to inactivate MAPKKs and to block downstream signal transduction, as well as induction of effector cytokines (IL-2, IL-4, and interferon gamma). These cytokines are required for the normal functions of CD4+ T cells, including cellular proliferation and the support of cellular and humoral immune responses. Specific inhibitors of the proteolytic activity of anthrax LT similarly block its inhibitory effect on the function of CD4+ T cells, confirming the direct effect of anthrax LT on these pathways. Dose-response experiments reveal that inhibitory effects of anthrax LT on IL-2 production are evident even at very low levels of anthrax LT (1-10 ng/ml), indicating that IL-2 production is a very sensitive marker for toxin activity.
Conclusions: We describe here the identification of cytokines produced by T cells that are sensitive markers for the activity of anthrax LT in human cells, establishing the basis for physiologically relevant anthrax LT bioassays for the characterization of potential therapeutics targeting anthrax LT.
| Sigma Xi Outstanding Poster Award - 2005 FDA Science Forum |
The Use of Near-Infrared Chemical Imaging (NIR-CI) for the Indentification of Micro-organisms of Concern in Food
J. Dubois1 , E. N. Lewis2 , F. S. Fry3 , E. M. Calvey3 , 1JIFSAN, University of Maryland, College Park, MD and Spectral Dimensions Inc., 3416 Olandwood Court, Suite 210, Olney, MD 20832, 2Spectral Dimensions Inc., 3416 Olandwood Court, Suite 210, Olney, MD 20832, 3CFSAN, 5100 Paint Branch Parkway, College Park, MD 20740
Introduction: Near-infrared chemical imaging (NIR-CI) is investigated as a tool for the high-throughput analysis of self-contained microbial identification test cards for micro-organisms of concern in food.
Methods: In this initial work, an NIR-CI system operating in the spectral range 1000-2450 nm was used to acquire NIR chemical images of bacterial cells deposited on a "card" containing both the calibration and test samples.
Results: Results indicate that some bacteria can be identified from differences observed at unique wavelengths, and that a standard operating procedure may be developed for a particular reference database to differentiate, and hence identify, the various organisms it contains using discrete wavelengths. A Partial Least Squares (PLS) model was developed to identify a particular organism of concern on the "card" without a need to know the taxonomic identity of the complete complement of bacteria present in the bacterial matrix.
Conclusions: This NIR-CI and test card technique mimics a system operating with a food-specific reference database for the detection of an organism of concern in a target food.
Effects of Food Matrices and Processing on Detection of the B Subunit of Ricin Using Enzyme-Linked Immunosorbent Assays
G. M. Orlowski1 , E. A.E. Garber2 , 1JIFSAN, UMD, College Park, MD, 2CFSAN, FDA, College Park, MD
Approximately 1 billion pounds of castor beans are produced annually. Castor beans are used as a source of oil, medicinal products, protein feed, ornamental landscaping and jewelry, and as a source of sebacic acid. Castor beans are also the source of the toxin ricin. Ricin is a protein dimer made up of an A subunit and a B subunit. The non-toxic B subunit was used as a surrogate to study the effects of food preparation on the detection of ricin.
Method:
Commercially available ELISA kits were used to examine qualitative and quantitative detection of the B subunit of ricin spiked at 0, 50, 125, 250, 500 and 1000 ppb in dairy products, juice, phosphate buffered saline, pasta, baked goods, and phosphate buffered saline (PBS). Associated with the preparation of these food matrices were the effects of freezing, baking, and boiling. Analysis of the data were based on a best fit exponential curve analysis (R-squared value of 0.98) of standards consisting of 0, 10, 50, 125, 250, and 500 ppb subunit B.
Results:
The highest levels of recovery were observed with uncooked pasta, followed by ice cream, infant formula, and juice. Cooking reduced the detection of the B subunit of ricin while freezing had little effect on the detection. The data showed a good correlation with the amount of subunit B spiked into the food, although the recovery varied with the level of spiking.
Detection Of Ricin In Food Matrices Using Electrochemiluminescence (ECL)-based Technology
E. A.E. Garber1 , T. W. O'Brien2 , G. Sigal3 , 1CFSAN, FDA, College Park, MD, 2Tetracore Inc., Gaithersburg, MD, 3Meso Scale Diagnostics, LLC, Gaithersburg, MD
Background: Ricin is a potent ribosome inactivating protein (RIP-2) present in beans of the castor plant, Ricinus communis. Recent events of food tampering have made the detection of ricin a serious concern.
Methods: Samples of juice, dairy products, soda, vegetables, bakery products, chocolate, and condiments were spiked with varying concentrations of ricin and analyzed using the 96-well format, Sector PRTM 100 electrochemiluminescence (ECL) detector manufactured by Meso Scale Diagnostics (MSD). Assay configurations including monoclonal or a polyclonal antibodies, and whether the samples and detector antibody were added sequentially or in combination in the capture step, were compared.
Results: The ECL-based detector was able to detect ricin in all samples at concentrations orders of magnitude less than what would be considered a health concern. Using a polyclonal antibody preparation, 0.04 ng/ml ricin was detected in analytical samples prepared from several foods. The simultaneous incubation of the samples with the detector antibody decreased the assay time considerably while still enabling the detection of ricin at levels considerably less than that which might pose a health concern.
Conclusions: ECL-based detection of ricin provides a sensitive, rapid one-hour alternative to ELISA technology for the detection of ricin in food.
Rapid Detection of Enterotoxigenic Escherichia coli - a Primary Agent of Infectious Diarrhea
M.A. Grant, Pacific Regional Laboratory Northwest, Bothell, WA
Enterotoxigenic E. coli (ETEC) is a leading cause of travelers diarrhea and causes over 300,000 deaths among children in developing nations annually. A method was developed for multiplex real-time PCR detection of ST and LT genes in ETEC. Contaminated produce is a common vehicle of ETEC infection and studies with laboratory-spiked produce indicated as few as 10 target cells could be detected. Several other foods representing varieties known to have caused outbreaks were spiked and enriched for 4 or 6 hours. Both ST and LT genes could be detected (Ct values ranging from 25.2-41.1) with the exception of hot sauce, in which a high acid content interferred with rapid detection. A procedure to allow enumeration of ETEC in <28 hours was also developed and is described.
Effect of Sample Preparation and Preenrichment Media on the Recovery of Salmonella from Cantaloupes, Mangoes, and Tomatoes.
T. S. Hammack1 , M. L. Johnson2 , A. P. Jacobson1 , W. H. Andrews1 , 1CFSAN, FDA, College Park, MD, 2CFSAN, FDA, College Park, MD (retired)
The relative effectiveness of buffered peptone water (BPW), lactose (LAC) broth, and Universal Preenrichment (UP) broth for the recovery of Salmonella from fruit rinses, whole fruit, and comminuted fruit was determined. In the first phase, the relative effectiveness of rinse and soak methods for the recovery of Salmonella from surface-contaminated cantaloupes, mangoes, and tomatoes was examined. Fruit were surface contaminated with Salmonella, aged, and then rinsed with portions of BPW, LAC broth, or UP broth. Portions of each rinse were preenriched in its respective broth. Individual whole fruit, in their remaining broth rinses (soak method), and the fruit rinse/broths (rinse method) were incubated overnight. The BAM Salmonella culture method was followed. Soaking was significantly more effective (p < 0.05) than rinsing for the analysis of fruit. The 3 broths were equivalent for cantaloupes and mangoes for both methods. For tomatoes, LAC broth was significantly less effective than BPW and UP broth (p < 0.05). In the second phase, the relative effectiveness of LAC broth, BPW, and UP broth for the recovery of Salmonella from comminuted fruit was examined. Test portions (25 g) were preenriched in the 3 broths. The BAM Salmonella culture method was followed. For cantaloupes, LAC broth was significantly less effective (p < 0.05) than UP broth and BPW. For mangoes, there were no significant differences among the broths, but BPW recovered a larger number of S.-positive test portions than did either LAC broth or UP broth. For tomatoes, there were no significant differences among the 3 broths.
Isolation of Yersinia pestis from Food using Immunomagnetic Capture.
D. E. Hanes, M. L. Saylor, M. H. Kothary, M. E. Lorenzo, B. D. Tall, CFSAN, FDA, Laurel, MD
Heightened concerns about bioterrorism have required that the FDA strengthen their methods for the isolation and identification of pathogens not traditionally associated with foodborne disease. One such pathogen is Yersinia pestis (Yp), the causative agent of plague. This study examines the use of the Pathatrix system of immunomagnetic capture for the isolation of Yp from foods. Immunomagnetic beads were coated with rabbit polyclonal antiserum raised to whole cells of Yp strain A1122. Food samples spiked with A1122 were placed in sterile stomacher bags and diluted 1:10 with Butterfield's phosphate dilution buffer. The samples were placed in the Pathatrix apparatus and circulating tubing placed into the stomacher bags. Antibody-coated immunobeads were introduced into the tubing and magnetically bound in place. The food samples were circulated past the immunobeads for 30 min at 28°C, after which the immunobeads were harvested and washed with 0.85% saline. Aliquots of the immunobeads were directly plated onto Sheep Blood Agar (SBA) and Yersinia selective agar (CIN), and the cultures incubated for 48 h at 28°C. Immunobeads were also inoculated into Brain Heart Infusion Broth as an enrichment culture. Following 24 h of incubation, the enrichment cultures was plated onto CIN and SBA and the plates incubated at 28°C for 48 h. Identity of suspect colonies subcultured from direct plating and enrichments were confirmed using lysis by an Yp specific phage and by PCR. PCR analysis was also performed on aliquots of the immunobeads as a rapid screening method for the presence of Yp. Yp was recovered from both the direct plating and the enrichment cultures on CIN and SBA. Colonies of Yp were more evident on CIN however there was a 10-100 fold decrease in recovery on CIN when compared to SBA. Yp was also detected in all samples using PCR analysis. These results indicate that immunomagnetic capture can be used to isolate and identify Yp from foods. These studies also will facilitate the development of a rapid method for the identification of Yp in food using immunocapture combined with PCR.
Comparison of Multi-Locus Sequence Typing, Pulsed Field Gel Electrophoresis, and Antibiotic Susceptibility Profiles for Characterization of Salmonella enterica serotype Newport Isolates from Human and Food Animal Infections and Retail Meat
H. C. Harbottle, S. Zhao, D. G. White, P. F. McDermott, R. D. Walker, CVM, FDA, Laurel, MD
Background: Several Salmonella enterica serotypes are a major source of food borne illness in the United States accounting for over 1.4 million cases of human illness yearly. Multi-drug resistant phenotypes of serotype Newport (MDR-AmpC) have emerged in animals and humans and have become a major public health problem. Pulsed field gel electrophoresis (PFGE) and antimicrobial susceptibility profiling methods are commonly used methods for studying microbial epidemiology and trends in antibiotic resistance of bacteria.
Methods: In this study, 82 S. serotype Newport isolates from human and animal infections and retail meat were analyzed by PFGE, antimicrobial susceptibility profiles, and multi-locus sequence typing (MLST) of seven genes, aroC, dnaN, hemD, hisD, purE, sucA, and thrA.
Results: PFGE profiling resulted in 44 fingerprint types, and five of these fingerprints were indistinguishable among animal and human isolates. Antimicrobial susceptibility testing results show that 37.8% of the 82 S. serotype Newport isolates were considered to be MDR-AmpC and 21 resistance profiles were identified. MLST resulted in 20 sequence types, with one sequence type encompassing 52.4% of the strains.
Conclusion: These results demonstrate that the MLST scheme employed did not add any discriminatory power to the profiling of these isolates, and discriminated poorly between isolates that were separated well by PFGE. In this study PFGE and antimicrobial susceptibility profiling were the most useful tools for discriminating between isolates and tracking Salmonella enterica serotype Newport infections in humans and animals.
E. coli resistant to third-generation cephalosporins show decreased cefquinome susceptibility
J. R. Hayes1 , L. L. English2 , P. J. Carter2 , T. A. Proescholdt2 ,P. F. McDermott2 , D. G. White2 , 1CVM, FDA, Rockville, MD, 2CVM, FDA, Laurel, MD
Background: Cephalosporins are an important class of antimicrobial agents in both human and veterinary medicine. The potential impact of cephalosporin use in the animal production environment on the prevalence of resistance in human clinical isolates is a public health concern.
Methods: To evaluate the prevalence of cephalosporin-resistant organisms on retail meats, 967 raw turkey, pork, beef, and chicken samples purchased from retailers in Iowa were surveyed for the presence of ceftazidime-resistant E. coli. These isolates were tested for susceptibility to a battery of other cephalosporins.
Results: Ceftazidime-resistant E. coli were isolated from 32% of all samples, with a prevalence of 8 to 63% within a given meat type. Minimum inhibitory concentration (MIC) distributions to the 4th generation cephalosporin, cefquinome, were similar across all meat types, ranging from ¡Ü0.06 to 1 µg/ml. The proportion of isolates that possessed resistant or intermediate MICs to the extended-spectrum cephalosporin, ceftiofur, ranged from 0% at the lowest observed cefquinome MIC to 100% of those at the highest MIC, with a mean value of 89% across all meat types at the highest cefquinome MIC. Increases in MICs to cefotaxime were similarly observed with a mean value of 56% across all meat types of those at the highest cefquinome MIC.
Conclusions: The cefquinome susceptibility profile of E. coli from retail meat products showed an absence of overt resistance to fourth-generation cephalosporins. However, increases in cefquinome MIC values were associated with resistance to other third-generation cephalosporins.
Comparison of antimicrobial susceptibility profiles among Salmonella spp. recovered from retail poultry, NARMS 2002 vs. 2003.
S. K. Hubert, S. L. Ayers, A. Glenn, E. Hall-Robinson, P. F. McDermott, L. A. Walker, T. A. Proescholdt, S. Zhao, S. Friedman, J. Abbott, R. D. Walker, D. G. White, CVM, FDA, Laurel, MD
Background: The Retail Meat component of the National Antimicrobial Resistance Monitoring System (NARMS) is a collaborative effort between CDC, FoodNet and FDA. This program monitors antimicrobial susceptibility patterns among select foodborne bacterial pathogens and commensal organisms.
Methods: Salmonella were recovered from a monthly sampling of chicken breasts, ground turkey, ground beef and pork chops purchased from grocery stores in 6 participating FoodNet sites (CT, GA, MD, MN, OR, TN) in 2002 and an additional 2 sites in 2003 (CA and NY). Salmonella isolates were serotyped and tested against a panel of 16 antimicrobials following CLSI standards.
Results: Salmonella contamination of ground turkey increased from 11.5% in 2002 to 13.3% in 2003. There was a decrease in Salmonella contamination of chicken breasts between 2002 and 2003, 9.7% vs. 9.3% respectively. Commonly observed resistance phenotypes among Salmonella isolates recovered from ground turkey during 2002 and 2003 were to streptomycin (38% vs. 46%), tetracycline (55% vs. 40%), and sulfamethoxazole (20% vs. 33%). Resistance among isolates recovered from chicken breasts was most often observed to ampicillin (17% vs. 35%), cephalothin (13% vs. 30%), and tetracycline (33% vs. 29%) between 2002 and 2003, respectively. There were increases in the number of chicken isolates resistant to ceftiofur (10% vs. 26.5%) between 2002 and 2003. No resistance was observed to amikacin, ceftriaxone, or ciprofloxacin among poultry isolates.
Conclusions: The first 2 years of retail meat testing in NARMS showed comparable data among Salmonella antimicrobial susceptibilities, with the exception of ß -lactam antimicrobials where increases in resistance were observed.
Whole genome optical maps of E. coli O157:H7 and related pathogens: investigating strain diversity for microbial forensic applications
S. A. Jackson1 , M. L. Kotewicz1 , W. Jiang2 , J. Henkhaus2 , A. Briska2 , C. W. Dykes2 , J. E. LeClerc1 , T. A. Cebula1 , 1CFSAN,FDA, Laurel MD, 2OpGen, Inc Madison WI
Background: In order to develop forensic tools that will allow the detection, identification, and tracing of pathogenic strains of Escherichia coli O157:H7, optical mapping of whole genomes has been used to compare individual strains. Enteric bacteria have had sufficient evolutionary time to accumulate unique sequence signatures and chromosomal changes. These markers should make individual strain identification possible.
Methods: Single molecules of intact bacterial chromosomes were immobilized on a derivatized glass surface and digested with the restriction enzyme BamH I. Restricted chromosomes were fluorescently stained and photographed using a fluorescent microscope interfaced with a digital camera. Image analysis was performed to quantitate the length of each restriction fragment. Whole chromosome optical maps were assembled from approximately 30 independent molecules.
Results: Seven enteric genomes were optically mapped. Large inversions, insertions, and deletions were observed by optical mapping and in many cases have been confirmed by other molecular techniques. These polymorphisms demonstrate the extent of diversity present within the O157:H7 pathogen group. Comparisons with maps from other E. coli serotypes indicates that optical mapping can also provide identifiers for distinguishing pathogenic groups within E. coli.
