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Final Report Abstracts


Before they graduate, each Commissioner's Fellow must prepare a final preceptor-approved project abstract that summarizes the work they've preformed during their two-year CFP fellowship. Fellows abstracts from the Classes of 2011, 2012, 2013, and 2014 can be found below.

Class of 2014

Yu-Ching Cheng, Ph.D. (Nicolette Borek, Ph.D., Preceptor) CTP Fellow; FDA project: Patterns of smokeless tobacco product use among US adults: descriptive findings from the Population Assessment of Tobacco and Health (PATH) Study

The PATH Study is a collaborative effort by the Center for Tobacco Products, FDA and the National Institute on Drug Abuse, NIH to monitor and assess tobacco product use, attitudes, biomarkers and health outcomes. A nationally representative sample of 45,971 youth, young adults and adults aged 12 years and over are participating in the study. The sample includes current, former, and never users of tobacco products. The goal of this research project is to characterize the pattern of use for smokeless tobacco (SLT) products using the national-representative data from the PATH study. We analyzed data from 32,320 adults (aged 18 years and above) in Wave 1 of the PATH Study, to assess the use of SLT products, including moist snuff, dip, chewing tobacco, and pouched or loose snus. Findings from this project, along with data from future waves of the PATH study, will help to better understand the trajectories of SLT use in adults over time and to inform FDA’s regulatory actions related to tobacco products.

Yu-Yi Hsu, Ph.D. (Ram Tiwari, Ph.D., Preceptor) CDER Fellow; FDA project: Hierarchical Bayes Approach for Subgroup Analysis

To better understanding the difference between treatment effects among the demographic subgroups including subgroups defined by sex, age, race and ethnicity, FDA published the action plan to enhance the collection and availability of demographic subgroup data (2014) and suggested to improve the subgroup data collection reporting and analysis quality as one of the three priorities. We use the hierarchical Bayes (HB) to describe data with group factor that could have interaction with treatment effects. The HB approach has been applied to linear regression models (LMs) and linear mixed-effects models (LMMs) with categorical group covariates. A decision procedure for consistency assessment is suggested based on pre-selected decision criterion, using either the posterior probability or the Bayes factor. The decision relies on the concept of having treatment effects in all subgroups consistently in the same positive direction. We also discuss the details of model implementation, including the benefit of using informative half-Cauchy prior for one of the variance parameters that describes the consistency of treatment effects across the subgroups. The decision-making procedures and their properties are illustrated using a simulated example with the normally distributed response and repeated measurements.

Wojciech Jankowski, Ph.D. (Zuben Sauna, Ph.D., Preceptor) CBER Fellow; FDA project: Development and evaluation of novel strategies for characterizing high-order therapeutic protein structure during drug development and manufacture 

Recombinant therapeutic proteins are an important component of the pharmaceutical industry. In 2013, worldwide sales of all biopharmaceuticals reached US $140 billon, and are projected to constitute 20 percent of the overall pharmaceutical market by 2017. Moreover, the impact of these medications is deeper than these numbers alone suggest, because protein-drugs often address unmet medical needs, provide cures, or permit the management of complex diseases thereby improving the quality of life. Protein therapeutics have a number of advantages over small-molecule drugs. They offer highly specific and complex set of functions that small chemical compounds cannot replicate, e.g. enzymatic activity which can be used to correct genetic defects. These molecules however sometimes do not make good drugs and it is becoming increasingly common to engineer protein molecules to improve protein stability, activity, or binding properties. These changes involve the introduction of neo-sequences that do not exist in nature and have the potential to elicit an immune response to the protein. The FDA's "Guidance for Industry - Immunogenicity Assessment for Therapeutic Protein Products" identifies changes in protein sequence as one of the risk factors for immunogenicity.

Recently, Novo Nordisk discontinued development of Vatreptacog alfa following Phase III clinical trials due to the development of anti-drug antibodies. Vatreptacog alfa was intended as an improved bypassing agent providing safe, rapid and sustained resolution of bleeds in patients with inhibitors. Structurally, Vatreptacog alfa was >99% homologous to native FVIIa, with only three amino acid substitutions. We use a broad range of methods that include in silico assessments, computerized modeling, and in vivo and ex vivo assays along with clinical information from the Vatreptacog alfa phase III trial to understand the mechanisms of immunogenicity, particularly the role of an individual patient’s genetics, and use this information to eliminate or reduce the incidence of the immune response to the therapeutic. In this proof-of-principle study, we have generated several peptides corresponding to the immunogenic and de-immunized region of Vetreptacog alfa and evaluated its ability to bind soluble human leukocyte antigen (HLA) proteins. The initial data have confirmed that our in-silico predictions correspond very well to the in-vitro results (reduced binding to a panel of HLA variants by over an order of magnitude). We have also expressed and purified Vatreptacog alfa and demonstrated increased activity when compared to NovoSeven (the currently licensed product) at the same protein concentrations. We have generated over 20 alternative de-immunized Vetreptacog alfa variants and evaluated each of them for activity. All constructs retain enzymatic activity despite the modifications in their primary amino acids sequence. We are in the process of generation of stable cell lines that will be used for protein expression, purification and detailed characterization. In the future, these methods could be used by both industry and regulatory agencies during all stages of the development and review of recombinant therapeutic proteins.