Conclusions: Optical maps of E. coli O157:H7 genomes demonstrated individual characteristics for each genome examined. Optical genome maps are a powerful means for microbial strain identification and will aid in strain attribution for microbial forensic applications.
Microbial forensics of foodborne pathogens: An FDA Collaboration with the Department of Homeland Security
T. A. Cebula, E. W. Brown, P. Davis, K. Dudley, A. Hayford, S. A. Jackson, J. Jean-Giles, M. L. Kotewicz, B. Li, M. K. Mammel, T. J. Mays, K. L. McCutchan, A. Mukherjee, I. R. Patel, W. L. Payne, A. Perlloni, D. Roberson, J. E. LeClerc, OARSA, CFSAN, Laurel, MD 20708
Background: Microbial forensics is a new scientific discipline that seeks to analyze and interpret microbial data in order to establish a link between a microbe and a biocrime or bioterrorist event. For forensic investigation of foodborne pathogens, tools that will allow the detection, identification, and tracing of pathogens such as Escherichia coli O157:H7, Shigella, and Salmonella are needed. To accomplish this, the Office of Applied Research and Safety Assessment of FDA's Center for Food Safety and Applied Nutrition is engaged in collaboration with the Department of Homeland Security's National Bioforensic Analysis Center (NBFAC).
Methods: Research was directed at developing methods that would establish attribution of a suspect strain of E. coli O157:H7 for its assignment to a source of known origin with a high degree of scientific certainty.
Results: A reference collection of E. coli O157:H7 isolates was established and validated by microbiological and molecular assays. In silico genomic comparisons, MLST analyses, DNA microarray strategies, and optical mapping of whole chromosomes were used to discover polymorphisms that would serve individual strain discrimination. Cell phenotyping using the Biolog PM system provided further markers to differentiate the E. coli O157:H7 pathogenic group.
Conclusions: The results established that isolates of foodborne pathogens can likely be identified at the strain level without the need for whole genome sequencing. More significantly, these approaches may be used to measure the individuality of a strain against the overall diversity within its pathogen group, a prerequisite for any bioforensics investigation.
Recovery of Bacillus anthracis from two artificially contaminated foods using FERN methodology
S. M. Madson1 , E. D. Gonzales1 , L. T. Michel1 , Z. A. Miller1 , P. L. Dexter1 , M. Z. Thomas1 , K. S. Kreuzer1 , C. L. Burns1 , J. Sofos2 , 1FDA, 2Colorado State University
BACKGROUND: A method for use by Food Emergency Response Network (FERN) laboratories to detect Bacillus anthracis in foods was developed by the Center for Food Safety and Applied Nutrition (CFSAN). This study evaluated this method for use in rehydrated infant formula and canned tuna salad.
METHODS: Six samples (plus controls) of each product were inoculated with B. anthracis (Pasteur strain, ATCC4229) spores at low (100), medium (102), and high (105) cfu/mL or g levels. Heated (65°C for 30 minutes) and unheated sample homogenates were screened using real-time PCR (rt-PCR) and by spread-plating onto selected media. Sample aliquots were also enriched overnight in trypticase soy broth with polymixin B, plated onto the same media, and screened using rt-PCR.
RESULTS: Presence of the organism was confirmed in all inoculated enrichments except for two samples of infant formula inoculated with the low level. At high inoculum, the organism was detected with rt-PCR and confirmed culturally after direct plating of all samples of both products. At low levels, the organism was not recovered by direct plating and rt-PCR. Two of six infant formula samples inoculated at medium levels were positive by rt-PCR, while direct plating found five of six positive. The medium inoculum level in tuna salad was recovered in four of six samples by plating and none by rt-PCR.
CONCLUSIONS: Enrichment allowed recovery of even low levels (1 spore/ml or g) of the organism, while rt-PCR detected the organism if high levels were present. The results of this study should improve FDA's readiness to deal with a possible terrorist attack.
Tracking E. coli O157:H7 strains by genomic comparisons
M. K. Mammel, A. Hayford, E. W. Brown, J. E. LeClerc, T. A. Cebula, OARSA, FDA, Laurel, MD
Background: It is imperative to identify single nucleotide polymorphisms (SNPs) that serve as molecular markers for closely related strains of Escherichia coli O157:H7 if one wishes to differentiate these strains.
Methods: We compared the published sequences of E. coli O157:H7 EDL933 and Sakai strains, E. coli K-12 (MG1655), and E. coli CFT073 to find regions of genetic variation. A BLAST database was constructed for each gene from all four genomes and each gene was blasted against the database to find matches. Matched genes were aligned and evaluated for informative SNP sites, which showed a difference between the two O157:H7 sequences and were flanked by conserved sequences suitable for primer binding. Seventeen regions containing candidate SNPs were sequenced in a set of 16 independent isolates of E. coli O157:H7 and ten closely related serotypes. Pyrosequencing assays for each verified SNP were used to test 75 E. coli O157:H7 strains.
Results: Five useful SNPs for distinguishing the test strains were identified. The results of pyrosequencing assays on these SNP sites divided the strain collection into four groups. Analysis of roi, a prophage gene encoding a DNA binding protein, was also used as a molecular marker. In strains carrying the prophage gene, roi sequences fell into three diverged allele types, which further discriminated strains within the groups.
Conclusions: The discovery of informative sites by in silico genomic comparisons and assay of a diverse set of test strains formulates an effective approach for identifying molecular signatures for individual strains of E. coli O157:H7.
Temperature Affects on Lateral Flow Devices Used in the Detection of Ricin in Food Matrices
M.A. McLaughlin, CFSAN, FDA, College Park, MD
Ricin, the toxin derived from the castor bean (Ricinus communis), is a potential threat to the security of the US food supply. The ability to rapidly and reliably detect the presence of this select agent in foods is essential to the FDA's food security mission. This project involved the effect of temperature on the operation of lateral flow devices (LFDs) adopted for use by our Agency. We have studied the sensitivity of these strips at 4o, 20o, 37o and 43o C. Evaluations of these devices have found that the sensitivity is substantially increased when developed at 37oC. The sensitivity of these LFD's are listed in the company literature as 50 ppb. In our labs we have been able to demonstrate reliable LOD's down to 20 ppb at room temperature. We have found that when incubated at 37oC during development, the sensitivity can decrease to the 5 ppb range, a 4-fold increase in sensitivity. In contrast, at 4oC the sensitivity suffers so that only by using a strip reader can you detect the agent at 50 ppb. This study demonstrates that temperature can have a significant effect on the operation of LFD's and that this should be taken into consideration when conducting these tests in the field.
Recovery of Enterobacter sakazakii and Klibsiella pneumonia from infant formula by filtration and cultivation on a unique infrared-transparent hydrophilic membrane filter
M. M.. Mossoba, S. F. Al-khaldi, F. S. Fry, CFSAN, FDA, College Park, MD
Introduction: Enterobacter sakazakii has been associated with neonate deaths and outbreaks of a rare form of infant meningitis and other diseases. Reported mortality rates were high and ranged from 40-80% among immuno-compromised infants. Therefore, the presence of E. sakazakii in powdered infant formula milk (IMF) is of particular concern. Several methods for the isolation of E. sakazakii from dehydrated powdered infant formula are currently available.
Methods: In the present study, a new reagent-free infrared (IR) microspectroscopic procedure was developed that can identify presumptive E. sakazakii colonies four days sooner than the 2002 FDA/CFSAN method, and hence is potentially an excellent screening tool.
Results: The IR procedure entails the use of a novel disposable IR-transparent hydrophilic (IR-TH) membrane filter that can be used (i) to filter aqueous bacterial suspensions, as well as (ii) to grow bacterial colonies when the membrane is placed over BHI agar medium and incubated at 37°C. Because the levels of E. sakazakii in IFM products are low ranging from 0.36 to 66.0 cells/100g, samples were inoculated during pre-enrichment at similar levels. A mixture of two bacterial species were used in this study E. sakazakii and Klibsiella pneumonia (reportedly another potential contaminant) to inoculate the infant formula. A 100 µl portion of a diluted reconstituted IMF sample was filtered under vacuum. Subsequently, the IR-TH membrane filter was placed on a BHI agar plate and incubated at 37°C. The incubation times used varied between 4 hrs and overnight (16 hrs). An alternative approach was also explored in which contact deposition microarray deposition was used to print and subsequently grow microarrays of microcolonies on IR-TH membrane filters.
Conclusions: Because this unique membrane is transparent to infrared light, isolated microcolonies (or arrays of microcolonies) of bacterial cells could then be rapidly fingerprinted by IR microspectroscopy. IR data on the inoculation, recovery, and identification of a mixture of E. sakazakii and K. pneumonia are reported here.
An assessment of preharvest source tracking and transverse movement of Salmonella Heidelberg population on a turkey grow-out facility
R. Nayak1 , T. M. Stewart2 , C. E. Cerniglia1 , 1NCTR, FDA, Jefferson, AR, 2University of Tennessee, Memphis, TN
Background: An understanding on the origin and movement of Salmonella on turkey farms can be useful in implementing HACCP-based critical control measures to interrupt the transmission cycles.
Methods: A total of 110 S. Heidelberg strains were isolated from various sources on a turkey farm. The genetic diversity was mapped by XbaI-PFGE restriction profiles. The similarity matrix and clustering dendrogram type were calculated using the Dice band matching coefficient and UPGMA algorithm, respectively.
Results: Heidelberg genotypes were clustered (>80% homology) in four groups (G1-G4). Identical G1 and G3 genotypes originated from turkey cecae, litters, swabs and drinkers, suggesting that the birds were shedding Salmonella in the pens and functioning as a "vector" in cross colonizing pens. Furthermore, identical G1 genotypes were observed in differently sampled pens and locations of the facility, suggesting transverse movement of Heidelberg genotypes between pens and within the facility. Most G2 genotypes originated from turkeys at weeks 2, 10 and 18, suggesting persistence of Heidelberg during the grow-out period. Overall, distinct genotypes from G1, G3 and G4 were mostly isolated from weeks 10 and 18 and that of G2 from week 2, suggesting prevalence of certain genotypes depends on the stage of the production cycle, and genotypes may undergo changes in their genetic clonality with the grow-out period.
Conclusions: Heidelberg strains appeared to originate from the birds and cross-contaminate other birds, drinkers, and litter samples by direct and/or indirect contact. Dissemination of Heidelberg serovars can be source specific and may depend on the stage of production cycle.
Salmonella population dynamics in preharvest turkey environment: In vitro antimicrobial susceptibility phenotypes of multi-drug resistant serovars
R. Nayak1 , T. M. Stewart2 , S. L. Foley3 , C. E. Cerniglia1 , 1NCTR, FDA, Jefferson, AR, 2University of Tennessee, Memphis, TN, 3University of Central Arkansas, Conway, AR
Background: The widespread use of antibiotics in the human and veterinary medicine may contribute to the emergence of multi-drug resistant Salmonella.
Methods: Salmonella serovars (n=145) were isolated from 4 turkey flocks (F5-F8). The antimicrobial susceptibility profile of 111 Heidelberg, 14 Senftenberg, 10 Muenster, 3 Anatum, 2 Worthington and 5 unidentifiable "roughs" were determined by disk diffusion assay (DDA) and "manual" broth dilution assay (BDA). The efficacy of these methods was compared with the SensiTitre (ST) method.
Results: All serovars were resistant to erythromycin, novobiocin, rifampin and sulfamethoxazole. In F5, Heidelberg showed additional resistance to gentamicin, spectinomycin, streptomycin and tetracycline, while Anatum were resistant to streptomycin and tetracycline. In F6, Senftenberg were resistant to cephalothin and spectinomycin. Salmonella was undetected in F7. In F8, "roughs" and Muenster were resistant to gentamicin, spectinomycin, streptomycin and tetracycline. A high degree of correlation (>90%) was observed between DDA/ST and BDA/ST for all antibiotic with the exception of kanamycin (49% between DDA and ST) and tetracycline (77% between BDA and ST). This deviation may be due to variation in the potency of each antibiotic or the solubility and diffusion properties of antibiotics. Antibiogram showed that 80% of the isolates were resistant to 4 to 5 antibiotics, while remaining 20% were resistant to 6 to 12 antibiotics.
Conclusions: Most S. Heidelberg strains showed resistance profile similar to multi-drug resistant S. Typhimurium DT104, suggesting the possibility of the "emergence" of S. Heidelberg as the next generation multi-drug resistant Salmonella with the potential to caused food borne outbreaks.
Detection of Staphylococcal Enterotoxins in Various Matrices with Commercial Kits
C. W. Noah1 , J. N. Sofos2 , 1FDA Denver District Laboratory, Denver CO 80225, 2Colorado State University, Fort Collins, CO 80523
Four commercial rapid immunoassay-based test procedures were evaluated for potential use in detecting Staphylococcal enterotoxins in foods. The foods chosen were liquids (i.e., bottled water, orange juice, milk, soft drink and infant formula) that could be used as agents for a bioterrorism event, as well as solid food products (i.e., cheese, pork chops and coleslaw). The procedure tested included: the TECRA Staphylococcal Enterotoxin Visual Immunoassay (SET-TECRA), which qualitatively detects enterotoxins A, B, C1, C2, C3, D, and E concurrently; the Oxoid Staphylococcal Enterotoxin Reverse Passive Latex Agglutination (SET-RPLA) kit, which qualitatively detects enterotoxins A, B, C, and D individually; and two lateral flow devices (SET-LFD) which specifically and qualitatively detect enterotoxin B [one manufactured by the Department of Defense (DOD), and the other by Tetracore SEB Biothreat Alert (Tetracore)]. Post-extraction samples (liquids and solids) were centrifuged, filtered or pH adjusted according to the specific method protocol and then inoculated with enterotoxin. Samples of cheese, coleslaw, and pork chops were also inoculated before extraction (pre-extracted) to test for the efficiencies of detection of the extraction procedures compared to post-extraction detection results. Overall, the SET-TECRA ELISA demonstrated slightly better sensitivity in detection than the SET-RPLA. Of all the enterotoxin types, C1 and D were more easily detected using both the SET-TECRA and the SET-RPLA tests. The SET-LFD kits were the least sensitive of the tests, only detecting enterotoxin B. The SET-LFD test kit produced by the DOD was significantly more sensitive than the Tetracore SET-LFD test kit.
A real time PCR Assay for the Detection of Toxigenic Vibrio cholerae
G. M. Blackstone1 , M. D. Bowen2 , P. McCready3 , M. C. Vickery4 , J. L. Nordstrom1 , R. Meyer2 , A. DePaola1 , 1FDA, 2CDC, 3LLNL, 4Cepheid
Vibrio cholerae, the etiological agent of cholera, causes severe diarrheal disease in thousands of people each year in developing countries. It is the subject of extensive testing of shrimp produced and exported from these countries. Strains of toxigenic V. cholerae contain DNA from the filamentous bacteriophage CTXΦ, which carries the ctxAB genes encoding cholera toxin (CT). FDA currently has a conventional PCR assay for the detection of CT producing strains of V. cholerae; however, we report the development of a real time PCR assay to detect CT producing strains more rapidly and with greater sensitivity. Real time PCR imparts an added degree of specificity since it requires the annealing of two target-specific primers plus an internal probe in order to generate a positive signal. This assay was tested against isolated DNA from soil samples collected from various locations across the US, eukaryotic DNA isolated from human, mosquito, flea, Drosophila, chicken, murine, bovine, feline and canine sources, and prokaryotic DNA from Bacillus spp, Candida albicans, Erwinia herbicola, E. coli, and representative strains of Vibrio spp. Only Vibrio strains known to contain the ctx gene generated a fluorescent signal, confirming the specificity of the assay. In addition, the assay was found quantitative across a five-log dynamic range down to 1 colony forming unit per reaction in pure culture. To test the robustness of this assay, oyster, sediment, and seawater, from Mobile Bay, AL and respective spiked controls were analyzed by real time PCR and traditional culture methods. No toxigenic V. cholerae strains were identified in the thirteen environmental samples, nor among any of the control DNA samples known to lack the ctx gene. This assay provides a highly specific, sensitive, and rapid detection (less than 1h) of toxigenic strains of V. cholerae.
Comparative genomic analysis of E. coli O157:H7 using a novel high-density, high-throughput DNA microarray platform
S. A. Jackson1 , M. K. Mammel1 , I. R. Patel1 , T. Albert2 , J. E. LeClerc1 , T. A. Cebula1 , 1OARSA, DMB, Laurel, MD, 2Nimblegen Systems, Inc., Madison, WI
Background: Tiling arrays are a novel DNA microarray-based platform that utilizes an overlapping probe design for the detection of different types of DNA polymorphisms, including single nucleotide changes. We have used tiling arrays to interrogate the genomic diversity that exists among E. coli O157:H7 pathogens.
Methods: DNA tiling arrays were used to interrogate genomic diversity among a diverse collection of E. coli O157:H7 isolates. The high density tiling arrays are capable of interrogating relatively large sections of bacterial genomes (~1%) and are informative on the type (SNPs, indels, etc.) and precise locations of polymorphisms occurring in the test strains.
Results: This tiling strategy has been effective in detecting changes at ~120 individual loci in 60 kb from each of 40 unique isolates of E. coli O157:H7. We demonstrate the usefulness of tiling arrays to detect deletions, substitutions, and SNPs within the genomic regions analyzed. These polymorphisms serve as biomarkers that can be utilized to differentiate individual strains.
Conclusions: The tiling strategy described here provides a rapid and semi-high throughput technique for interrogating relatively large regions of bacterial genomes for novel polymorphisms. Tiling arrays provide a genomic profile for individual strains and, taken together, may be used to assess the overall genomic diversity within the E. coli O157:H7 pathogen group.
Bovine Immune Globulins are an Effective Post-Exposure Prophylaxis against Anthrax Parenteral & Aerosol Spore Challenge in Mice
J. Mellquist-Riemenschneider 1 , K. Stabler 2 , T. Sathiyaseelan 2 , K. Haffer 2 , J. Jiao 2 , M. Porter 2, V. Grippe 1 , G. Lee 1 , T. Merkel 3 ,B. Golding 3 , J. Robl 2 , 1 CBER, FDA, Bethesda, MD , 2 Hematech Sioux Falls, SD, 3 CBER, FDA, Bethesda, MD
Background: We have developed a novel platform with transgenic cattle that express human polyclonal antibodies and plan to develop antibodies against various bioterrorism agents, including anthrax. As a first step at characterizing anti-anthrax antibodies developed in a bovine system, we have generated and characterized purified bovine anti-anthrax immune globulin (IgG) and have shown that they protect against different routes of spore challenge.
Methods: Cattle were immunized with various anthrax antigens. Serum was assayed by ELISA and toxin neutralization assay (TNA) and the IgG were purified. A/J mice were challenged by the intraperitoneal (IP) or aerosol route with approximately 1x10 6 or 1-5x10 6 Sterne strain spores, respectively. Approximately 4h after challenge, mice were treated with bovine IgG against anthrax or a negative control IgG +/- Ciprofloxacin (Cipro).
Results: The immunized cattle showed robust antibody responses by ELISA and the TNA revealed that bovine antibodies have high neutralizing activity. Mice that were challenged by IP injection and treated with anti-PA IgG showed a 90% protection, when combined with Cipro treatment. In contrast, a negative control IgG given with Cipro treatment showed only 50% protection. Mice that were challenged by aerosol exposure showed 90% protection when anti-PA IgG was administered with Cipro, whereas only 10% of mice treated with a negative control IgG survived.
Conclusions: Our studies show that bovine IgG can neutralize anthrax toxin, in a cell-based TNA and in mice challenged with anthrax by different routes. IgG purified from bovine serum have the potential to be efficacious therapeutics against bioterrorism agents such as anthrax. Ongoing studies are characterizing similar anti-anthrax human IgG.
Detection of Peanut Protein in Chocolate using Mass Spectrometry
K. J. Shefcheck, J. H. Callahan, S. M. Musser, CFSAN, FDA, College Park, MD 20740
Background: Dark chocolate has been a problematic matrix for peanut allergen detection. We propose to develop a method using mass spectrometry to detect peanut protein in dark chocolate using peanut allergens as markers.
Methods: Six dark chocolate samples containing known concentrations of peanut protein were used: A (10 ppm), B (100 ppm), C (50 ppm), D (20 ppm), E (0 ppm) and F (2 ppm). One hundred milligrams of each chocolate sample was incubated for 2 days in 2.5 mL of 8 ug/mL trypsin in 50 mM ammonium bicarbonate at 37 °C with vigorous shaking. The resulting mixture was acidified and centrifuged at 8000 rpm for 30 minutes. The supernatant was filtered through a 100 kDa MWCO centrifugal filter and further purified with Strata-X multimode solid phase extraction media. The peptides were eluted off the SPE media with 500 uL of 70% acetonitrile, 0.1% formic acid. The samples were characterized using RP-HPLC-MSMS.
Results: We used four peptides from Ara h 1 (606.7, 688.9, 786.9, 870.0) as markers for peanut protein. Using an Ara h 1 standard and tandem mass spectrometry on a Q-TOF, three secondary marker product ions were identified for each peptide. Multiple reaction monitoring (MRM) on a triple quadrupole was then used to detect and confirm peanut protein in chocolate. Using MRM, the four digest peptides were observed in the samples A, B, C, and D. Additional experimentation is underway to decrease the detection limit of this method using additional cleanup and nanoflow LC/MS.
An Experimental Model Assessing Chemical-Microbial Interactive Neurobehavioral Toxicity
T. Sobotka1 , D. Quander1 , S. Long1 , M. Smith1 , C. N. Barton2 , L. H. Garthoff1 , 1FDA, CFSAN, OARSA, MOD-I, Laurel MD, 2FDA, CFSAN, OSAS, College Park MD
Background: Chemical toxicity may be influenced by interaction with common bacterial toxins. This proof-of-principle study assesses neurotoxic interactions of lipopolysaccharide (LPS) and deoxynivalenol (DON).
Methods: Male rats were given two ip injections, the first with saline or LPS (83 mcg/kg) and the second one hour later with saline or DON at 2.5, 5 or 10 mg/kg. Body weight, food consumption, saccharin preference, activity, auditory startle, pre-pulse startle inhibition, and spatial water maze performance were measured to index neurobehavioral toxicity.
Results: Overt signs of toxicity including hyperalgesia, stilted gait, paralysis, and death were exhibited by 2/10 rats in the LPS/DON5 and 2/10 in the LPS/DON10 groups. Preliminary data analysis indicated body weights and food consumption to be decreased by DON and LPS with no differences between individual or combined dosing. Saccharin preference was decreased by LPS and DON alone or in combination, but combined dosing produced a more persistent effect. Motor activity decreased after either DON or LPS with no differences between individual or combined dosing. LPS alone had no apparent effect on auditory startle but DON markedly increased startle responding in dose related fashion. This DON effect was diminished when combined with LPS exposure. Prepulse inhibition of startle and spatial water maze performance, including swim distance, time to escape and swim speed, appeared unaffected by any experimental treatment.
Conclusions: LPS and DON administered in combination may interact synergistically to precipitate clinical neurotoxicity and prolong depression of saccharin preference but their toxic interaction does not appear to disrupt primary behavioral functions.
Anthrax lethal toxin alters chemokine release by human lung microvascular endothelial cells.
A. D. Steele, J. M. Warfel, F. D'Agnillo, CBER, FDA, Bethesda, MD
Vascular endothelial dysfunction is thought to play a prominent role in anthrax pathogenesis. In this study, we examined the effect of anthrax lethal toxin (LT) on chemokine production and leukocyte recruitment in endothelial cell cultures. Primary human lung microvascular endothelial cells were treated with anthrax lethal factor (LF), protective antigen (PA), or both (LT) in the presence or absence of tumor necrosis factor alpha (TNFα). Chemokines were measured by ELISA and an antibody-based protein array was utilized to analyze 120 cytokines, chemokines, and growth factors. Neutrophil and monocyte transmigration across endothelial monolayers grown on 3µm transwell inserts was also examined. As expected, TNFα increased the expression of chemokines including CCL2 (MCP-1), CCL5 (RANTES), and CXCL8 (IL-8). LT dramatically inhibited cytokine-induced CXCL8 release by 4 hours and CCL2 and CCL5 release by 24 hours. Treatment with LF, PA, or the combination of an inactive LF mutant and PA did not inhibit cytokine-induced chemokine expression. Interestingly, LT alone decreased the production of CXCL8 along with other cytokines. Of note, LT inhibited the release of tissue inhibitor of metalloproteinase (TIMP-2) which could be significant given the metalloproteinase activity of LT. LT also decreased monocyte and neutrophil transmigration through TNFα-stimulated endothelial cells. These findings suggest that LT may play a prominent role in the progressive stages of anthrax infection by disrupting endothelial inflammatory activation and impairing the ability to combat infection.
Characterization of Virulence Factors Expressed by Vibrio alginolyticus Isolates Obtained from Teleosts and Elasmobranchs Housed in a Public Aquarium and Aquaculture Facility.
B. D. Tall1 , M. H. Kothary1 , B. A. McCardell1 , S. Zhao1 , J. Abbott1 , P. Whittaker2 , S. K. Curtis2 , J. Arnold3 , HACU Intern Team1 , JIFSAN Intern Team1 , 1U.S. FDA, Laurel, MD 20708, 2U.S. FDA, College Park, MD 20740, 3Nat. Aquarium, Baltimore, MD 21202.
Diseases caused by Vibrio species have greatly forged the management practices associated with aquaculture and public health. Of primary concern is the potential for the transmission of zoonotic pathogens from aquacultured seafoods to humans. One species, Vibrio alginolyticus, causes illness in a variety of marine seafood species; and can also cause septicemia, wound infections and gastroenteritis in humans. A study was initiated to reveal the types of virulence factors expressed by 23 isolates obtained from various fish species and sources. The organisms were isolated and identified using standard methods as well as API20E and MIDI microbial identifications systems. The strains were characterized further by pulsed field gel electrophoresis (PFGE) and plasmid analyses. Additional information was collected on surface hydrophobicity of the organisms; and the expression of hemagglutinins, hemolysins, cytotoxins, enterotoxins, proteases, and siderophores. PFGE analysis using SwaI revealed 22 different genotypes among the strains. 91% of the strains possessed plasmids. The cell surfaces of all strains, except for one, were hydrophilic; and 66% of the strains could agglutinate sheep erythrocytes (RBCs). Only one isolate was hemolytic for sheep, chicken and rabbit RBCs. Another single isolate was hemolytic for chicken, rabbit, guinea pig, and calf RBCs. All other isolates were hemolytic for both chicken and rabbit RBCs. Only one isolate exhibited caseinolytic activity, while all isolates produced siderophores. Results using Chinese hamster ovary (CHO) cells in tissue culture showed that some of the strains produced factors that elongated CHO cells, but only a few isolates produced factors that caused rounding and/or death of the cells. In summary, these results demonstrate that the pathogenic V. alginolyticus strains isolated from ill fish are genetically diverse; that most of the strains express multiple virulence factors, including hemagglutinins, hemolysin(s), and siderophores; and that the organisms also produce other factors that are cytotonic and cytotoxic toward CHO cells.
| Sigma Xi Outstanding Student Poster Award - 2005 FDA Science Forum - Nicholas Fico |
Determination of Unknown Toxicants in Flour Tortilla by Sequential Extraction: Preliminary Report
S. Trujillo1 , N. Fico2 , H. Njapau1 , C. R. Warner3 , B. J. Canas1 , D. L. Park1 , 1Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Plant and Dairy Foods, Division of Natural Products, 5100 Paint Branch Parkway, College Park, MD 20740, 2JIFSAN, University of Maryland, 0220 Symons Hall, College Park, MD 20742, 3Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Food Additive Safety, Division of Chemistry Research & Environmental Review, 5100 Paint Branch Parkway, College Park, MD 20740
Background: Foodborne outbreaks of unknown origin occur frequently. In the U.S. 68% of the reported foodborne-disease outbreaks from 1993 to 1997 were of unknown etiology. Although the majority of foodborne outbreaks are associated with the presence of pathogenic microorganisms, some of the outbreaks may be caused by chemical substances.
Methods: Flour tortillas associated with a foodborne outbreak, tortillas from the same company as those associated with the outbreak, and two tortillas from a local supermarket were tested for toxic components using sequential chemical extraction and partitions and enzymic digestion. Fractions from each set were subjected to toxicological screening using Bacillus megaterium (NRRL B1368) and brine shrimp (Artemia spp.) assays.
Results: After enzymic digestion, the toxicity of the outbreak tortillas was eliminated, whereas the toxic effects were only partially removed in fraction from control tortillas, in the Bacillus megaterium assay. High mortality was observed in the acid/base treatment and enzymic digestion fractions of outbreak-associated and control tortillas in the brine shrimp assay. Low toxic potentials were evident in organic and methanolic fractions. Propionic acid from the antimycotic calcium propionate in the outbreak-associated tortillas has been found at levels five to ten times the amounts found in locally purchased tortillas. Also, the outbreak-associated tortillas have the equivalent of 1 to 3 ppm potassium bromate.
Conclusions: Preliminary results indicate that no distinct differences in potentially toxic components between the outbreak-associated and control tortillas were observed. Further investigation is required to determine the cause of the outbreak and the apparent toxic potentials in both control and outbreak-associated tortillas.
CFSAN Outbreak Surveillance Database
C. Copty, E. Elliot, J. Guzewich, G. Henderson, T. Hill, K. Klontz, P. McCarthy, S. McGarry, M. Ross, J. Sanders, D. Street, B. Timbo, K. Vierk, CFSAN, FDA, College Park, MD
Background:
Each year multiple foodborne illness events associated with FDA-regulated products are reported.
Methods:
A model foodborne illness surveillance system was developed by the Epidemiology and Emergency Coordination and Response teams to efficiently capture and retrieve information related to foodborne illness. Events are reported to FDA by CDC and State/Local Health Departments and are entered into the database if an FDA-regulated product is implicated in causing human illness. Epi Info™ (version 3.2.2) and SAS™ (version 8.02) software are used to enter and conduct statistical analysis of outbreak data.
Results:
In 2004, a total of 40 events were classified as outbreak-related, with an estimated 1,801 illnesses, 65 hospitalizations and 4 deaths. Bacterial infection accounted for 65% of the outbreaks, followed by chemical or toxic agents (15%), parasitic infection (7.5%), viral infection (5%), and unknown (7.5%). The majority of illnesses reported were due to bacterial infection (71.9%). Salmonella was the leading bacterial pathogen and accounted for 45% of the illnesses, 33% of hospitalizations, and 25% of the deaths. Produce accounted for the greatest number of both outbreaks (32%) and illnesses (60%). Seafood accounted for 25% of the outbreaks, followed by dairy (15%), and processed foods (12.5%).
Conclusion:
Using EpiInfo™ software, we were able to relatively quickly develop a useful database for collecting information from outbreaks. This outbreak database allows for timely and efficient data extraction for research purposes, trend analysis, and insight into the factors that are related to foodborne illness that is needed in the regulatory setting.
Anthrax Lethal Toxin Induces Human Endothelial Barrier Dysfunction
J. M. Warfel, A. D. Steele, F. D'Agnillo, CBER, FDA, Bethesda, MD
Background: Systemic anthrax infection is characterized by hemorrhages and pleural effusions, suggesting the involvement of altered vascular permeability in the disease pathogenesis. We examined the effect of anthrax lethal toxin (LT), a major virulence factor of Bacillus anthracis, on the barrier function of primary human lung microvascular endothelial cells. We also examined the distribution patterns of cytoskeletal actin and vascular endothelial-cadherin (VE-cadherin), both involved in barrier function regulation.
Methods: Endothelial monolayers cultured on porous membrane inserts were treated with varying concentrations of lethal factor (LF), protective antigen (PA), or both (LT). Barrier function was assessed by measurement of transendothelial electrical resistance (TEER) and permeability to fluorescently labeled albumin. F-actin and VE-cadherin distribution was monitored by immunofluorescence microscopy.
Results: LT induced a concentration- and time-dependent decrease in TEER that correlated with increased permeability to fluorescently labeled albumin. LT also produced a marked increase in central actin stress fibers and significantly altered VE-cadherin distribution. Treatment with LF, PA, or the combination of an inactive LF mutant and PA did not alter barrier function or the distribution of actin or VE-cadherin. LT-induced barrier dysfunction was not dependent on endothelial apoptosis or necrosis. Treatment with chemical inhibitors of the p38, JNK and ERK signaling pathways, either individually or in combination, did not reproduce the effects of LT.
Conclusions: The present findings support a possible role for LT-induced barrier dysfunction in the vascular permeability changes accompanying systemic anthrax infection.
Quantitative determination of thallium binding to Prussian Blue
Y. S. Yang1 , J. J. Progar2 , C. R. Brownell1 , N. Sadrieh3 , J. C. May2 , E. Leutzinger3 , D. A. Place3 , E. P. Duffy3 , F. Houn3 , S. A. Loewke3 , M. M. Nasr3 , A. S. Hussain3 , M. A. Khan1 , R. C. Lyon1 , P. J. Faustino1 , 1CDER, FDA, Silver Spring, MD, 2CBER, FDA, Rockville, MD, 3CDER, FDA, Rockville, MD
Background: Prussian Blue (PB) is the Active Pharmaceutical Ingredient (API) of Radiogardase which is the first approved drug product for treatment of known or suspected internal contamination with radioactive cesium, thallium, or non-radioactive thallium. The purpose of this study is to determine the in vitro thallium binding capacity of PB, especially to focus on the effect of pH and the physiochemical properties of PB.
Methods: PB (0.1 g per 50 mL) was added to buffered solutions (pH 1.0 to 9.0) containing different concentrations of thallium ranging from 600 to 1500 ppm. Samples were incubated at 37oC in a water bath shaker for 30 min to 24 hrs. Following incubation, each sample was filtered. The free thallium was analyzed using a validated Inductively Coupled Plasma method.