M. Shahjahan Kabir, Ph.D. (Irshad M. Sulaiman, MSc, MPhil, Ph.D., Preceptor) ORA Fellow; FDA project: Development of diagnostic method for rapid detection and differentiation of Bacillus cereus, B. thuringiensis and other closely related species

With the introduction of Food Safety Modernization Act in 2011, the U. S. Food and Drug Administration have prioritized its mission to promote the prevention-based approach for the control and prevention foodborne disease. A critical component of this approach involves optimization and improvement of recovery and DNA-based typing methods for the human-pathogenic microorganisms causing foodborne illness. Bacillus cereus is one of the prominent foodborne pathogen that can infect raw and processed foods and cause diarrhea in humans. Several species of genus Bacillus are considered to be closely related to B. cereus including B. thuringiensis, and has been linked to foodborne outbreaks and sporadic cases of gastroenteritis in humans worldwide. This project yielded additional strategies for the optimal growth and recovery of Bacillus species isolated from foods, cosmetics, and environment samples. A lack of specificity and selectivity with traditional media (MYP and PEMBA) was evident. Nevertheless, the chromomeric media (Bacara and Brilliance) were found more specific for the growth and enumeration of B. cereus and B. thuringiensis isolates. Thus, the chromogenic media are can be used for testing foods and other official samples for Bacillus spp. of public health importance.

Geoffrey Kilili, Ph.D. (Christine Karbiwnyk, Ph.D., Preceptor) ORA Fellow; FDA project: Aptamer based molecular tools for the rapid capture concentration and purification of foodborne pathogens

Aptamers are 3D-structure forming single-stranded DNA or RNA oligonucleotides that have emerged as a more desirable alternative to antibodies. In this report, a series of ssDNA aptamers against the foodborne pathogen E. coli O157:H7 was developed using a modified whole bacterium-based Systematic Evolution of Ligands by EXponential enrichment (SELEX) method. After approximately 10 cycles of selection, the highly enriched pool of oligonucleotides was subjected to next generation sequencing and homology clustering. The top 3 representative sequences were then selected for further characterization using binding assays, differential chromogenic agar and MALDI-TOF MS Biotyper analysis. In addition, 3 published DNA aptamers indicated as specific to E. coli O157:H7 were also included in our study. Results showed that one of the three aptamers selected in this study and one of the three aptamer sequences obtained from literature demonstrated high specificity, selectivity and affinity for whole cell E. coli O157:H7. In addition to the selection of a new DNA aptamer specific to E. coli O157:H7, our results call to question the authenticity of published aptamer sequences and the need for caution when considering them for further applications. In addition, we present an optimized and improved method for the generation of single stranded DNA (ssDNA) for SELEX applications. The most popular method recommends the use of 150 mM NaOH to elute the desired ssDNA strand using the biotin-streptavidin capture system. However, we found that 10 - 20 mM NaOH eluted ssDNA had approximately 4x less dsDNA and streptavidin contamination. Moreover, analysis of ssDNA generated from different streptavidin bead types suggested that various bead chemistries may have different NaOH tolerance limits. Our results not only casts doubt on the validity of using these high molarity NaOH concentrations, but further suggests that care should be taken to empirically determine the optimal NaOH concentrations applicable for each kind of streptavidin bead chemistry.

Catherine McCollum, Ph.D. (Suzanne Hill, Ph.D., Preceptor) CFSAN Fellow; FDA project: A Scientifically Based Guidance to Facilitate Efficiency of Environmental Reviews of Food Contact Notifications involving Antimicrobial Compounds

Assessment of the environmental impacts of Food Contact Substances (FCSs) in compliance to the National Environmental Policy Act (NEPA) necessitates careful and detailed examination. NEPA, passed by Congress in 1969, requires all federal agencies to conduct systematic and interdisciplinary analyses to explore and understand possible environmental risks when reviewing proposed actions (42 USC § 4321). This project was designed to accurately analyze the environmental impact(s) of antimicrobial substances used during food processing and paper/packaging manufacturing. Antimicrobial agents and stabilizers used in their manufacture, typically high in phosphates, may potentially contribute to eutrophication, a process by which a water body acquires a high concentration of nutrients, thereby promoting an excessive growth of algae. The surplus algae compete with other aquatic organisms for available oxygen and sunlight. Fate and transport, and ecotoxicity of single components of antimicrobial agents have been evaluated; however, a comprehensive assessment of how continued use of these antimicrobial FCSs affects the environment has not been conducted. Hence, it is critical that regulatory reviewers are equipped with the most current data and methodologies to ensure that FDA, when complying with NEPA, evaluates environmental impacts using current and relevant science. FDA has not issued guidance regarding the preparation of these documents, there is variability in the quality of the analysis, and extensive deficiencies requiring revision of the documents are common. Hence, the research and analyses conducted in this project will help streamline the pre-market approval process for FDA, and provide direction for reviewing environmental assessments.

Yasith S. Nanayakkara, Ph.D. (Sean W. Linder, Ph.D., Preceptor) ORA Fellow; FDA project: Use of metal nanoparticles with Surface Enhanced Raman Spectroscopy (SERS) for qualitative and quantitative analysis of contaminants within FDA regulated products

Surface Enhanced Raman Spectroscopy (SERS) is a powerful analytical technique. In this project we aimed to: 1) Evaluate the potential of SERS as a detection methodology for chemical contaminants or adulterants in FDA regulated products. 2) Experimentally determine the enhancement factors and limits of detection for specific chemical contaminants. 3) Evaluate the performance limits of SERS on a model analytes [malachite green (MG), brilliant green (BG) and gentian violet (GV] in seafood. A method was developed to extract MG, BG and GV from fish tissues rapidly and the subsequent spectra of MG, BG and GV were highly specific. The method seems to be rapid (<2 hours); however, a number of factors identified that mitigate the quantification. Since both native and leuco forms of MG, BG and GV can be present in parts per million (ppm) levels in fish tissues, regulatory methodology should be sensitive and specific enough to identify at such levels. The established FDA methods, i.e. liquid chromatography – mass spectroscopy and liquid chromatography- UV visible spectroscopy, are sensitive and specific enough to address these issues; however, the analysis time using the established methods is more than 10 hours and the instrumentation is very expensive. In contrast, the SERS method has shorter analysis times as well as a high degree of specificity. With more controlled environment and parameters SERS has potential to achieve the same levels of sensitivity; however, controlling these parameters is challenging. SERS technology is promising, but has limitations related to quantitation for regulatory analysis and reviews.