Results: PB was found to have the highest binding capacity (679 mg/g) at pH 7.5. The binding capacity decreased with reduction in pH to a minimum at pH 1.0. It was also found that the binding capacity and binding rate were related to the state of hydration and the particle size of PB. Significant variation of thallium binding from batch to batch was also observed among the APIs and the DPs.
Conclusions: The pH, the state of hydration and particle size of PB had a significant effect on the PB thallium binding. These findings can be utilized for medical product characterization to evaluate efficacy and predict drug product quality under certain manufacturing and storage conditions.
Development of a Lyophilized Reagent Format Real-time PCR Assay for the Simultaneous Indentification of Shiga-toxin Producing Escherichia coli (STEC) and E. coli O157:H7 with Incorporation of an Internal Control
K. C. Jinneman1 , K. J. Yoshitomi1 , S. D. Weagant2 , G. M. Blackstone3 , 1FDA, ORA, PRL-NW/SPRC, Bothell, WA, 2FDA, ORA, PRL-NW, Bothell, WA, 3FDA, CFSAN, OS, GCSL, Dauphin Island, AL
Background: Real-time PCR is an essential technology towards achieving rapid and sensitive detection of foodborne pathogens. A multiplex real-time PCR assay which is easy to run and provides reliable results has been developed to detect both STEC and E. coli O157:H7, a human pathogen capable of causing serious illness such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS).
Methods: The 4-plex assay was designed to detect three gene targets, two Shiga-toxin genes (stx1 and stx2) and a unique mutation at +93 of the ß-glucuronidase gene (uidA) of E. coli O157:H7, in addition to a unique DNA sequence serving as an internal control (IC) target. A test panel of 138 pure culture isolates consisting of STEC, non-STEC E. coli, enterohemorrhagic E. coli (EHEC), and non-E. coli species were evaluated. The assay was then converted into a convenient lyophilized bead containing all primers, probes, and control DNA, and further evaluated.
Results: Assay specificity was 100% for all gene targets. Average cycle thresholds were 17.51, 17.09, 21.03, and 19.72 for stx1, stx2, uidA, and IC targets, respectively, for positive samples. Positive amplification of the IC assures amplification was possible in the sample, preventing false negative EHEC reporting. Assay specificity for pure culture isolates remained unchanged between the use of wet reagents and the lyophilized assay.
Conclusions: This novel assay simultaneously identifies STEC and the E. coli O157:H7 serotype in a simple, ready-to-use bead format. Quality, rapid molecular methods provide an invaluable tool to address threats of bioterrorism and routinely safeguard our nation's food supply.
Real-time PCR Technology Transfer to FDA and State FERN Laboratories Through Laboratory Training and Test Samples
K. J. Yoshitomi1 , K. C. Jinneman1 , G. M. Blackstone2 , T. M. Bozicevich3 , A. R. Datta4 , 1FDA, ORA, PRL-NW/SPRC, Bothell, WA, 2FDA, CFSAN, OS, GCSL, Dauphin Island, AL, 3FDA, ORA, DHRD, Rockville, MD, 4FDA, ORA, DFS, Rockville, MD
Background: Scientists from ORA, CFSAN, and State public health laboratories were trained in real-time PCR techniques for the detection of bacterial pathogens from food matrices. The training course was followed up by practice samples completed at the participant's home laboratory.
Methods: A blinded panel of 14 pure culture DNA templates were administered to students to assess proficiency in performing the EHEC FERN Real-time PCR protocol. The assay detects the presence of STEC and E. coli O157:H7 by targeting stx1, stx2 and a conserved SNP in the uidA gene. Students were provided with a frozen master mix containing all PCR components (n=27) or lyophilized beads (n=14) that were re-hydrated immediately prior to use. A portion of the participants (n=12) also tested 24h food enrichments for the presence of gene targets after template preparation.
Results: Tests on the panel using reagent solution resulted in false positives 1.85%, 0.82%, and 1.01% for stx1, stx2, and uidA, respectively. False negatives were reported at 2.31%, 1.23%, and 6.35% for the same genes, respectively. Use of lyophilized reagent beads which also included an internal control improved reporting of correct results. There were only 1.59% false positives for stx2 and 0.4% false negatives for the internal control. Tests on the food enrichments using beads were 100% accurate.
Conclusions: Rugged methods, hands-on training, and follow-up practice samples ensured transfer of advanced technology to our field laboratories. This further strengthens the ability of FDA and FERN Laboratories to become proficient in advanced methods and respond to food related emergencies.
FDA's Most Wanted Flies
G.C. Ziobro, CFSAN, FDA, College Park, MD
Wanted posters for the following twelve species of flies will be displayed for:
House fly [Musca domestica L.], Stable fly [Stomoxys calcitrans (L.)], Little house fly [Fannia canicularis (L.)], Latrine fly [Fannia scalaris (Fabricius)], Cosmopolitan blue bottle fly [Calliphora vicina Robineau-Desvoidy], Holarctic blue bottle fly [Calliphora vomitoria (L.)], Cochliomyia macellaria (Fabricius) [Secondary screwworm], Oriental latrine fly [Chrysomya megacephala (Fabricius)], Blue bottle fly Cynomyopsis cadaverina [Robineau-Desvoidy], Green bottle fly [Phaenicia sericata (Meigen)], Black blow fly [Phormia regina (Meigen)], and the Redtailed flesh fly [Sarcophaga haemorrhoidalis (Fallen)].
These species are known carriers of human pathogenic diseases. They make up over half of the known vectors of insanitation of concern to the Agency. The presence of any of these species in an FDA regulated facility may lead to a 402(a)(4) charge, "adulterated [because] it has been prepared, packed, or held under insanitary conditions whereby it may have become contaminated with filth, or whereby it may have been rendered injurious to health." In order to initiate enforcement actions, the presence of these insects must be confirmed through the collection of specimens both inside and outside the facility, direct observations made of their landing on product, their means of entering and existing the facility noted, their movements from outside to inside and vice versa directly observed, and the lab identify the collected samples to the species level using the appropriate taxonomic keys.
DNA Markers for Identifying Humans: How Many SNPs?
R.L. Bernstein, San Francisco District, FDA
DNA typing potentially can identify uniquely every individual in a population. Single nucleotide polymorphisms (SNPs) occur widely in genomic DNA and distinguish individual genomes from each other. A SNP is a basepair location in the genome where more than one type of basepair can occur, when comparing among individuals in a population. Each SNP is usually biallelic (only two types known in the population) and neutral (type of basepair does not affect fitness). Over 20 million SNPs occur in the human genome.
Results show that surprisingly few SNPs must be measured in order to distinguish uniquely every human on the planet. By using more informative SNPs (minor allele frequencies at least 0.05), calculations demonstrate that only 20-100 particular SNPs need be measured. SNPs are measured in individuals as haplotypes in the diploid state. Equations of the Hardy-Weinberg equilibrium help to predict diploid SNP genotypes. Of course, the chosen SNPs should be unlinked in order to define their own haplotypes and assort independently, a feature of the genome of 2900 Mb encouraged by choosing SNPs spaced at least 20 Mb apart throughout.
This very small number of well-chosen SNPs leads to the design of a single standard DNA microarray to measure SNPs by probe hybridization. The microarray can easily incorporate numerous controls for correct interpretations of the hybridization reactions. Such a standard DNA microarray technique facilitates the practical DNA identification of all individuals in a population. The principle can be extended to using DNA for marking lots and shipments of foods or drugs for identification.
Acute hemodynamic and hemolytic effects of intravenously administered ethylene glycol in the pig: Implications for ethylene oxide sterilization residues standard (ISO 10993-7)
R. Brown, D. Wray-Cahen, M. Stratmeyer, CDRH, FDA, Laurel, MD
Background: Patients undergoing medical procedures with ethylene oxide (EO)-sterilized medical devices can be exposed to ethylene glycol (EG), a hydrolytic breakdown product of EO. A preliminary study was conducted to examine the acute hemodynamic and hemolytic effects of intravenously administered EG in anesthetized pigs to better define the dose-response relationship for the acute parenteral effects.
Methods: Anesthetized (isoflurane) male pigs were instrumented with blood flow probes around the renal and carotid arteries. Mean arterial pressure (MAP) was measured invasively with an indwelling catheter. Venous blood was drawn 5 minutes prior to injection of EG and at 5 minute intervals following injection for measurement of plasma free hemoglobin. Arterial blood gases were determined using an iStat analyzer.
Results: Bolus administration of a 125 mg/kg dose of EG in a 5 ml total volume (over 1 minute) resulted in a dramatic hemodynamic changes (decrease in mean arterial pressure, carotid and renal blood flow) immediately after injection. Gross hemolysis was seen in plasma 5 minutes after intravenous administration of the compound. In contrast, administration of the same dose of EG in a 50 ml volume produced no adverse effects. No change in arterial blood pH was seen after injection of either concentration of EG.
Conclusion: The short-term parenteral Tolerable Intake (TI) value for EG residues on medical devices should take into account both dose and concentration. Consequently, the draft ISO 10993-7 standard for EG residues on medical devices was amended to caution the user to account for both EG dose and concentration.
Clinical Data Presentations for Orthopedic Device Applications
B.D. Buch, M.D., FDA/DGRND/ODE/ORDB
This poster demonstrates recommended essential elements of general clinical data presentation formats for premarket notifications (510(k)s), investigational device exemption (IDE) annual progress reports, premarket approval (PMA) applications, and annual and post-approval study reports for orthopedic implant devices. Using these formats help ensure consistency and understanding between FDA and sponsors when discussing and presenting clinical data and will conserve FDA and industry resources to facilitate timely review.
The data presentation formats described help standardize presentations to facilitate review of Orthopedic Devices Branch (ORDB) submissions. The descriptions and definitions used in this document are commonly used in ORDB but may not be applicable to submissions in other product areas
Effect of Sorbitol on Bioequivalence of Ranitidine Oral Solutions
A. Straughn1 , N. Sadrieh2 , C. Yates1 , B. Meibohm1 , M. Chen2 , A. S. Hussain2 , 1Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis, TN 38163, 2Office of Pharmaceutical Science, CDER, FDA, Rockville, MD 20852
Background: Formulation excipients may affect the drug bioavailability. In a previous study, we showed that 5 gm sorbitol significantly reduced the bioavailability of ranitidine compared with 5 gm of sucrose. The present study evaluates the minimal amount of sorbitol required to affect the bioequivalence outcome from an oral solution of ranitidine HCl.
Methods: Sixteen normal volunteers each randomly received, at weekly intervals, one of the following aqueous solutions (10 mL):
Tx 1 = 150 mg ranitidine and 5 gm sorbitol
Tx 2 = 150 mg ranitidine and 2.5 gm sorbitol
Tx 3 = 150 mg ranitidine and 1.25 gm sorbitol
Tx 4 = 150 mg ranitidine
Blood samples were collected pre-dose and then serially for 12 hours post dose. Plasma was assayed by an HPLC method with UV detection.
Results: The mean AUCinf for Tx 1, Tx 2, Tx 3 and Tx 4 was 1475, 2061, 2577 and 2678 ng*hr/mL, respectively. The corresponding Cmax was 238, 362, 469 and 490 ng/mL. Using Tx 4 as a reference, the relative ratio for AUCinf was 0.55, 0.77 and 0.96 for Tx 1, Tx 2 and Tx 3, respectively; and the corresponding ratio for Cmax was 0.48, 0.74 and 0.96. Only Tx 3 was bioequivalent to Tx 4 based on the 90% confidence limit of 80-125%.
Conclusions: Bioequivalence of an oral solution of ranitidine is not affected by the presence of sorbitol at levels less than or equal to 1.25 gm.
Effects of zearalenone on in utero development of rats
T. F.X.. Collins1 , R. L. Sprando1 , T. N.. Black1 , N. Olejnik1 , R. M.. Eppley2 , H. Z.. Alam3 , D. I.. Ruggles3 , 1CFSAN, FDA, Laurel, MD, 2CFSAN, FDA, College Park, MD, 3CFSAN, College Park, MD
Background: The estrogenic mycotoxin zearalenone (ZE) is one of the five most common contaminants of cereal grains in the U.S. and Canada.
Methods: ZE (0, 1, 2, 4, or 8 mg/kg) was given by gavage once daily on gestation days (GD) 6-19. Each animal was examined daily. Feed and water consumption were measured regularly. Reproductive parameters were measured and blood was taken for endocrine analysis at cesarean section on GD 20. Fixed fetuses were later examined for skeletal or soft-tissue development.
Results: In all treated groups, ZE produced significant, dose-related decreases in maternal feed consumption and body weight gain. Fetal body weight was significantly decreased in males and females in all treated groups (dose-related at 4 and 8 mg/kg). Anogenital index was significantly increased in male and female fetuses from all treated groups; greater increases were seen in males than in females. At 8 mg/kg, ZE produced significant decreases in viability of male and female fetuses and retarded skeletal development. Soft-tissue development was not affected. Maternal liver-body weight ratios were significantly increased at 4 and 8 mg/kg. Organ-brain weight ratios for liver, heart, spleen, kidneys, and ovaries were significantly decreased at 4 and 8 mg/kg. In addition, kidney-brain weight ratios were decreased at 2 mg/kg. At 8 mg/kg, estradiol was significantly decreased and prolactin was significantly increased.
Conclusions: There was no NOEL for maternal or fetal toxicity. ZE was not teratogenic.
The PEPFAR Program: Bioequivalence Issues for Fixed-Dose Combination and Co-Packaged Drug Products for Treatment of HIV
B. M. Davit1 , K. S. Reynolds2 , 1OGD/OPS/CDER, FDA, Rockville, MD, 2OCPB/CDER, FDA, Rockville, MD
Background: The President's Emergency Plan for AIDS Relief (PEPFAR) will provide resources of $15 billion over 5 years to 15 countries. Antiretroviral (ARV) drugs that receive either full or tentative FDA approval can be procured under PEPFAR. The FDA encourages development of (1) simplified HIV treatment regimens, such as fixed-dose combinations (FDCs) or co-packaged drugs; and (2) generic versions of existing FDA-approved single-ingredient ARV drug products. For most PEPFAR applications, the pivotal clinical study is a bioequivalence (BE) study.
Methods: The FDA encourages innovator and generic domestic and foreign firms to submit applications under the PEPFAR program. The FDA developed a Guidance for Industry to assist sponsors developing FDCs and co-packaged ARV therapies. FDA personnel frequently interact with firms and regulatory agencies and provide expedited review of ARV products.
Results: The following emerge as significant regulatory and scientific issues: (i) appropriate drug combinations for FDCs and co-packaged products; (ii) whether to submit as a new drug or generic; (iii) whether to use a US or non US-reference drug; (iv) appropriate BE study design; (vi) short review timelines; (vii) interactions with foreign regulatory agencies and governments; (viii) legal considerations.
Conclusions: Following a series of meetings throughout 2004, domestic and foreign companies submitted applications to the FDA under the PEPFAR program. To date, two products have been approved, one for a new co-packaged product, and one for a new generic. The rapid review of ARV drugs should facilitate the availability of quality and cost-effective ARV therapies in countries targeted for PEPFAR resources.
Estimating Intake of Flavors: Is there a "better" method?
M. J. DiNovi, D. L. Doell, A. J. Edwards, J. J. Mihalov, CFSAN, FDA, College Park, MD
Flavors are defined as substances that are intentionally added to food to achieve a specific odor or taste. They can be natural or synthetic, where synthetics may or may not be chemically identical to a naturally-occurring substance. The use of flavors in food is typically self-limiting, controlled by the intensity required for organoleptic characteristics. This results in low food concentrations, and hence, low probable intakes. The estimate of probable intake is a key component in the safety assessment of flavors at FDA and internationally. Both FDA and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) are using the "maximized survey-derived daily intake" (MSDI) method to estimate the dietary intake of flavors. This method is based on the annual quantity of a flavor produced. Recent publications have created a controversy in Europe over the methods used to estimate intake by suggesting that the MSDI method is not conservative enough to assess the intake of flavors for the "high" level consumers. These papers advise that the "Theoretical Added Maximum Daily Intake" method (TAMDI), which assumes that an individual will consume a fixed amount of flavored food containing the flavor at a specified upper use level on a daily basis, is a better method to reflect the intake of flavors. This poster analyzes the MSDI method used by FDA and JECFA, and explores alternative methods, including the TAMDI method, to estimate the intake of flavors, comparing the strengths, weaknesses, and assumptions of each model, and the relevance to safety evaluation.
THE PRODUCT QUALITY RESEARCH INSTITUTE IMPURITY CHARACTERIZATION AND QUANTIFICATION BEST PRACTICES SURVEY
P. J. Faustino1 , C. C. Chan2 , J. Carrano3 , M. Gosnell4 , Z. Q. Gu1 , A. Maule5 , K. Sigvardson6 , Y. F. Zhang7 , 1CDER, FDA, 2Eli Lilly, Canada, 3Wyeth Pharmaceuticals, 4Alkermes Inc, 53M Pharmaceuticals, 6Schering-Plough Research Institute, 7Pfizer Inc
Background: The Product Quality Research Institute, (PQRI) is a consortium of industry, academia, FDA and the USP that was formed to conduct research to generate scientific information to support regulatory policy. PQRI's research mission is to identify and address potential gaps between scientific knowledge and regulatory policy to reduce regulatory burden. The PQRI Impurity Working Group (IWG) was established to examine issues surrounding impurity testing in drug substances.
Methods: The IWG developed a survey seeking information from pharmaceutical companies regarding their practices in structural elucidation and quantification of impurities prior to agency submission. The survey consisted of 25 questions that were divided into 4 sections: structural characterization/elucidation, quantification, regulation and demographics.
Results: Results of the survey indicate there is a shift to increased utilization of analytical resources and advanced technology during the development process. The survey indicates that analytical resources and advanced technology are allocated based on scientific need at the early stages of development. Respondents from small and large companies indicated no need for a formal guidance regarding drug substance impurities at early stages (IND) of development. Survey results indicated a good balance between science and regulatory requirements during each phase of drug development.
Conclusions: The survey provided a better understanding of the application and utilization of resources at each stage of drug development. Additionally the survey clarified the need for additional regulatory guidance. In conclusion, collaborative research on regulatory issues by organizations like PQRI, can impact the safety, medical utility and industrialization dimensions of the "critical path."
Iontophoretic Delivery of Anesthetic and Analgesic Drugs: Design Evolution and Regulatory Challenges
R. S. Harapanhalli1 , R. Agarwal1 , J. Boal1 , K. Lee2 , P. Love3 , E. Duffy1 , 1CDER, FDA, Rockville, MD, 2CDRH, FDA, Rockville, MD, 3OCP, FDA, Rockville, MD
Background: Iontophoresis is an electrically driven process of transporting ionic species across the skin.
Methods: We reviewed both the literature and the approved NDAs and 510(k)s for iontophoretic drug delivery systems.
Results: Lidocaine, tetracaine, epinephrine, and fentanyl have been delivered iontophoretically to achieve local dermal anesthesia or systemic analgesia. Innovations in design have resulted in fully integrated, compact, programmable, single-unit iontophoretic patches. Increasing complexity in design has paralleled the complexity of scientific and regulatory approaches to review and inspection of these products. The Office of Combination Products (OCP) designated the jurisdiction of these products to CDER as the primary mode of action is due to drugs. A close interaction between CDER and CDRH was essential to the approval of these products as drug delivery is governed by Ohm's and Faraday's laws and thus is dependant on the drug's ionic charge, molecular weight, and concentration, as well as skin conductivity, applied current, and the presence of competing ions in the formulation. The OCP streamlined the inter-center consultative and collaborative review process and published draft guidance on current good manufacturing practices for Combination Products.
Conclusion: The review and inspection of regulatory submissions containing complex, integrated, iontophoretic products requires a concerted effort and high level of interaction between CDER and CDRH in the interpretation of scientific and regulatory requirements. OCP will play a pivotal role as a facilitator in this interaction.
Comparability Protocols for Small Molecule Drugs in NDAs and Supplemental NDAs: Regulatory Issues and Challenges
R. S. Harapanhalli, S. Moore, E. P. Duffy, CDER, FDA, Rockville, MD
Introduction: A comparability protocol (CP) is a well-defined, detailed written plan that describes proposed change(s), prospectively specifies tests, studies, analytical procedures and acceptance criteria that will be achieved to demonstrate the CMC changes and requests reporting categories for the change(s). FDA is encouraging manufacturers to use a CP to facilitate future implementation and reporting of CMC changes, which could result in downgraded reporting and regulatory requirements, enabling product movement into distribution sooner.
Methods: Comparability protocols submitted in recent NDAs and sNDAs were reviewed for their suitability and rigor to support the proposed changes.
Results: In light of draft guidance for Industry published in 2003 describing basic elements of a CP and how to use it, the types of changes proposed in recent CPs included changes in drug substance synthesis, new vendors of the drug substance, drug substance specifications, quality of the excipients, drug product manufacturing site, order of addition of components in the manufacture of the drug product, vendors of device part of the combination products, analytical procedures, and container closure systems. While some were adequate in their original form, others were deficient and needed additional provisions to be able to demonstrate equivalence, whereas some others were deemed inappropriate based on the complexity and level of risk deciphered from the pharmaceutical development reports.
Conclusion: The CP approach to regulatory filing has received wide interest. The CP process has been a learning experience for both the manufacturers and the Agency. A specific, risk-based and well-described CP that provides for recurring and/or foreseeable post-approval changes is likely to gain significant regulatory relief.
Methods of Manufacture That Remove Contaminants From Fish Oils: information compiled through the GRAS (generally recognized as safe) notification program
A. J. Edwards, E. Garcia, C. A.. Hendrickson, CFSAN, FDA, College Park, MD
Introduction: Through the GRAS notification program (62 FR 18938; April 17, 1997), a manufacturer or individual may voluntarily notify FDA of their determination that an ingredient such as fish oil is GRAS for its intended use in foods. FDA has received several GRAS notices for fish oils obtained from a variety of fish species. Fish oils are of interest in human health as rich dietary sources of omega-3 and omega-6 fatty acids; however, they are also potential dietary sources of environmental contaminants. Published literature and information contained in GRAS notices show evidence of contamination of fish with heavy metals, pesticides, polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), dioxins, and dioxin-like compounds.
Method/Results: On a case-by-case basis, the GRAS notices for fish oil have addressed chemistry issues relevant to the safety of these ingredients, including source, estimated dietary exposure, method of manufacture, fatty acid composition, analyses for contaminants, and quality assurance specifications. OFAS has compiled data from the published literature regarding the removal of environmental contaminants through various oil refining steps and additional purification processes. For purposes of reference, this compilation includes safety assessment information from both national and international government agencies and other expert bodies.
Conclusion: This poster presents an overview of chemistry issues regarding the safety of fish oils as ingredients in foods and the role of purification processes used during manufacturing to remove contaminants from fish oils.
Guidance for Industry and FDA Staff: Non-Clinical Engineering Tests and Recommended Labeling for Intravascular Stents and Associated Delivery Systems
V.M. Holt, CDRH, FDA, Rockville, MD
OSEL/Division of Solid and Fluid Mechanics, ODE/Division of Cardiovascular Devices/ Interventional Cardiology Devices Branch & Peripheral Vascular Devices Branch
Background: Intravascular Stents and their Associated Delivery Systems are class III devices, which require a premarket approval application (PMA). These devices consist of a metallic tube called a stent that is mounted and attached to the end of a long thin flexible tube (stent delivery catheter).
The stent is used in patients who have a narrowing in their arteries (blood vessels supplying blood to the heart) caused by atherosclerosis -- the collection of fatty substances such as cholesterol that forms "plaque" along the lining of the arteries.
A delivery catheter is inserted into a blood vessel in the arm or groin, and advanced within the vessel, to the narrowed section of the coronary artery. The stent is deployed within the narrowed artery to open the artery. The delivery catheter is removed and the stent remains permanently implanted within the artery, acting as a support (scaffold) for the newly opened section of the vessel.
Methods: The guidance entitled, Non-Clinical Engineering Tests and Recommended Labeling for Intravascular Stents and Associated Delivery Systems describes FDA's recommendations for performance characteristics, testing, design, voluntary standards, and labeling for the intravascular stents and associated delivery systems. The guidance document was a multidisciplinary collaborative effort involving experts in cardiology, biocompatibility, mechanical and biomedical engineering, material science, and statistics from CDRH's Office of Device Evaluation and Office of Science and Engineering Laboratories.
Points to Consider in the Design of Nonclinical and Clinical Evaluations of Products Intended to Repair or Replace Articular Cartilage
A. D. Kaiser1 , R. D. McFarland2 , S. M. Dawisha1 , S. Leibenhaut2 , 1CDRH, FDA, Rockville, MD, 2CBER, FDA, Rockville, MD
Damage to articular cartilage, particularly in the knee, that results in pain and altered function affects thousands of people in the US at a cost of millions of dollars each year. Current treatment options range from conservative therapies, e.g., pain medication and rehabilitation, to surgical procedures, e.g., microfracture or autologous chondrocyte implantation. Because existing therapies may not result in durable clinical responses, companies are developing products to repair or replace the damaged cartilage. These products fall into three broad categories:
Depending on the components of a specific product, the lead regulatory jurisdiction may be in CDRH or CBER. In order to define a transparent regulatory environment governed by a consistent set of expectations, staff in the two Centers have collaborated to identify points to consider when developing the various non-clinical and clinical parameters that should be evaluated in the analysis of the safety and effectiveness of these products. It is anticipated that this information will form the basis of an inter-Center guidance document that will assist sponsors in the preparation of regulatory submissions and enhance the consistent regulation of articular cartilage repair or replacement products between the two Centers.
Visual Analog Compared with Categorical Scale for Acute Pain Measurement
M. Katzper, CDER, FDA, Rockville, MD
Background: Visual Analog Scales (VAS) and Categorical scales (CAT) are both used in submissions requiring acute pain measurement. The question arises whether results will differ depending on the scale used.
Methods: Acute pain studied submitted to the FDA were examined. Two dental extraction studies in which pain was measured on both visual analog and categorical scales simultaneously were selected for analysis. Placebo and multiple drugs were used in the studies. Pain responses were obtained at multiple times. JMP was used to examine all the simultaneous VAS and CAT responses.
Results: We found an excellent correspondence between measurements using the unconstrained visual analog scale and a 4 point categorical scale pain in the aggregated data of both dental extraction pain model studies. Only in the range of VAS responses identified as the transition range for each CAT score was there was an overlap between measured VAS scores and chosen CAT categories.
Conclusions: Visual analog as well as categorical scales are concordant in determining acute pain and may be used interchangeably. There is ambiguity in the transition range, which itself may drift. A combined metric scale for pain measurement that provides the subject with multiple cues may improve communication and concordance between scales for individual pain determination.
LABELING OF DRUGS THAT INTERACT WITH ORAL CONTRACEPTIVES
M. J. Kim1 , E. Castillo1 , V. R. Jarugula1 , A. Parekh1 , L. A. Furlong2 , J. P. Hunt1 , H. J. Malinowski1 , 1DPEII, OCPB, CDER, FDA, Rockville, MD, 2OND, CDER, FDA, Rockville, MD
BACKGROUND: Labeling should clearly describe drug-drug interactions (DDI) between oral contraceptives (OCs) and other drugs. The purpose of this study was to survey the labels of drugs that interact with OCs.
METHODS: Drugs known or suspected to interact with OCs were identified using DRUGDEX®. The following keywords were used: combination contraceptives, levonorgestrel, and norethindrone. Additional drugs were identified from a literature search. Labels were obtained from the Physicians' Desk Reference.
RESULTS: Our search identified 61 drugs. Twenty-six labels (43%) contained general language about DDI of OCs and 35 (57%) specified interaction with estrogen/progestin components of OCs. Four labels described increased OC exposure but only 1 of these 4 labels recommended a dose reduction of OC. Two labels contained no recommendation about OC dose adjustment while 1 label recommended 'considering an increase in OC exposure when OCs are prescribed'. Of 21 labels describing decrease in OC exposure, 16 recommended additional/different forms of contraception while 8 did not specify any dose adjustment. Four labels recommended a general precaution. Two labels reported unknown clinical significance of decreased OC exposure. Three labels reported a lack of DDI with OCs by evaluating the inhibition of ovulation.
CONCLUSIONS: Label recommendations for OCs regarding DDI vary considerably. DDI between a drug and a specific OC may not apply to all OCs because of different progestins in OCs.
Guidance for Industry on the Development of Decorporation Agents for Internal Radioactive Contamination
J. G. Beitz, S. Biade, Y. M. Choi, L. Cress, S. S. Farr, C. John, T. G. Kokate, A. A. Laniyonu, M. Mathis, D. A. Place, R. Raman, P. A. Stewart, O. H. Suleiman, M. Welch, R. J. Yaes, CDER, FDA, Rockville, MD
Radioactive contamination can occur as a result of nuclear accidents, criminal events or terrorist actions. Internal radioactive contamination occurs when radioactive material is ingested, inhaled, or absorbed from a contaminated wound.
Decorporation agents are medical products that increase the rate of elimination or excretion of radioactive contaminants. The efficacy of most decorporation agents cannot be tested in humans because the occurrence of radioactive contamination is rare and it would be unethical to deliberately test humans with radioactive materials for investigational purposes. For such instances, the Animal Efficacy Rule (21CFR Part 314 Subpart I) may be invoked to support marketing approval of decorporation agents. To that end, this document was developed to provide specific guidance to Industry on the timing and type of preclinical and clinical safety studies, on development of specific CMC in vitro tests to predict effectiveness, and on the commitments for appropriate post-marketing clinical studies.
Bioequivalence of Highly Variable Drugs in Generic Drug Applications
D. Patel, B. M. Davit, S. H. Haidar, L. X. Yu, D. Conner, Office of Generic Drugs, CDER, FDA
Background: Drug products that are highly variable (HV) in the pharmacokinetic (PK) parameters AUC and Cmax can fail to meet the FDA's bioequivalence (BE) limits of 80-125% when the usual number of subjects are enrolled in a BE study. Generic drug applicants claim that applying the usual BE criteria to these drugs places a high economic burden on them since they believe that they have to use large numbers of subjects to successfully demonstrate BE for their products. We evaluated the scope of this problem by reviewing BE studies of generic HV drugs.
Methods: We collected data from 212 fasting and fed BE studies in 115 Abbreviated New Drug Applications (ANDAs) submitted in 2003. We analyzed the ANOVA Root Mean Square Errors (RMSEs), point estimates and 90% confidence intervals (CIs) for generic/innovator AUC and Cmax ratios. Drugs were considered HV if Cmax and/or AUC RMSE > 0.3.
Results: All of the 212 studies reviewed successfully met FDA's BE limits. Cmax was HV more frequently than AUC. Higher PK variability occurred in fed BE studies. Only 14 studies of HV drugs used > 50 subjects, and the maximum number of subjects used in any of these studies was 78. The width of the 90% CI narrowed as number of subjects increased.
Conclusion: In 2003, 15.5% of all BE studies submitted in ANDAs were for drugs that met the HV criteria. It appeared that many of the successful BE studies of HV drugs did not need excessively large number of subjects; however, research is continuing to confirm this observation.
COMPLEXITIES OF THE BOTANICAL NOMENCLATURE SYSTEM IN TRADITIONAL CHINESE MEDICINE (TCM): LESSON LEARNED FROM ARISTOLOCHIA AND IMPORTANCE OF THE PHARMACEUTICAL NAME
K. M. Wu, J. G. Farrelly, DAVDP, CDER, FDA
Botanicals used in TCM have diverse culture/historical backgrounds and are described based on complex nomenclature systems. By using aristolochiaceae family as an example, at least three categories of nomenclature could be identified: (1) one-to-one: (one plant part from one species): herb guan mutong refers to the root of Aristolochia manshuriensis (Chinese species name [CSN], dongbei madouling), (2) multiple-to-one (multiple plant parts from the same species serve as different herbs): three herbs, madouling, qingmuxiang and tianxianteng, derived from the fruit, root and stem of Aristolochia debilis/contorta (CSN/guang fangji), (3) one-to-multiple: (one herb refers to multiple species): herb fangji refers to the root of either Aristolochia fangchi (CSN/guang fangji), Stephania tetrandra (CSN/fen fangji or han fangji), or Cocculus trilobus/orbiculatus (CSN/mu fangji); the first belongs to a different family (Aristolochiaceae) than the latter two (Menispermaceae), and only the first contains aristolochic acid, the controversial but rather lethal renotoxin. To complicate the issue further, mutong (Akebia quinata/trifoliate, Lardizabalaceae) can be substituted for by guan mutong (Aristolochia manshuriensis) or chuan mutong (Clematis armandi/Montana, Ranunculaceae); and mu fangji (Cocculus trilobus/orbiculatus) by guang fangji (Aristolochia fangji) or hanzhong fangji (Aristolochia heterophylla) in practice, thereby increasing the risk of exposing aristolochia species to patients. To avoid these and other confusions, we wish to emphasize the importance of a pharmaceutical name, which defines the species name, the plant part, and sometimes the special process performed on the herb (e.g., Radix Rehmanniaeg glutinosae conquitae), for botanical supplement or drug product development. It is hoped that by following the pharmaceutical name, toxic botanicals can be effectively identified and substitution or adulteration avoided.
Bioequivalence Study Recommendations Guidance, a Critical Path Initiative.
W. P. Adams, P. M. Sathe, D. Holovac, L. X. Yu, CDER, FDA, Rockville, MD
Background: In March 2004 the Agency report Innovation/Stagnation: Challenge and Opportunity on the Critical Path to New Medical Products noted that new methods are needed to improve efficiency along the critical path from laboratory concept to commercial product. Based on the report, a Bioequivalence (BE) Study Recommendations Guidance was proposed to efficiently disseminate BE information. This guidance is perceived as a dynamic document to include a growing list of product-specific BE recommendations.