Laura Ricles, Ph.D. (Steven S. Oh, Ph.D., Preceptor) CBER Fellow; FDA project: Evaluation of additive manufactured products: Current scientific trends, regulatory challenges, and enhancement of FDA review

Additive manufacturing, or 3D printing, involves layer-by-layer deposition of materials with precise spatial control and resolution. This technology is driving innovations in human medical products, including drug, device and biologic. Additive manufactured medical products currently marketed in the United States are mostly devices. As such, the Agency is recently gaining experience in regulating additive manufactured medical devices and has not yet approved any 3D printed biological products. Thus, a rigorous investigation of additive manufacturing technologies and additive manufactured products would be of great value to the Agency. Therefore, the goal of this project was to gather data on additive manufactured products that are regulated by the FDA in order to identify important criteria used to evaluate such products and define current regulatory challenges. In addition, this project aimed to assist the FDA with evaluating additive manufactured biological products (e.g., cellular products, tissue-engineered products) and providing regulatory guidance to stakeholders in developing safe and effective additive manufactured biological products for treating human diseases and conditions. These goals were achieved by conducting a rigorous analysis of FDA internal and public databases and the current scientific literature. In addition, the effects of printing conditions and post-processing steps on the bioburden and biocompatibility of additive manufactured samples were evaluated. The data collected from this project contributed substantially to the Agency’s understanding of the landscape of cleared and/or approved products manufactured using additive manufacturing and the research and development efforts being undertaken with the printing of biologics. The outcomes from this project have been instrumental in ensuring the FDA is aware of the current state-of-the-art and future directions in the field of additive manufacturing. In addition, inter-Center activities and regulatory review work supported communication among CBER, CDRH, and CDER and contributed to guiding FDA regulatory review processes.

Silvia Secelean, Ph.D. (Donna Williams-Hill, Ph.D., Preceptor) ORA Fellow; FDA project: Developing a Whole Genome Sequence Database for Improved Identification of Mycobacteria spp. Isolated from FDA Regulated Foods and Cosmetics

Due to increased reports of non - tuberculosis Mycobacteria (NTM) infections, FDA needs accurate genetic data to perform epidemiological traceback on these outbreaks. Whole Genome Sequencing (WGS) was identified as the optimal means to perform these studies, particularly for its advantages over conventional mycobacterial identification methods: it is a rapid, accurate, and cost effective method. WGS enables considerably better analysis and outbreak investigations than would be otherwise possible. In healthy individuals NTM rarely causes disease, usually occurring after traumatic injuries or cosmetic procedures. NTM infections can become critical in elderly patients, in very young children, and in patients with conditions debilitating the immune system. The goal of this study was to develop a database to assist in WGS identification of NTMs. Highly purified DNA was obtained from NTM strains and sequenced using the de novo assembly technique to build a WGS database that accurately and rapidly identifies the source of NTM in foods and cosmetics.

We obtained DNA from a total of 21 strains of NTM, most cultured in the laboratory, and performed sequencing and assembly by using a combination of technologies: sequencing by synthesis using the Illumina MiSeq, and the CLC Genomic Workbench software to assemble de novo the high-throughput sequencing obtained from the Illumina MiSeq sequencer. In addition, we performed post assembly analyses, including genome description, genome mapping description, and statistical analysis. In this study we generated a preliminary genome assembly database, only. To obtain de novo, true and complete whole genome sequencing database on NTM, further analysis is needed to assess accuracy and to update the database through greater computational analysis using computing resources not available at this time.

Eric Stevens, Ph.D. (Peter Evans, Ph.D, M.P.H., Preceptor) CFSAN Fellow; FDA project: The role of Whole Genome Sequencing in foodborne illness source attribution and its impact on regulatory science

In 2011, the Food and Drug Administration (FDA) along with the Center for Disease Control (CDC) and the United States Department of Agriculture’s (USDA) Food Safety and Inspection Service (FSIS) created the Interagency Food Safety Analytics Collaboration (IFSAC), whose goal is to improve coordination of federal food safety analytic efforts with a focus on food safety topics related to foodborne illness attribution. In 2013, IFSAC completed a Phase I study that looked at Salmonella Enteritidis (SE) source attribution in eggs. The study used a combined analytic method that best estimated SE illnesses attributed to shell eggs and other food commodities (chicken, beef, and produce) using data collected between 2002 and 2009. Phase II began in early 2015 with the goal of applying whole genome sequencing (WGS) to better attribute SE illnesses among different food commodities. WGS data was collected from both environmental and food isolates that had been deposited in FDA’s GenomeTrakr to see if the whole-genome sequences of Salmonella that came from shell eggs could be differentiated from other food commodities. In total, the raw reads from 100 SE isolates (50 from shell eggs, 48 from chicken, 1 cilantro, and 1 chocolate pie) were analyzed. Unfortunately, phylogenetic analysis did not reveal clear and distinct separation between SE isolates derived from egg and chicken; however, smaller clusters made up of five or ten isolates derived from solely chicken or egg were seen. These initial results suggest that while SE that comes from shell eggs is not completely genetically distinct from other food commodities (i.e. chicken), this work may ultimately provide information in which the sequence data from a clinical case of salmonellosis could help inform the epidemiologic analysis by suggesting a potential source of the illness by geographic region or time period. This study also emphasizes the critical importance of combining epidemiological data with the WGS to improve the tracking and tracing and foodborne outbreaks.

Peter Tobin, Ph.D. (Brendan O’Leary, Ph.D., Preceptor) CDRH Fellow; FDA project: Development of a Quality Management System for the Office of In Vitro Diagnostics and Radiological Health

Quality Management Systems (QMSs) are a set of customer and systems focused management techniques that have been used successfully in both manufacturing and service organizations to continuously improve product quality and consistency since the 1950’s. Additionally, QMSs are an important aspect of regulatory science integral to FDA current good manufacturing practices regulation for food, drugs, biologics, and devices. This Commissioner’s Fellowship project encompassed helping to develop a QMS for the Office of In Vitro Diagnostics and Radiological Health (OIR) in order to facilitate regulatory process improvement and improve the consistency of regulatory decision making. Quality management practices were piloted for the Clinical Laboratory Improvement Amendments (CLIA) program while acting as the CLIA program lead. Office-wide projects included developing and implementing a new document control system for OIR and leading a team of division representatives to enhance ISO 9001 capability within OIR. Additionally, new procedures and SharePoint-based tools were developed to allow staff to more easily access training resources and manage records.

Todd A. Townsend, Ph.D. (Mugimane (Manju) G. Manjanatha, Ph.D., Preceptor) NCTR Fellow; FDA project: Modification of the Comet Assay for in vitro and in vivo Assessment of the Global DNA Methylation Status

Assays that determine DNA damage are extremely important in predicting the carcinogenicity of drugs, physical and biological agents of interest to the FDA. The induction of DNA damage by a genotoxic agent is implicated in a variety of chronic human diseases and cancer, and it is becoming increasingly clear that epigenetic effects (such as DNA methylation) also play major roles. DNA methylation provides a stable gene silencing mechanism that, along with histone modification, plays an important role in regulating gene expression, carcinogenesis, and maintaining genome stability. Although enormous progress had been made in using DNA methylation markers in cancer diagnosis and prognosis, the development of a sensitive and fast method for assessing DNA methylation that can utilize single cells isolated from any tissue was determined to be of high value for the field and was lacking at the start of this Project.

The Comet assay is a sensitive and simple technique traditionally used to detect DNA damage in single cells, which can be modified to detect more complex DNA lesions. In this Project we established unique tools and novel methods to combine the Comet assay with restriction enzymes for evaluating global DNA methylation status in individual cells in a high-throughput, cost effective manner to address this regulatory challenge. The successful application of this novel technology will aid hazard identification and risk characterization of FDA-regulated products, while providing utility for investigating epigenetic modes of action of agents in target organs, since the assay is amenable to cells in culture or nucleated cells from any tissue.

Xia Xu, Ph.D. (Paul Morin, Ph.D., Preceptor) ORA Fellow; FDA project: Apply next-generation sequencing technology to subtype Listeria monocytogenes isolates from fish-processing facilities

Listeriosis is the third leading cause of death from infections caused by foodborne pathogens. Listerial infection occurs through ingestion of foods contaminated with Listeria monocytogenes. In food-processing facilities, long-term presence of L. monocytogenes poses a considerable challenge in controlling the transmission of this foodborne pathogen. My regulatory research project involved the application of a novel technology, Next-Generation Sequencing (NGS), to subtype genomes of L. monocytogenes that were derived from fish-processing facilities. These fish-processing facilities have had continuous L. monocytogenes contamination issues over a period of almost 10 years. Our hypothesis was that NGS is a valuable tool for subtyping L. monocytogenes in a regulatory laboratory at FDA. To test this hypothesis, genome libraries of L. monocytogenes strains were prepared by utilizing the Illumina Nextera XT prep kit and sequencing them (2x250bp) using the Illumina MiSeq sequencer. Raw reads of genomic sequences were imported into the CLC Genomics Workbench for bioinformatics data analysis. Several workflows (pipelines) have been built for whole genome sequencing (WGS) data analysis. All the genomic sequence data were characterized with overall high sequencing coverage, high percentages of reads mapping to their best matches, low sequencing gaps, and differing amounts of variants. Based on our phylogenetic analysis, strains of L. monocytogenes can persist in a fish-processing facility for many years. These workflows and SNP-based phylogenetic analysis derived from this study can easily be applied to other important foodborne pathogens, laboratory internal quality control and outbreak investigations. Our study indicates that NGS is a valuable subtyping tool for analyzing L. monocytogenes genomes and that the CLC Genomics Workbench software is very useful for bioinformatics data analysis in a regulatory laboratory.

Jia Yao, Ph.D. (Yongbin Zhang (NCTR) and Dr. Ikram Elayan (CDER), Preceptors) NCTR Fellow; FDA project: Mechanistic Toxicological Evaluation of Engineered Nanomaterial Using a Human Stem Cell Model 