Methods: Office of Generic Drugs (OGD) has begun a program to develop BE recommendations for products for which no recommendations have previously been available. The guidance will also include previously available recommendations. The guidance will provide applicants with the information on the design and conduct of BE and dissolution studies that meet the rigorous standards of FDA. BE recommendations follow the March 2003 Bioavailability and Bioequivalence Studies for Orally Administered Drug Products-General Considerations Guidance, when applicable.
Results: A BE Study Recommendations Guidance is being developed for posting on the FDA/CDER internet. The document is anticipated to initially include over two dozen orally administered drug products. Recommendations regarding fasting/fed studies, moieties measured, waiver strengths, and dissolution will be made.
Conclusions: The guidance will provide product-specific study recommendations on new and existing products. It will assist the applicant and others in providing readily available uniform study recommendations. It will also increase the efficiency and uniformity of review by OGD.
Regulatory Aspects of Total Product Life Cycle
E. D. Hausman, S. S. Altaie, S. I. Gutman, OIVD, CDRH, FDA, Rockville, MD
Total Product Life Cycle (TPLC) is an integrated product development scheme and a conceptual framework for assessing a variety of industrial and clinical models. For the manufacturer TPLC is the market-driven evolution of a device, drug, or biologic from initial conception, through pre-market development, to widespread market use, and finally to obsolescence and replacement by subsequent generations of products.
At the regulatory arena, the Center for Devices and Radiological Health (CDRH), and the Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD) are spearheading the metamorphosis of device regulation by incorporating TPLC as a global quality and process improvement initiative. Under this regulatory philosophy all phases of the regulatory life cycle are integrated, and all stakeholders (e.g., industrial, scientific, marketing, and regulatory) share responsibility in efficiently evaluating medical devices. Subsequently, compartmentalization of information, process, and decision-making are decreased, and efficiency is optimized.
From the regulatory perspective, at OIVD, the previous practice of having pre-market review and post-market surveillance viewed as independent and dichotomous facets of regulatory responsibility has been replaced with an integrated approach. In Vitro Diagnostic devices are appropriately viewed existing in a pipeline of innovation and constant improvement. This creates a very different regulatory challenge than regulating a pharmaceutical with a long patent life and few, if any, modifications.
OIVD represents the first office-wide integrated approach of this cradle-to-grave format. The Office engages in multiple activities that were historically spread out over different offices, and much of the staff is cross-trained and engages in both pre-market and post-market activities. The Office still performs pre-market review for determination of reasonable probability of safety and effectiveness, but a significant percentage of the staff regularly engages in various aspects of post-market surveillance, which was formerly under the aegis of a separate office.
Several mechanisms help with the collection and integration of post-market information. The MedWatch system (FDA Safety Information and Adverse Event Reporting Program) and the Medical Device Reporting regulations are two anchor mechanisms for the public to notify the FDA of device-related issues. The Manufacturer and User Facility Device Experience system is a database consisting of all voluntary reports since June 1993, user facility reports since 1991, distributor reports since 1993, and manufacturer reports since August 1996 and contains information on medical devices that may have malfunctioned or caused a death or serious injury. The Office also engages in compliance activities such as inspections and device evaluation and recall activities.
CDRH started utilizing TPLC as a new regulatory framework in early 2000. TPLC is now one of the core elements in the Center's strategic goals. Center-wide "Score Cards" were developed in 2002 to link the strategic goals to Key Results Areas in the strategic path. In 2003 the Center started defining the key indicators for each Key Results Area to measure performance of everyone in the Center, thereby creating "line of site" activity tracking to support the mission and vision of the Center as a whole.
Recommended Exposure Schedules for Indoor Tanning: Explorations to improve current FDA guidance
S. A. Miller, S. G. Coelho, B. Z. Zmudzka, H. F. Bushar, J. Z. Beer, CDRH, FDA, Rockville, MD
FDA regulates manufacturers of sunlamp products using a performance standard promulgated in 1979 and amended in 1985. To provide manufacturers with guidance on developing recommended exposure schedules for these products, FDA published a policy letter in 1986. This guidance was based on current knowledge (available at the time) of the effects of repeated exposures of ultraviolet radiation to tan human skin. Scientific progress in this area indicates that the 1986 guidance could be improved so that the cumulative dose received would be substantially reduced without compromising the desired effect.
We conducted a pilot study on six subjects. The goal of the pilot study was to experiment with variations on 3 different exposure schedules so that we could fine-tune them to ultimately set them for the main study. The three exposure schedules will be subsequently tested on a total of 40 subjects using two different sunlamps. Changes in skin color were measured subjectively - by trained observers - and objectively - by using a Minolta chromameter to measure the ΔE value in L*a*b* color space. The changes in skin color obtained through the use of the different exposure schedules were compared with measured color changes published in a 2000 study which evaluated currently-used exposure schedules. Preliminary results indicate that cumulative doses can be reduced by a factor of 3 to 4 compared with the current FDA recommended guidance. This research was supported in part by the FDA Office of Women's Health.
A key to the genera of commonly imported seaweeds
L.A. Solorzano, San Francisco District
A dichotomous key has been developed to facilitate the genus-level identification of commonly encountered, minimally processed, marine macroalgae (seaweeds). This key is based on samples collected by FDA San Francisco District personnel, on products purchased through the internet, mail order, at local markets, and on herbarium specimens. The key works best with fresh frozen seaweeds or with
dried seaweeds which have been rehydrated in water.
Detection of 10 Infectious Units of Pathologic Prion Protein From Scrapie Infected Hamster Brain Homogenates Using Real-time Immuno-PCR (IPCR)
J. Barletta, Ph.D.1 , D. C. Edelman, M.S.2 , W. E. Highsmith, Ph.D.3 , N. T. Constantine, Ph.D.4 , 1CDER, FDA, Rockville, MD, 2University of Maryland Baltimore, Baltimore, MD, 3Mayo Clinic, Rochester, MN, 4University of Maryland, Baltimore, MD
Purpose: The detection of the pathologic prion protein (PrPSc), implicated in transmissible spongiform encephalopathies, is the only method of diagnosing prion diseases. Presently, the Western Blot or ELISA format test is officially used to screen the brain stem in cattle for the presence of PrPSc. The development of an assay that can detect femtogram/mL levels of PrPSc is believed to be required to detect infection in blood at pre-clinical stages of the disease.
Methods: The immuno-polymerase chain reaction (IPCR) is a technique whereby exponential amplification ability of the PCR is coupled to the detection of proteins by antibodies in an ELISA format. We incorporated a modified real-time IPCR method capable of exponential amplification after immuno-capture of PrPSc for the detection of hamster recombinant PrPSc, and Proteinase K (PK)-digested scrapie infected hamster brain homogenates.
Results: The real-time IPCR method detected recombinant hamster PrPC with an analytical sensitivity of 100 ag/mL. PK-digested scrapie infected hamster brain homogenates diluted from 10-2 to 10-8 (approximately 1-10 infectious units) exhibited a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than levels detected by Western Blot or ELISA.
Conclusions: Although not yet a standardized protocol, real-time IPCR exhibits unparalleled analytical sensitivity for the detection of proteins and distinguishes the method as a testing system capable of detecting PrPSc in the pre-clinical phase of infection.Further, our study indicates that unless complete PK-digestion of PrPC in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.
(Experimental work was performed at the University of Maryland, Baltimore, MD)
Barletta J.M., Edelman D.C., Highsmith W.E., Constantine N.T. Detection of 10 Infectious Units of Pathologic Prion Protein (PrPSc) from Scrapie Infected Hamster Brain Homogenates Using Real-Time Immuno-PCR. J. Virol Methods. Accepted and in press, March 2005.
Development of a Broth Microdilution Susceptibility Testing Method for Campylobacter
S. M. Bodeis-Jones, R. D. Walker, P. F. McDermott, CVM, FDA, Laurel, MD
Background. Prior to June 2004, the only Clinical and Laboratory Standards Institute (CLSI/NCCLS) approved antimicrobial susceptibility testing method for Campylobacter was agar dilution. While this method is highly reproducible, it is labor intensive and not practical for routine testing of a single isolate.
Methods. Two 10-laboratory studies were performed to develop quality control guidelines for broth microdilution testing of C. jejuni ATCC 33560 against fourteen antimicrobials: azithromycin, chloramphenicol, ciprofloxacin, clarithromycin, clindamycin, doxycycline, erythromycin, florfenicol, gentamicin, levofloxacin, meropenem, nalidixic acid, telithromycin and tetracycline. The method requires cation-adjusted Mueller-Hinton broth supplemented with 2-5% laked horse blood and growth in a humid microaerophilic environment of 10% CO2, 5% O2, and 85% N2 at 36°C for 48 hours or 42°C for 24 hours.
Results. Quality control ranges were established for all fourteen agents at both temperatures. The proposed ranges encompass the observed modal or bimodal MIC + 2 log2 dilutions, and > 95% of the observed values.
Conclusion. This represents the first validated in vitro susceptibility testing method for Campylobacter using a broth microdilution format. The availability of this method will make routine susceptibility testing of Campylobacter more practical and feasible for research, clinical laboratories and surveillance programs.
Dissolution of Atropine Sulfate tablets and of extended release Hyoscyamine Sulfate tablets or capsules
U.R. Cieri, ORA, FDA, Philadelphia, PA
Background : The Belladonna alkaloids Atropine Sulfate and Hyoscyamine Sulfate have identical molecular formulas but different stereo configurations. Hyoscyamine sulfate is considered to possess greater anticholinergic activity because it is composed almost entirely of the levo isomer. Current official methods for the analysis of tablets containing one or both of these alkaloids do not include dissolution testing.
Method : In this proposed procedure , dissolution testing is carried out with USP Apparatus 2 (paddles) at a speed of 100 rpm using 500 mL of water at 37 C as dissolution medium. Analytical determinations were made by HPLC with a 7.5 cm long C18 column. The mobile phase is prepared as indicated ; 2.5 g of 1-octanesulfonic acid , sodium salt is dissolved in 1000 mL of water already containing 2.5 mL of phosphoric acid ; 500 mL of this solutions is mixed with 500 mL of methanol. Injection volume is 200 uL , detection by UV absorbance at 220 nm. Reference solutions of appropriate strength are prepared in water.
Results : For a sample of Atropine sulfate tablets labeled to contain 0.4 mg per unit , testing was done after 30 minutes. Average % dissolution for 6 tablets was 95.1. For a sample of extended release Hyoscyamine Sulfate tablets labeled to contain 0.375 mg per unit , testing was done after 1 , 3 , 6 and 8 hours. Average % dissolution for 6 tablets were 23.6 after 1 hour , 42.8 after 3 hours , 75.4 after 6 hours and 85.1 after 8 hours. A similar testing scheme was used with a sample of Hyoscyamine Sulfate capsules labeled to contain 0.375 mg per unit. Average % dissolution for 6 capsules were 26.3 after 1 hour, 52.7 after 3 hours , 76.9 after 6 hours and 91.9 after 8 hours.
Conclusions : The proposed method appears to be very appropriate for dissolution testing of Atropine Sulfate and Hyoscyamine sulfate tablets . It is recommended that more products be tested.
A Novel Quantitative PCR Internal Control System Adaptable to any Standard or Real-Time PCR Assay
M. C. L.. Vickery1 , G. M. Blackstone2 , A. DePaola2 , B. Burkhardt2 , 1CePheid. Inc., 2FDA, Dauphin Island, AL
Real-time PCR is an evolving technology which allows the rapid and sensitive detection/enumeration of specific nucleic acid sequences. The quantitative abilities of real-time PCR depend upon the linear relationship that exists between the initial template copy number and the partial PCR cycle at which a reaction is observed to enter exponential amplification [cycle threshold (Ct)]. Reaction inhibition can therefore negatively affect the quantitative ability and detection sensitivity of a real-time assay. Inclusion of a quantitative internal control (IC) in a real-time PCR assay allows the identification of false negatives, but also may be used to adjust quantitative data when partial reaction inhibition/enhancement occurs, causing a shift in the expected Ct value of the IC. While several types of internal controls were recently developed for use in real-time PCR, to our knowledge, all of these are either instrument-platform-specific or require some level of assay-specific primer or probe sequence customization for use in different assays. We developed a novel internal control system which uses an exogenous nucleic acid with a computer-generated pseudo-randomized (i.e., randomized with specific design parameters) sequence. Amplicons generated by PCR with this IC have no significant homology to any naturally-occurring or synthetic DNA sequences, and the system provides a choice of amplicon sizes and fluorescent reporters. The design allows for ease of incorporation into virtually any existing standard or real-time PCR on any instrument platform, regardless of the organism or nucleic acid sequence being targeted. In the present study, in order to evaluate the flexibility of this system, it was tested with numerous individual and multiplex real-time PCR and RT-PCR assays. These assays targeted various bacterial and viral pathogens. The IC was incorporated into each assay with only minor adjustments to the IC reagent concentrations, and the sensitivity and specificity of each assay was maintained without significant adjustments to any of the existing reaction conditions, demonstrating the universal adaptability of the IC system.
Prioritizing sources of variability in genomic microarray data (OSHC inter-center project)
D. Ranamukhaarachchi1 , R. Puri2 , J. C. Fuscoe3 , S. Morris3 , G. A. Pennello4 , J. Han2 , T. Han3 , K. Simon4 , S. J. Wang5 , D. Mendrick6 , T. Martinsky7 , R. Elespuru1 , 1CDRH, FDA, Silver Spring MD, 2CBER, FDA, Bethesda MD, 3NCTR, FDA, Jefferson AR, 4CDRH, FDA, Rockville MD, 5CDER, FDA, Rockville MD, 6Gene Logic, Gaithersburg MD, 7TeleChem/Arrayit.com, Sunnyvale CA
Background: FDA is preparing to regulate microarray-based diagnostic devices and to evaluate microarray-based data relevant to the safety and efficacy of diverse products. This project seeks to prioritize sources of variability, and to facilitate product review by focusing regulatory attention on the most critical areas, consistent with the Critical Path Initiative.
Methods: 4 FDA Genomics Labs (CDRH, CBER, NCTR, CVM) are participating in an inter-lab validation exercise examining the following variables: microarray surfaces and chemistries; microarray printing, organization, and feature location; microarray hybridization and processing; sample RNA isolation and labeling; microarray and RNA stability; performance of commercial high density chips. The biological systems are gamma irradiated and unirradiated cell cultures.
Results: 1. A fractional factorial statistical design was created, allowing the efficient comparison of diverse variables. 2. Known gamma irradiation-induced genes and several controls were selected, oligonucleotide probes were designed, and labeling options were tested for different microarray surfaces. 3. A microarray printing design was created that addresses elements of feature location and intra-array variability. 4. Pilot experiments were performed with TK6 cells exposed to different doses of gamma radiation. 5. RNA was isolated from irradiated and unirradiated cells. Genomic profiles of cells irradiated at different doses were generated and compared with unirradiated controls, using our oligonucleotide arrays. Data are still being evaluated.
Conclusions: Experiments are in progress. An inter-center team has been created that is working to debate options and resolve technical issues as they arise. This team includes laboratory and review scientists, statisticians, and external experts
Feasibility Testing and Development of a Rapid Detection and Identification System for Mollicutes Using a Microarray-Based Assay
J. George1 , D. Volokhov1 , S. Liu1 , P. Ikonomi2 , C. Anderson1 , V. E. Chizhikov1 , 1FDA, Rockville, MD, 2ATCC, Manassas, VA
PURPOSE:
Mollicutes contamination is a common problem in research and production cell lines. Traditional systems of detecting these contaminants are resource intensive; use of a rapid detection system would provide a major advantage to maintaining safety and cell function. The results here describe a PCR/Microarray method with the potential to improve the safety of biological products.
METHODS:
Two versions of the microarray method were developed. The first version utilizes short oligonucleotide species-specific probes (20-30 nts in length) for identification of more than 60 species of Mycoplasma, Spiroplasma, and Ureaplasma. The second version employs oligoprobes specific to human pathogens and common cell cultures contaminants among Mollicutes species. The assay procedure includes PCR amplification of 16s-23s rRNA intergenic spacer region (IGS) and subsequent DNA microarray analysis with T7 polymerase transcribed single-stranded RNA molecules from the PCR amplicons.
RESULTS:
Broadly specific primers targeting the IGS were used to amplify 280 strains of Acholeplasma, Entomoplasma, Mycoplasma, Mesoplasma, Spiroplasma, and Ureaplasma. Sequence analysis of Mollicutes IGS revealed unique signature sequences suitable for Mollicutes species identification using microarray technology. Nested primer sensitivity testing resulted in successful amplification of approximately one genomic copy of Mollicutes DNA. Based on genetic divergence in the IGS region, two versions of the microchip were capable of identifying their target Mollicutes species.