Titanium dioxide (TiO2) has been used in a broad spectrum of consumer products, including food, cosmetics and various medical products. Increased use of nano-scale TiO2 in recent years has raised concern regarding their safety. In the current study, we characterized TiO2 nanoparticles with different crystal structures and particle sizes through electron microscopy, diffraction and scattering techniques. Subsequently, we determined the impact of TiO2 nanoparticles on cell viability using LDH, ATP assays, and the adipogenic differentiation capacity using Oil red O Staining assay in human mesenchymal stem cells (hMSCs). We further investigated whether the impact of TiO2 nanoparticles was associated with specific particle size and/or crystal structure. Data revealed that TiO2 nanoparticles exhibited minimal acute (up to 72 hours exposure) cytotoxicity in hMSCs. There was a size- and crystal structuredependent inhibition of hMSC adipogenic differentiation (21 days) by TiO2 nanoparticles. Cellular uptake and media “stripping” studies indicated that the inhibition of hMSC adipogenesis was likely due to direct cellular response to TiO2 nanoparticles instead of a “charcoal-stripping” effect of TiO2 leading to depleted growth factors in the culture media. Additional exploratory gene expression array analyses suggested that TiO2 nanoparticles inhibit hMSC adipogenesis by down-regulating key genes involved in adipogenesis promotion, including FGF2, IRS1, CEBPA, CEBPB, and ACACB, etc. Findings from this study indicate that TiO2 nanoparticles, while exhibiting minimal acute cytotoxicity, may impost long-term impact on hMSC adipogenic differentiation.

Shadia Zaman, Ph.D. (Darrell Abernethy, Ph.D., Preceptor) CDER Fellow; FDA project: Predicting Cardiac Adverse Events Associated with Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitors (TKIs) are targeted agents that have greatly improved the survival of cancer patients; however, their long-term use leads to toxicity such as cardiac adverse events associated with left ventricular dysfunction. Our hypothesis is that tyrosine kinase inhibitors are targeting proteins in cardiac myocytes leading to left ventricular dysfunction. To identify the mechanism of cardiac adverse event, the PredicTox program was established to build a systems pharmacology model for TKI-associated cardiotoxicity. PredicTox is a public-private partnership that is working on building a knowledge environment to integrate data acquired during TKI drug development and data existing in publicly available databases to learn the mechanism of cardiac adverse events and build prediction models for adverse events. For the PredicTox project, I built an application ontology that will be used for data integration and to discover the genes and biological processes associated with the adverse event. Ontology is a standardized vocabulary to define terms and the semantic relationship between terms for a specific domain of knowledge. They are useful in data integration because biomedical data in different databases are annotated using different vocabularies. Ontologies can be used by a computer program to integrate data using the semantic relationship defined in the ontology. The application ontology that I built will help integrate data across different levels of biological complexity starting from the molecular to the organismal level and will aid in building the systems-level model of TKI-associated cardiotoxicity.

In addition to building the ontology, I investigated the mechanism of cardiac adverse events by examining kinome screens of 22 FDA approved TKIs and built a database of their target profile. These 22 TKIs were linked to cardiac adverse events associated with left ventricular function which were obtained by analyzing FDA Adverse Event Reporting System and FDA Drug Labels. An analysis of this database revealed a set of genes that are targeted more by TKIs that have cardiotoxicity. Gene Ontology Term Enrichment Analysis was performed on this set which demonstrated that they were enriched at a P value of < 10-8 for biological processes involved in regulation of MAPK cascade, vascular endothelial growth factor signaling pathway and regulation of phospholipase activity. Overall, my project advanced our understanding of TKI-associated cardiotoxicity and helped towards building the PredicTox knowledge environment. My work advanced FDA’s mission in its regulatory science priority area of “Modernize Toxicology to Enhance Product Safety.”

Class of 2013

Aaron Bandremer, M.S. (Stephen Torosian, Ph.D, Preceptor) ORA Fellow; FDA project: Development of a field capable device designed to reduce environmental sampling analysis times from 5 days to hours

Effective food safety monitoring is challenged by the necessity to identify human infection capable microorganisms at sufficient infective doses and consumption levels. While improvements have been made to current technologies offering increased sensitivity and decreased sample time, nanotechnology offers the potential for dramatic improvements. Promising new nanotechnology offers the ability to coat a material with a highly conductive co-polymer that does not interfere with the ability of attached receptors (e.g. antibodies) to bind to targets. Such a technology has been incorporated into a biosensor that offers real time environmental monitoring in a field deployable format. As the target binds to the receptor, a detectable change in the electrical properties of the conductive polymer is recorded and correlated to the concentration of the target. We have focused our work on the detection of the pathogenic E.coli strain O157:H7 and have developed a system capable of low level detection in under 30mins.

Aschalew Z. Bekele, Ph.D. (Sangeeta Khare, Ph.D., Preceptor) NCTR Fellow; FDA project: Evaluation of the antimicrobial effects of nanoparticles on gut microbes

Subsequent to the recent advances in nanotechnology, the use of silver nanoparticles (AgNP) as alternative biocide agents has gained momentum. There are now several consumer products on the market that incorporate AgNP as dietary supplements, food packages, hand sanitizers, cosmetics and several household items. It is expected that this widespread use of AgNP may ultimately result in increased human health risk. The most likely targeted site is the gastrointestinal tract following ingestion. Although effects of silver on host cells is well documented, however, the effect of AgNP on the commensal gut microbes (collectively known as microbiome and virome) that play significant roles in host nutrition, immunity and occurrence of diseases is not documented in detail. The objective of this study was two-fold; the first part was to address the virus inactivating properties of AgNP, and the second part was aimed to assess the effect of AgNP on intestinal microbiome.