CONCLUSIONS:
Sequence data has shown the 16S-23S rRNA IGS region to be a suitable marker for Mollicutes detection and identification. These PCR primers are very sensitive and highly specific. Evaluation of the microarray chips by hybridization with their appropriate target samples has demonstrated the feasibility of this method. These results demonstrate the potential of this highly sensitive and specific PCR/microarray system for detection and speciation of Mollicutes contamination in biological products and research applications.
Evaluation of multiple NAT and serologic assays for detection of HIV variants in Cameroonian blood samples
J. Hu1 , S. Lee1 , S. Kerby1 , O. Wood1 , A. Machuca1 , S. Daniel1 , M. Rios1 , A. Bih2 , L. Zekeng2 , I. K. Hewlett1 , 1CBER, FDA, Rockville, MD, 2Cameroon Ministry of Health, Yaounde, Cameroon
Background: Several serologic assays have been licensed for detection of antibodies against HIV in blood donors. Two NAT assays have also recently been licensed for early detection of HIV in blood donor samples. However, the sensitivity of these assays for detection of HIV variants has not been fully established. We evaluated a number of commercially available assays for their ability to detect HIV in samples from Cameroon where a number of diverse HIV strains are known to exist.
Methods: 239 plasma samples from two blood donation sites in Cameroon were tested in this study. Of these 149 were determined to be positive and 90 negative based on a rapid antibody test in routine use in the country. In our study we used 2 qualitative NAT assays, 1 quantitative NAT assay, an HIV-1 p24 antigen assay, 3 licensed antibody assays and an IFA. All reactive samples were further tested by a Western blot assay.
Results: Of the 149 samples previously determined to be positive,131 were confirmed for their HIV status by all assays. 4 samples were negative by all methods and determined to be negative. 14 were discordant among the various EIA and NAT assays. 5/14 were confirmed as positive based on Western blot, 4 had indeterminate band patterns, and 5 were negative by WBlot and NAT. 2/90 negative samples were positive by 2 EIAs and 1 NAT assay and confirmed by Western blot. 6 additional samples were reactive on at least one of the EIA tests but only one had an indeterminate pattern, all others being negative by WBlot. All samples except one were negative by the HIV-1 p24 antigen assay.
Conclusions: Our results indicate that although HIV assays currently in use in the US are highly sensitive for detection of viral variants, some samples may not be detected by all assays. The results suggest that surveillance studies to determine test performance with viral variants in areas where they are prevalent are a useful means to the impact of viral diversity and evolution on assay performance. Genetic sequencing is being performed on plasma viral RNA isolated from the discordant samples to determine if they could represent new variants.
Use of Standards in the Review of Medical Devices
C. Ho, D. Jensen, F. Lacy, N. Muni, S. Reilly, E. Mallis, CDRH, FDA, Rockville, MD
The U.S. Food and Drug Administration's (FDA) Center for Devices and Radiological Health uses a myriad of standards in order to facilitate the review of premarket submissions of medical devices. The benefits of using standards in this manner include providing a set of common requirements and test protocols to the device manufacturer, thus reducing the manufacturer's need to 're-invent the wheel' for each new bench test to ensure safety and effectiveness of the device. Further, with the present trend towards international harmonization of standards, tests performed in accordance with an international standard may be acceptable to several countries. However, there are instances when FDA does not agree with a few, or many, provisions in a standard. This article aims to clarify the approaches taken by FDA to balance or resolve disagreements. One approach begins with the recognition of only some provisions of a standard, or more commonly, excluding those parts of a standard that are unacceptable to FDA. Other approaches include working with the Standards Development Organizations in order that the standard can be revised to include language more agreeable to all parties involved. Specific examples will be presented on medical devices, such as ECG cables and connectors, and non-invasive blood pressure monitors.
Differentiation of Generic and Innovator Pharmaceutical Products Using Near Infrared Spectroscopy
S. Jenney1 , A. R. Bryant1 , A. L. Hoskins1 , S. H. Colson1 , R. L. Scott1 , B. J. Westenberger2 , J. A. Spencer2 , 1CDER, FDA, DPA, Silver Spring, MD, 2CDER, FDA, DPA, St. Louis, MO
Background: This study was undertaken to evaluate the ability of near infrared reflectance spectroscopy (NIR) to differentiate between generic and innovator products during bioequivalence studies. The goal is to replace the time-consuming and labor intensive traditional methods currently being used (FT-IR, TGA, and Physical Characteristics) with a faster spectroscopic procedure.
Methods: Ten official bioequivalence sample sets, consisting of an innovator and one or more generic products, received for testing were evaluated simultaneously by the traditional methods and NIR. Tableted samples were scanned directly (non-destructive) and after surface sanding (destructive) to remove any effects from coatings. NIR spectral data were processed using the chemometric tools PCA (principal component analysis) and SIMCA (soft independent modeling of class analogy).
Results: In all 10 cases, PCA treatment of the NIR spectral data enabled differentiation of the generic product from the innovator's product. The traditional methods were also able to distinguish generic from innovator products but there were numerous instances where one or more of the traditional methods were unable to make an unequivocal distinction. Additional comparisons of the NIR spectra were made using the SIMCA technique to determine similarities or differences between the innovator and generic samples.
Conclusions: These results show the potential use of NIR spectroscopy as an alternative method for rapidly differentiating innovator and generic pharmaceutical products.
A Three-Phase Evaluation of the Feasibility to Extend the Storage Life of In-House Prepared Plates of Xylose-Lysine-Desoxycholate and Hektoen Enteric Agar Media for Salmonella Recovery in Foods
J. A. Kinney1 , T. B. Bickell1 , E. W. Laster1 , C. Ramirez1 , J. N. Sofos2 , 1FDA, Denver District Laboratory, Denver CO 80225, 2Colorado State University, Ft. Collins, CO 80523
Xylose-Lysine-Desoxycholate (XLD) and Hektoen Enteric (HE) agar media are used for detection of Salmonella in FDA regulated products. The current Bacteriological Analytical Manual (BAM) Online states that both agar media should be used within one day after preparation; commercially available agar plates have a storage life of at least four weeks. A three-phase study was conducted to compare Salmonella recovery with freshly prepared and plates stored under refrigeration (4oC) . Results of the first phase indicated no differences (p>0.05) in colony counts of Salmonella Gaminara recovered with freshly prepared or stored (60 days) plates. Phase two indicated no difference in colony counts recovered by fresh and stored (45 days) plates for 20 different Salmonella serotypes. The final phase was conducted with thirty samples of food products that initially had tested Salmonella negative. The samples were inoculated at <70 CFU/g with S. Gaminara or S. Aberdeen and analyzed according to BAM using fresh and stored (45 days) agar plates. There were no observable differences in agar plate appearance, colony size and density or morphology between fresh and stored agar plates. Isolates from fresh and stored XLD and HE agar plates were confirmed as Salmonella with biochemical tests. The results indicated that in-house pre-prepared and refrigerated (4oC) XLD and HE agar plates were as effective as one-day old plates in Salmonella recovery. Storage of plates for 45 days at 4oC did not affect the appearance of the agar or the colonies formed, and allowed similar colony recoveries as freshly prepared plates.
A Sampling Strategy for Monitoring Adverse Event Coding Errors in e-AER Submissions
Q. H. Li, Y. Tsong, S. Machado, CDER, FDA, Rockville, MD
In post-marketing electronic Adverse Event Report (e-AER) submissions, the adverse events have been coded by manufactures using MedDRA. To ensure the quality of the coding in FDA's post-marketing adverse event database, 100% checking was implemented for e-AER submissions. However, 100% check was inefficient and costly. We developed a three-stage cost-efficient feed-back controlling sampling strategy. Using this strategy, the database error rate can be controlled under 1%. This sampling plan is sensitive to the change of the report error rate and can adjust sampling rate accordingly. Simulation studies are conducted to exam the performance of this sampling plan.
Development of Digoxigenin Labeled ipaH DNA Probe for Shigella spp. Detection
W. S.. Lin, C. Cheng, K. T.. Van, PRL-SW, ORA, FDA
The FDA Pacific Regional Laboratory - Southwest has developed two-step enrichment and real-time PCR procedures with high sensitivity (< 1 CFU/100 mL) and high specificity to detect Shigella spp. in fresh produce. With such high sensitivity of newly developed real-time procedure, Isolation of Shigella from enrichment becomes a must not just to validate real-time PCR positive results but also to proof the presence of viable Shigella in violative samples for taking any legal action.
Because the similar morphological appearance, it is always a challenge to isolate low cell number of Shigella from high number of background flora on McConkey or other agar plates. The enrichment does not guarantee the Shigella cells will be increased enough for visual selection. Therefore, a selection tool such as digoxigenin (DIG) labeled DNA probe is a feasible choice to identify Shigella among crowded background flora.
A 100-bp fragment within 181-bp fragment of Shigella specific ipaH gene amplified by real-time PCR is chosen to label with DIG using random primed DNA labeling method. The DIG labeled probe is then used to identify Shigella colonies among high background flora on the plates.
The DIG labeled probe has been tested with colony blots of 43 Shigella strains and 62 non-Shigella in house strains. The DIG probe hybridized with all 43 Shigella strains plus 3 enteroinvasive E. coli strains. But did not hybridized with the remaining non-Shigella strains. The results of this study showed the DIG labeled ipaH DNA probe has great potential to identify Shigella colonies among crowded background flora on the agar plates.
Potential Impurities Generated from Ethylene Oxide Sterilization Process
H. S. Khorshidi, Y. Lu, CDER, FDA, Rockville, MD
Ethylene oxide (EtO) treatment is a common process for the sterilization of the packaging components. The technology involves exposure of the packaging components to ethylene oxide gas followed by an aeration process. It has been found that the residual ethylene oxide if trapped in the container/closure system could react with the active drug substance (mainly in solution formulations) and result in the formation of unexpected impurity(s). Such an EtO-drug substance impurity has been observed in a commercial product. Particularly, if the drug substance contains some nucleophilic functional groups or moieties, such as acid anions, alkoxide anions and any neutral molecule that has an unshared electron pair, a SN2 nucleophilic reaction of the drug substance with the ethylene oxide will occur. The EtO-drug substance impurity with a mass increase of 44 (-CH2CH2O-) will be formed. To verify the origin of these impurities (interaction between the drug substance and ethylene oxide or its residuals), a solution of the drug substance may be designed to react with each of the chemicals such as carbon dioxide, ethylene oxide, formaldehyde, ethylene chlorohydrin, ethylene glycol, and isopropyl alcohol under the stressed condition (70oC for couple of hours). To avoid the formation of these impurities, the aeration time of the packaging components should be evaluated to control the ethylene oxide residual level in the container/closure system. Alternatively, gamma irradiation or heat sterilization rather than EtO sterilization could be considered.
Normal Flow Virus Filtration - Detection and Assessment of Endpoint in Bioprocessing
S. Lute1 , G. Bolton2 , M. Cabatingan2 , D. LaCasse2 , M. Rubino3 , M. Bailey3 , K. Brorson1 , 1DMA, CDER, FDA, Bethesda, MD, 2Millipore Corp., Bedford, MA, 3Eli Lilly & Co., Indianapolis, IN
The breakthrough of a model virus, bacteriophage φX174, through Viresolve NFP filters was studied using both commercial process fluids and model feedstreams. The results indicate that: (1) φX174 is a reasonable model for a mammalian parvovirus (MMV) in virus filtration studies; (2) φX174 LRV shows a better correlation with percent flow decline than with volume processed under a variety of conditions; (3) While the extent of decline in virus LRV is dependent on the mechanism of filter fouling, the fouling mechanisms operative in a viral validation study are representative of those likely to be found under actual production conditions. Process impurities (DNA, φX174, and aggregated protein) can theoretically affect ΔLRV of a NFP filter during processing. We find that high levels of φX174 decrease ΔLRV, high levels of DNA lower ΔLRV, and protein aggregation and virus spike quality were found to have no effect on the ΔLRV. Thus, validation variables appear to have a clear impact on LRV loss and we believe that the LRV vs. flow decay relationship should be evaluated on a product specific basis.
Robustness of virus removal by Protein A chromatography is independent of media lifetime and mechanism of degradation
S. Lute1 , L. Norling2 , M. Hanson3 , Y. Xu4 , G. Blank2 , Q. Chen2 , K. Brorson1 , 1DMA, CDER, FDA, Bethesda, MD, 2Genentech Inc., So. San Francisco, CA, 3University of Maryland, Baltimore County, Baltimore, MD, 4Chiron Inc., Emeryville, CA
Chromatography media is often re-used in commercial manufacturing until performance begins to decline, up to 100-250 times. Establishment of useful media lifetime is critical because declining performance of the media may lead to undesirable outcomes such as low step yield and diminished virus or cell culture impurity removal. Our previous studies have shown that protein A unit operations clear viruses by allowing them to flow uninhibited through the column during Ab capture.
In this follow-up study, we used commercial process intermediates in scaled down studies with media from four vendors with four different backbone matrices (agarose derivative, porous glass, ceramic/polystyrene, and polystyrene/divinylbenzene) to determine if decay more catastrophic than ligand loss could impact viral clearance by protein A chromatography. We confirmed that uninhibited flow of endogenous retrovirus particles during loading is not impacted by loss of Ab capture capacity, compaction, bead size distortion or resin fouling caused by extensive cycling under forced degradation conditions. When repacked, the multiply-cycled resins also cleared three other viruses (SV40, X-MuLV, and MMV) comparably to naïve resins. Thus, virus clearance by protein A chromatography appears to be extremely robust.
Materials Issues in the Post-Market Experience of the Ferric Hyaluronan Adhesion Prevention Solution (Fe-HA).
M. D. Luu, I. S. Isayeva, K. Vorvolakos, D. V. Patwardhan, S. Das, CDRH, FDA, ROCKVILLE, MD
Background: There are approximately 5 million general and ob/gyn surgeries each year in the US. Studies have shown that 50-95% of patients develop post-operative abdominal-pelvic adhesions. We present a case of post-market incident involving Intergel®, a 0.5% ferric hyaluronic acid (Fe-HA) gel used as preventive abdominal pelvic adhesion barrier in women.
Methods: We synthesized Fe-HA gels using two different sources of HA and varied cross linking density and HA concentration. We measured HA molecular weight and molecular weight distribution by GPC, gel cross linking density by titration and gel viscosity at different shear rates and temperatures by viscometer. In addition we evaluated gels using DMA and FTIR.
Results: Preliminary experiments suggest that the Intergel® formulation procedure produces non-homogeneous Fe-HA gels due to poor control of cross linking uniformity. Phase separation of the Fe-HA gels into phases of low and high gel density was visually observed during formulation. Viscosity of the in-house freshly made Fe-HA gels differed widely from that of the original Intergel® at the end of its two-year shelf life. The FTIR spectra of an explanted patient tissue sample suggest post op residual Intergel®.
Conclusion: We believe that the lack of manufacturing control process for the percent cross linking of Fe-HA could lead to non uniform visco-elastic properties, and compromise its ability to spread and degrade in vivo. This could explain for the adverse events which include post-op pain, non infectious peritonitis and foreign body reactions.
EVALUATION OF K6/ODC TRANSGENIC MICE AS A DERMAL CARCINOGENICITY MODEL FOR ONCOGENIC DNA
T. J. Miller1 , P. Espandiari1 , R. Honchel1 , A. Knapton1 , J. Zhang1 , F. Sistare1 , L. Sheng2 , A. Lewis2 , K. Peden2 , J. Hanig1 , 1CDER, FDA, Silver Spring, MD, 2CBER, FDA, Rockville, MD
Transgenic mouse models can serve as sensitive, short-term alternatives to traditional rodent 2-year cancer bioassays. K6ODC transgenic mice develop epidermal tumors when exposed to carcinogens via the promotional stimulus of enhanced expression of ornithine decarboxylase in keratinocytes. K6ODC mice were used in a 36-week study to test their susceptibility to oncogenesis by a plasmid expressing the human T24-H-ras gene, which was developed to evaluate risks posed by residual DNA in vaccines produced in neoplastic cell substrates. Male K6ODC and C57BL/6 mice (15 per group) were treated with H-ras plasmid (2.5, 250, 25000 ng), vector DNA, or saline on scarified skin or by subcutaneous injection. One group of K6ODC mice received a single exposure of 7,12-dimethylbenz-[a]anthracene ([DMBA], 200 nM) dermally. Microscopy of skin, heart, lung, liver, and spleen were performed after unscheduled euthanization or at study completion. DMBA-treated K6ODC mice developed superficial papillomas by 6 weeks that increased in incidence to 25 weeks (9±0.69 tumors/mouse, < 3mm, 13/15 responded). No tumors were detected in other groups in either mouse strain. Microscopy of skin sections confirmed papillomas in DMBA treated animals with dermal/sebaceous gland hyperplasia and follicular dystrophy in all skin samples, characteristic of this strain. Tissue analysis of K6ODC mice revealed amyloid deposition and neutrophilic infiltration within liver, heart, and spleen, and splenic atrophy regardless of treatment. Pathology was not detected in C57BL/6 mice. After week 15, 94% (126/134) of the K6ODC mice developed unexpected dermal eczema (unrelated to either treatment regimen) requiring unscheduled euthanization. By week 32, six of the eight surviving K6ODC mice showed difficulty in mobility and loss of balance. In this study, the K6ODC failure to develop papillomas to oncogenic DNA, progressive adverse health, and decreased long-term survival suggest that K6ODC mice are an inadequate alternative model for oncogenic DNA and chemical carcinogenicity testing.