To address the first objective, variable sizes (10, 75, 110 nm) and doses (25, 50, and 100 µg ml-1) of AgNP were evaluated on feline calicivirus (FCV) and bacteriophage species (PP7, MS2, phiX174, PR772, T7-11303) that represented model for enteric viruses. The antiviral activities of the AgNP were evaluated by comparing the FCV TCID50, appearance of cytopathic effects (CPE), detection of viral capsid protein by western blotting, and the bacteriophages plaque forming units (PFU) ratio of viral suspension treated with AgNP to the untreated controls. The results showed that the 10 nm AgNP at 50 µg ml-1 and 100 µg ml-1 levels were able to completely inactivate FCV in a dose-dependent manner within two to four hours of exposure resulting in loss of up to 6 log10 TCID50 viral titer, absence of viral CPE in Crandell-Rees Feline Kidney cells (CRFK) and significant reduction in viral capsid protein. Similar trends were observed for inactivation of bacteriophages where the 10 nm AgNP resulted in modest reduction of PFU that ranged from 1 log10 to 3 log10 PFU. However, for both the FCV and the bacteriophages, no significant antiviral effect was seen for the 75 nm and 110 nm AgNP. Overall, these results indicate that antiviral effect of AgNP is size dependent. As we observed antimicrobial properties only by the smallest size of AgNP, in the second objective the effects of the 10 nm size AgNP to the non-human primate (Rhesus monkeys) gut microbes were assessed using fecal samples and next generation cultivation-independent 16S rRNA gene sequencing approaches. The Rhesus monkeys fecal microbiota was dominated by the phyla Firmicutes followed by Bacteriodetes, Proteobacteria and Verrucomicrobia. It was evident that the fecal microbial profiles shifted following exposure to low and medium doses (50 µg ml-1 and 25 µg ml-1) of the 10 nm AgNP. There was a notable increase in Proteobacteria (E. coli) and a decrease in Streptococcus (Lactobacilli) in AgNP treated a sample which warrants further investigation. Overall, it is evident that the small size AgNP could inactivate resident gut viruses and may lead to perturbations of the gut microbial ecosystem. Currently a study is under way to evaluate how this microbial shift may affect production of short chain fatty acids that is known to be involved in several inflammatory and metabolic diseases Taken together, the data generated in this study is expected to support the agency’s need to go beyond the current standards of testing for silver toxicity by including guidelines on the safety assessment of the commensal microbes.

Sarah Brittain, M.S. (Aric Kaiser, M.S. (CDRH), Elias Mallis, B.S. (CDRH), Mercedes Serabian, M.S., DABT (CBER), Preceptors) CDRH/CBER Fellow; FDA Project: Evaluation and assessment of FDA review of bone void filler devices with increased osteogenic properties without the use of biological factors

Regenerative medicine products in orthopedics have expanded to include a wide variety of therapeutic devices, including bone void filler (BVFs). These products are typically ceramic based (e.g., calcium phosphate, calcium sulfate, or bioactive glass) and are placed into bony voids or gaps and resorbed and replaced by healing bone. Increasingly, manufacturers are attempting to claim that calcium salt ceramic BVFs in the absence of such entities as cells or signaling molecules may elicit a more active biologic response. Rather than being passive osteoconductive scaffolds, they are claiming that they have active (osteoinductive or bioactive) properties. Recent studies have suggested that manufacturing and processing techniques may in fact produce physical structures and material properties which could induce a biological response after implantation of the BVF. As reviewers of regulatory submissions for these products, it is important to be able to understand the science behind these modifications when deciding if there are any questions related to safety and effectiveness and determining what testing is necessary to be able to validate these claims. The aim of this project was to use literature to identify processing techniques that might be implemented in an effort to increase the biologic response of materials commonly used in BVFs. The manufacturing techniques and methods for determining increased biological response (both in vitro and in vivo) were compared. Over the course of the project, it was identified that there is no standard method to evaluate these claims of increased biologic response. For the tests that were used, it was discovered that small changes in the test conditions may have a dramatic impact on the study results. As a result, this project expanded to also investigate the standardization of test methods related to one of these biologic responses, bioactivity, through collaboration with investigators at CDRH’s Office of Science and Engineering Laboratories (OSEL). A combination of the increased understanding of new calcium salt BVF technologies and standardization of methods to evaluate biologic response will aid in the future regulatory review of these regenerative medicine products.

Bryan Grabias, Ph.D. (Sanjai Kumar, Ph.D., Preceptor) CBER Fellow; FDA project: A Non-amplification, Oligonucleotide-based Sandwich Hybridization Assay for the Detection of Pathogens in Blood

Novel technologies for the sensitive and reliable detection of infectious agents in blood are still needed. While the standard method of nucleic acid-based pathogen detection generally relies on PCR amplification of target DNA or RNA, complex genome sequences can be resistant to amplification, due to factors such as secondary or tertiary structure, and the potential for nonspecific amplification or sample interference could result in false positive or false negative results. Here, we describe a novel nanoparticle-based sandwich hybridization assay (SHA) for the detection of Plasmodium falciparum and Babesia microti parasites without the need for amplification of target sequences in genomic DNA. A uniquely identifiable “barcoded” magnetic microbead and biotinylated silica nanoparticle are conjugated to either P. falciparum- or B. microti-specific 30-mer oligonucleotides corresponding to sequences of the 18S ribosomal gene. For each parasite, the magnetic microbead and silica nanoparticle bead sets hybridize to a unique but adjacent region in the genome. Parasite burden can then be quantified and analyzed upon the binding of an Avidin-PE fluorophore to the target capture complexes via a Bio-Plex 200 instrument. Determination of the analytical sensitivity of the SHA for short complementary oligonucleotide sequences revealed a limit of detection of 10-10 M for both P. falciparum and B. microti probe sets. Analytical sensitivity studies conducted by spiking human blood with known counts of parasites revealed that SHA can reliably detect up to 5 P. falciparum- or B. microti-infected red cells per mL of blood. In comparison, PCR can detect 100 P. falciparum- or 1000 B. microti-infected red cells per mL of spiked blood. Thus, SHA offers a 20-200 fold enhanced sensitivity for the detection of these two intraerythrocytic parasites of global public health significance. Preliminary tests of the sandwich hybridization assay with clinical samples have demonstrated 100% sensitivity. Cross-reactivity studies have demonstrated little to no nonspecific interaction between bead sets for both Plasmodium and Babesia parasites. Further validation of multiplex potential of the sandwich hybridization assay is currently under way. Cumulatively, these performance characteristics establish the value of the sandwich hybridization assay as a sensitive detection platform for blood donor screening.

Rita Humeniuk, Ph.D. (Dionna Green, M.D., Preceptor) CDER Fellow; FDA project: Application of quantitative tools in deriving pediatric dosing for medical countermeasure products

As a nation we remain vulnerable to threats posed by the intentional or accidental use of chemical, biological, radiological, and nuclear agents and emerging infectious diseases. Children in particular are designated an “at-risk” population. Therefore, it is essential that there are safe and effective medical countermeasures (MCMs) available for use in children. The aim of this project was to describe the utilization of regulatory pathways employed to grant marketing approval for MCMs, assess the existing gaps in dosing and approval of MCM products for the pediatric population, as well as evaluate the application of quantitative tools in deriving pediatric dosing for these products.

A sample of medical countermeasures was analyzed. For these products, the approval letter, FDA reviews, updated product label and any other relevant documents were identified and reviewed. The final assessment included the utilization of regulatory pathways employed to grant marketing approval for MCMs, pediatric approval status, analysis of time-lag between pediatric and adult approval, review timeline, and approach to pediatric dose selection. Based on the analysis of the sample of MCM products, we found that recent laws and regulations like the Pediatric Research Equity Act (PREA), Best Pharmaceutics for Children Act (BPCA) and Animal Rule have positively impacted pediatric MCM development. Neonates and infants remain the most commonly understudied subgroup of the pediatric population. Pharmacokinetic modeling and simulations, in particular the population-pharmacokinetic approach, but not the physiologically-based pharmacokinetic approach, were commonly employed to establish dosing of MCMs for the pediatric population.

Considerable progress has been made in providing age-appropriate dosage forms and drug doses for MCMs for pediatric patients. Providing dosing information for agents that in many instances cannot be tested in children is challenging, but innovative clinical pharmacology methods such as modeling and simulation have provided a scientific means of dose estimation. A continuing advocacy for pediatric and neonatal coverage and availability for MCM drugs is necessary for the foreseeable future.

April Marrone, Ph.D. (Frederick Beland, Ph.D. and Igor Pogribny, Ph.D. Preceptors) NCTR Fellow; FDA project: MicroRNA responses to human liver carcinogens: development and evaluation of an in vitro screening model system

Evaluating the carcinogenic risk of chemicals, including pharmaceuticals and hazardous food components and contaminants, is of great importance. Currently, it is estimated that more than 100,000 commercially manufactured chemicals are present in the human environment, and the majority of them have not been tested for their potential carcinogenicity. More importantly, many marketed drugs have not been tested according to current guidelines for carcinogenicity testing. In this study, we aim to develop a high-throughput in vitro screening assay using an epigenetic-biomarker approach to identify potentially toxic and/or carcinogenic substances. Specifically, we investigated microRNA alterations induced by the known genotoxic human carcinogen aflatoxin B1 (AFB1) and structurally similar AFB2 that exhibits a substantially lower genotoxic potential in human hepatic HepaRG cells. Low doses of drugs were used to minimize background changes caused by toxicity alone. The results revealed that treatment of HepaRG cells with AFB1 and AFB2 at the IC10 (90% survival) dose caused significant changes in levels of 10 microRNAs relative to control cells; 4 of these (miR-122, miR-134, miR-362-3p, and miR-410) were unique to AFB1 treatment based on levels in AFB2 treated cells. Single microRNA qRT-PCR assays verified that miR-134 and miR-410 were significantly up-regulated by low-dose AFB1 exposure, and miR-122 was significantly down-regulated. We also exposed cells to a higher dose (IC25) and found that miR-134, miR-410, and miR-122 levels were also altered in AFB1-treated cells. Further assessment of these microRNAs at both IC10 and IC25 doses revealed that miR-122 levels inversely correlate with exposure to AFB1 in a time- and dose-dependent manner. Additional gene expression profiling revealed down-regulation of miR-122 by AFB1 treatment or enforced down-regulation of miR-122 inhibited the expression of CYP2B6, CYP2C9, and CYP2C19. Our data suggest that miR-122 may be a sensitive biomarker for the evaluation of potential hepatotoxicants and carcinogens.

Aftin Ross, Ph.D. (Suzanne Schwartz, M.D., MBA, Preceptor) CDRH Fellow; FDA project: Protecting Health Care Workers from Airborne Transmitted Disease via the Use of Respiratory Protective Devices: A Gap Analysis of the State of Regulatory Science in the Context of Current Practice and Regulatory Policy to Inform Medical Countermeasures for Pandemic Preparedness

Medical countermeasures (MCMs) are drugs, biologics, and devices that can be used to safeguard public health against naturally occurring and manmade threats. Respiratory protective devices (RPDs) are an example of an MCM which play an important role in protecting health care workers (HCWs) employed in areas where patients may be infected with airborne transmitted diseases such as influenza and tuberculosis. In the event of an influenza pandemic (such as H1N1 in 2009), the need for RPDs may far outpace the supply. This results in device shortages which poses a serious public health problem. In anticipation of device shortages in a pandemic, several preparedness strategies for increasing and extending the RPD supply have been proposed. However, these approaches lead to additional challenges in regulatory science, regulatory policy, and healthcare practice which must be comprehensively explored in order to bolster pandemic preparedness. The broad objective of this project is to enhance preparedness in the event of an airborne transmitted disease pandemic.