Evaluation of Microarray Data Variability within Commercial Microarray Surfaces
M. Schneider1 , A. D. Nandanie2 , S. Lababidi3 , D. Ranamukhaarachchi2 , 1Butler University Indianapolis, IN, 2CDRH, FDA, Silver Spring, MD, 3CDRH, FDA, Rockville, MD
Gene expression-based microarray technology is still maturing to be included in regulatory perspectives for decision-making in public health. Generating quantitative data in expression microarray devices demands robust control standards for data normalization. This is relevant to FDA's critical path initiative, in that, producing consistently high quality microarray devices and proving the efficacy to determine the product that will have medical benefit are important aspects. Here, we evaluated data variability that exists within a given commercial microarray surface using invitro-transcribed mRNA with known abundance from Arabidopsis thaliana genes. We printed gene-specific oligonucleotide probes for a subset of 10 genes across the entire microarray surface. Fluorescence intensity signals of each corresponding gene hybridized onto the surface were determined at different times post hybridization. The results indicated that extensive data variability exists across the entire array surface. This data variability depended on the type of surface used: some commercial surfaces produced more variability compared with the other surfaces. Different hybridization times tested indicated that variability exists at every time point tested. This study confirms the requirement for positioning reference control standards representing the entire microarray surface for normalization of data.
One-sided and Two-sided Sequential Tests for Quality Assurance of Drug Products
Y. Tsong1 , M. Shen2 , 1CDER, FDA.Rockville, MD, 2CDER, FDA, Rockville, MD
In order to assure that the finished drug product is both safe and effective, quality assurance tests are often conducted to infer if the lot satisfies the required specifications of the chemical characteristics. Typically, the specifications are defined as a requirement as a limiting percentage(s) of lot outside a pre-specified limit(s). Parametric tolerance intervals and sampling acceptance rules are the most commonly used approaches. The concept and mathematics of the two approaches are different. The two approaches are exchangeable for one-sided specification. When using for two-sided specification, the two approaches become deviate from each other. When the two-sided specification is limiting the total percentage of lot outside the limits, the tolerance interval approach becomes conservative. On the other hand, when the two-sided specification is limiting a percentage outside either the lower or upper limits, the sampling acceptance approach becomes over-liberal. The properties are illustrated with examples.
Parametric Two-Stage Sequential Quality Assurance Test of Content Uniformity of Drug Products
M. Shen, Y. Tsong, CDER, FDA, Rockville, MD
The USP content uniformity sampling acceptance plan consists of a two-stage sampling plan with criteria on sample mean and number of out-of-range tablets was the standard for compendium. It is however often used mistakenly for lot quality assurance. A parametric tolerance interval procedure is proposed to test a two-sided specification that is equivalent to quality test of two one-sided hypotheses. Testing against a lower specification is to assure that the drug product is not under-doses for efficacy. On the other hand, testing against an upper specification is to assure that the drug product is not over-dosed for safety. The operating curves of the proposed procedure are compared with that of the USP test to illustrate the difference in acceptance rate against specified means and variances of the lot.
A Statistical Power Analysis for Average Bioequivalence Study Using Replicated Crossover Design: Case Study
D. S. Yim1 , S. M. Chung2 , S. J. Lau2 , 1UCSF, 2OCPB
Background: Replicated 4-way crossover study design for bioequivalence (RBE) has been recommended to characterize intrasubject variability since the FDA guidance for bioequivalence (BE) was published in 2001. This study was carried out to predict the statistical power for the average BE test results from a RBE study and to compare the statistical power between RBE and a conventional 2-way crossover study for BE (CBE) for a drug with relatively irregular oral absorption profile.
Methods: To analyze statistical power, 1,000 BE studies for each of RBE and CBE were simulated per situation of varying numbers of subjects. To generate 1,000 RBE datasets and 1,000 CBE datasets per situation, a non-replacement re-sampling method was applied to a RBE data in 65 subjects from an NDA for Drug X. The number of subjects per dataset for each situation increased by four starting from 16 to 64. The re-sampling and BE-test were performed using S-plus (Ver. 6.2) and SAS (Ver. 8.02), respectively. Percentage of the RBE versus CBE datasets which met the average BE criteria were compared through the situations.
Results: The statistical power to pass the BE criteria increased with the number of subjects per dataset, as expected. The power greater than 80% for both of AUC and Cmax could be achieved at 32 subjects for RBE and 64 patients for CBE.
Conclusions: Even for a drug having low bioavailability and an irregular oral absorption profile such as drug X, CBE method could present satisfactory results after doubling the number of subjects used for a RBE study.
Making FDA's Toxicological, Human Adverse Effect, and Chemical Data Available at Your Desktop
R. D. Benz1 , N. L. Kruhlak1 , M. E. LaVecchia2 , K. Sarig3 , A. Jaffrey4 , E. J. Matthews1 , J. E. Biles2 , D. W. Goldman3 , H. Trinh5 , J. F. Contrera1 , 1US Food and Drug Administration, Center for Drug Evaluation and Research (HFD-901), 5600 Fishers Lane, Rockville, MD 20857, 2US Food and Drug Administration, Center for Food Safety and Applied Nutrition (HFS-206), 5100 Paint Branch Parkway, College Park, MD 20740, 3GlobalNet Services, Inc., Suite 310, 11820 Parklawn Drive, Rockville, MD 20852, 4Caspian Sea, LLC, 307 Long Trail Terrace, Rockville, MD 20850, 5US Food and Drug Administration, Office of the Commissioner (HF-33), 5600 Fishers Lane, Rockville, MD 20857
In collaboration with FDA/CFSAN's Office of Food Additive Safety (OFAS), FDA's Office of Science and Health Coordination, GlobalNet Services, Inc., and Caspian Sea, LLC, FDA/CDER's Informatics and Computational Safety Analysis Staff (ICSAS) has created a Web-based, searchable, relational database to link FDA toxicological, adverse human effect, and chemical information databases and make them available to all FDA employees through the FDA Science FIRSt Intranet Website. This Webpage allows sharing at one location safety data from multiple FDA sources among all Agency components. The key field of the CACTVS software-based master database is the chemical structure which permits unambiguous chemical identification regardless of multiple names, synonyms, trade names, and identification numbers used. Structure-similarity searching is offered so that historical safety data on chemicals very similar to any of current interest can be readily obtained. Data can be retrieved from the master database in multiple formats, including Excel and formatted text. ICSAS compiled and maintains the chemical structure dictionary for all pharmaceutical products and the database components containing the results of their pharmaceutical toxicological studies and adverse human effect reports. OFAS compiled and maintains the chemical dictionary of all food additive products and the database containing their toxicological studies. GlobalNet Services, Inc. and Caspian Sea, LLC are providing software development expertise for this project. FDA's Office of Science and Health Coordination is supporting the server platform. Participation in this cross-Agency data sharing effort by other FDA Centers is invited.
Outreach in the Division of Biotechnology and GRAS Notice Review-Explaining FDA's Policy on Bioengineered Foods and the Consultation Process to Diverse Audiences
S.J. Carlson, CFSAN, Office of Food Additive Safety, Division of Biotechnology and GRAS Notice Review
The U.S. regulatory system for evaluating food safety is influential in the global marketplace. Consequently, the safety evaluation of bioengineered food is of interest to governments around the world. U.S. regulators have more than 10 years of experience in this arena. The Division of Biotechnology and GRAS Notice Review (DBGNR), Office of Food Additive Safety, hosts many international visitors each year who are interested in how FDA regulates bioengineered food. For the year 2004, representatives from 24 different countries participated in presentations given by DBGNR scientists. A diverse group of countries were represented ranging from Albania to Uruguay with participants from academia, regulatory agencies, legislative representatives, and non-governmental organizations. The international requests come to our division from a variety of sources, including the U.S. State Department and the U.S. Department of Agriculture. In addition to the international visitors, in 2004 we received four requests for presentations from domestic groups. These included university students and developers of small crops. The presentation given to these groups varies somewhat according to the interests of the audience. In our poster, we will present background information regarding the origins and affiliations of our audience. We will also present the elements of a typical biotechnology presentation given to interested visitors. These elements include:
Using the CFSAN Thesaurus to improve scientific communication
E. A. Reinhold1 , L. A. Strasser1 , D. M. Schmit1 , L. R. Dusold2 , 1CTS of Maryland, 2CFSAN, FDA, College Park, MD
BACKGROUND:
Information on the CFSAN Website http://www.cfsan.fda.gov contains specialized, technical vocabulary. Studies have shown that most users of online health information search for information about specific conditions. However, misspellings often result in search engine failure. Terms like Enterobacter sakazakii or acrylamide are often misspelled in our web search. We can leverage the 35+ years of expertise in the CFSAN Thesaurus to improve our communications with stakeholders.
METHOD:
RESULTS:
Lexicon users reported fewer misspellings in their documents. Users of the search with spell checking reported a higher rate of relevant search results when the original misspelled term would return few or no results.
CONCLUSION:
Lack of familiarity with technical vocabulary is an obstacle to public health communication. Our techniques utilizing the CFSAN Thesaurus in electronic and web applications expand access to technical vocabulary. This will help improve agency communications and stakeholder access to FDA information.
Committee for the Advancement of FDA Science (CAFDAS): The FDA Scientist's Liaison to the Commissioner's Office
N. Alderson1 , S. N. Ali2 , A. Debrabant3 , C. Elkins4 , R. M. Fahmy5 , J. V.S. Gobburu6 , M. Grant2 , J. Johannessen1 , M. Kulka7 , M. Major3 , M. Manjanatha4 , S. Stern8 , H. Trinh1 , R. Uppor6 , J. Ward5 , G. Wood7 , T. O. Woods8 , 1OC, FDA, Rockville, MD, 2ORA, FDA, Rockville, MD, 3CBER, FDA, Rockville, MD, 4NCTR, FDA, Rockville, MD, 5CVM, FDA, Rockville, MD, 6CDER, FDA, Rockville, MD, 7CFSAN, FDA, Rockville, MD, 8CDRH, FDA, Rockville, MD
The Committee for the Advancement of FDA Science (CAFDAS) serves as an internal advisory committee to the Commissioner, Associate Commissioner for Science, and the Senior Science Council. Functioning independently of center or discipline, CAFDAS addresses FDA-wide science issues from a working scientist's perspective. It is composed of two members from each Center and ORA who represent research, review and compliance aspects of the FDA mission; each member serves a three-year term. The primary objective of CAFDAS is to aid in enhancing the agency's science infrastructure by: serving as a medium for advancing ideas and concerns from scientists to senior FDA management regarding the state of science in FDA; providing comments to the Commissioner on science policy questions and planning; and offering practical suggestions and constructive solutions to achieve Agency-wide scientific excellence. CAFDAS activities include review of OSHC collaborative science project applications and summarizing leveraging information on competitive research funding sources/mechanisms. In cooperation with the Office of Science and Health Coordination (OSHC) and the Office of the Commissioner (OC), CAFDAS initiated "The Commissioner's Seminar Series." This internal FDA seminar series highlights FDA science pertaining to the "Critical Path" and promotes the recognition of mission-based science through plain-language presentations suitable for an audience with diverse backgrounds and experience. More information regarding CAFDAS' history, meeting minutes, leveraging activities and The Commissioner's Seminar Series is available at: http://first.fda.gov/cafdas.
Old Borax
E. Grundel, CFSAN/ONPLDS/DRAT, HFS-840, 5100 Paint Branch Parkway, College Park, Maryland 20740
Preparations are underway to observe the centennial of the law that would eventually lead to the establishment of the Food & Drug Administration. Much of the focus of these celebrations will center on the man largely responsible for that law: Dr. Harvey Washington Wiley. Who was he? Documents from his personal papers reveal aspects of his public and private life that give insights into someone who has achieved the status of a legend.
Role and Use of Guidance Documents and Standards for Submission of Applications for Combination and Human Cell, Tissue, and Cellular and Tissue-based Products to the Food and Drug Administration
D. S. Kaplan, V. M. Hitchins, CDRH, FDA, Rockville, MD 20850
Introduction: Combination and human cell, tissue, and cellular and tissue-based (tissue-engineered) products present unique challenges for FDA to ensure that they are safe and effective. Combination products containing natural materials have some degree of biological variability. Those products containing biological material may not be easily sterilized.
Current Regulatory Practice: Sponsors wishing to submit an application for combination and tissue-engineered products to the FDA should contact the FDA Office of Combination Products, which can assign an FDA Center to have primary jurisdiction for the review of the product, based on the primary mode of action. The Office of Combination Products can be reached at: (301) 427-1934 or by email: combination@fda.gov . The FDA is involved in writing guidances and voluntary national and international consensus standards to help assess safety and efficacy of FDA regulated products. For those products that will be reviewed by CDRH, CDRH recognized consensus standards may be considered in premarket applications. A complete listing of CDRH recognized consensus standards and guidance documents can be found at: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfStandards/search.cfm
Summary: The present guidances and standards do not adequately address all the unique challenges posed by these new combination products and tissue-engineered products. Thus, guidances and standards are being developed to address standardization of biological, chemical, physical and mechanical properties of natural materials used in combination and tissue engineered products.
THE FAILED PRECEPTS OF CARDIOTOCOGRAPHY IN RELATION TO ADVERSE OUTCOMES OF VACUUM ASSISTED DELIVERIES
B. S. Schifrin1 , D. Marinac-Dabic2 , R. A. Bright2 , T. Rubinstein1 , R. Harwell1 , R. Quintero1 , L. Lewis1 , 1Loma LInda School of Medicine, 2CDRH, FDA, Rockville, MD
Background: Despite the near universal application of cardiotocography (CTG) in intrapartum care, its pitfalls are considerable and its benefits continue to be debated. In theory, CTG is used to detect abnormal patterns (hypoxia) that will permit the timely removal ("rescue") of the endangered fetus in a timely way to avoid death or neurological injury. The study of CTG patterns associated with intrapartum fetal neurological injury, casts doubt on this theory.
Methods: In this case - control study, we compared obstetrical practice preceding Vacuum Assisted Delivery (VAD) of newborns with good outcome born in 4 local hospitals between 1997-1998 (n=192) and those with poor outcomes (n=179) mostly derived from malpractice cases between 1989-1998.
Results: The CTG analysis of infants with perinatal hypoxic-ischemic injury revealed in 21% of cases, a consistent, unique pattern of injury that does not conform to the usual patterns of fetal hypoxia. These patterns, characterized by a sudden change in baseline rate and variability most often appear in the second stage of labor in association with extraordinary expulsive efforts, frequent uterine contractions and occipito-posterior (OP) position. In the majority of these cases, the changes in CTG, though unpredictable, occurred under predictable clinical circumstances for which prevention is a realistic objective.
Conclusions: Understanding of these relationships has implications for the nomenclature of CTG patterns and the conduct of the expulsive phase of labor. The benefits of CTG will be fully realized when we use it, in addition to "rescue", to keep the fetus out of harm's way.
Neurological Implants with Lead Systems and Magnetic Resonance Imaging: Adverse Events and Prevention
R. G. Kaczmarek, N. A. Pressly, T. O. Woods, H. I. Bassen, M. Eudy, A. M. Ferriter, M. J. Hazes, W. Kainz, S. Lange, S. M. Malli, P. S. Ruggera, W. L. Scott, L. Zaremba, CDRH, FDA, Rockville, MD
Purpose: The purpose of this investigation is to ascertain the risks of Magnetic Resonance Imaging (MRI) scans performed on patients with neurological implants with lead systems, communicate to health care providers these risks and measures to reduce such risks.
Methods: FDA adverse event report systems, such as the Medical Device Reporting system (MDR), were searched for reports of adverse events related to MRI scans in patients with neurological implants with lead systems, such as deep brain stimulators. The medical and scientific literature was searched for published articles of relevance to this issue.
Results: Case reports received by FDA have documented the occurrence of serious injury, including permanent neurologic impairment, in patients with neurological implants during MRI scans. In vitro studies have provided compelling data that MRI scans can create electrical currents in leads, resulting in excessive heating. The literature search provided strong evidence that the use of neurological implants with lead systems and the total number of implanted lead recipients will increase substantially in the coming years, increasing the potential for adverse interactions with MRI scans. Risk reduction measures are warranted, including the use of alternative imaging modalities, design modifications in implants with leads that decrease their susceptibility to MR induced heating, and the careful adjustment of MRI scan parameters if MRI imaging is clinically essential.
Conclusions: MRI scans performed on patients with neurological implants with lead systems can create severe adverse effects. The communication to health care providers of risks and measures to reduce these risks is warranted.