This objective is first accomplished by examining the current usage of RPDs and challenges encountered in this usage in healthcare delivery (landscaping). Regulatory tools used in the landscape portion of the project included adverse event reports from the Manufacturer and User Device Experience Reports (MAUDE) system and in-depth interviews with healthcare facilities via the MedSun Product Safety Network. No adverse event reports for RPDs were found and an enhanced understanding of the selection and use of RPDs in healthcare delivery for both routine and pandemic scenarios was obtained. The next step was to identify and address regulatory science knowledge gaps related to the use of RPDs (influence factors and gaps). Here human-centered design (HCD), which is the process of developing solutions in the service of people, was leveraged to facilitate stakeholder discussion to identify key challenge areas and potential solutions as a part of a scientific summit meeting. RPD fit and training were the challenge areas/gaps that meeting stakeholders were most interested in addressing. The final part of the project was focused on utilizing the data/information collected to inform regulatory policies going forward (policy). Synthesis and analysis of the aggregate data as well as the creation of a stakeholder map were utilized in the generation of preliminary recommendations concerning harmonization of the premarket review of RPDs as a means to increase RPD pandemic preparedness.

Katherine Segars, Ph.D. (Irshad Sulaiman, MSc, MPhil, Ph.D., Preceptor) ORA Fellow; FDA project: Method development for rapid detection of Cronobacter sakazakii

Rapid detection and differentiation of human-pathogenic bacteria are critical to effecting successful public health programs. Current methodology frequently involves morphological analysis, biochemical profiling and genetic characterization. Cronobacter sakazakii is an emerging opportunistic pathogen which is known to express highly variable morphotypes, and has been historically challenging to differentiate from similar species. Due to its high fatality rate among the susceptible set of human populations, C. sakazakii is considered a top priority of the U.S. Food and Drug Administration. As such, continual evaluation and development of recovery methods are an important part of the agency’s mission. The aim of this research was to supplement available methodologies for rapid identification of Cronobacter sakazakii and related species. Variations in commonly used culture conditions were assessed for morphological and phenotypic impact on the bacteria. A multi-locus sequence typing (MLST) was used to differentiate highly similar Cronobacter isolates recovered from food and environmental samples. The results of this project clearly demonstrated that minor variations in growth conditions can significantly impact the morphology of this microorganism, and MLST is a strong platform for distinguishing between morphologically and genetically similar species and strains of Cronobacter. Thus, creating a comprehensive profile based on morphology, biological properties, and gene sequence is an effective way to identify and differentiate Cronobacter at the species and strain level.

Iwen Wu, Ph.D. (Mercedes Serabian (CBER), Pakwai Au (CBER), Aric Kaiser (CDRH), and Elias Mallis (CDRH), Preceptors) CBER/CDRH Fellow; FDA project: Evaluation of the Immunogenic Potential of Cell-based Regenerative Medicine Products

Cell-based regenerative medicine (RM) products are developed as a method of harnessing the ability of cells to restore, replace, or regenerate damaged tissues and organs. However, the administration of allogeneic cells to the human recipient can potentially elicit an immune response to these foreign cells. The consequence of such a response can be: 1) early clearance of the cells, 2) biological inactivation of the cells and/or 3) development of systemic/local immune-mediated adverse effects. Therefore, prior to administration of an allogeneic cell-based RM product in human subjects, it is important that this concern be addressed as part of the overall safety evaluation. 

Although there are a number of challenges to evaluating the immunogenicity profile of allogeneic cell-based RM products, assessment can be incorporated during different aspects of product evaluation. This can include extensive product characterization, comprehensive preclinical studies, and systematic clinical monitoring. Based on my analysis of Investigational New Drug (IND) submissions for allogeneic cell-based RM products submitted to the Office of Cellular, Tissue, and Gene Therapies in the Center for Biologics Evaluation and Research between 2008 and 2013, results showed that 36% of the INDs included in vitro experiments to evaluate immunogenicity, 20% included immunogenicity outcome assessments in the animal studies, and 68% included clinical monitoring of immunogenicity parameters in the clinical trial protocols. Additionally, mitigation strategies were employed for some clinical trials to address the ‘assumed’ immunogenic response to the respective cell product, such as: 1) prescreening of subjects (recipients) and HLA matching to cell donors, 2) administration of the cells into an ‘immune-privileged’ site (i.e., subretinal), 3) use of clinical immunosuppression regimens, and 4) use of immune isolation devices containing the cells. However, clinical monitoring for immunogenicity was not conducted across all of the INDs and results from monitoring were not consistently reported. Systematic collection and reporting of this clinical data would be helpful in better understanding the immunogenicity profile of this product class. Understanding the immunogenic potential of cell-based RM products upon administration to human subjects is a critical aspect of understanding the activity and ultimately, the efficacy of the product. 

Pin Zhang, Ph.D. (Elizabeth Sutkowski, Ph.D., Preceptor) CBER Fellow; FDA project: Analysis of safety profiles across licensed vaccines

During my two-year FDA Commissioner’s Fellowship, I have worked on a project entitled “Characterization of trends in adverse events across vaccines licensed for infectious disease indications,” Based upon an initial evaluation of the types of licensed vaccines that could be grouped and the data that are available in the FDA database, we decided to focus on licensed influenza vaccines. Seasonal inactivated trivalent influenza vaccine (TIV) contains three influenza viruses (A/H3N2, A/H1N1 and B [either Yamagata or Victoria lineage]). However, two influenza B lineages have co-circulated since 2001, often leading to mismatch between the predominant circulating B lineage and that contained in the vaccine. In order to address this issue, the World Health Organization issued a recommendation to include both B lineages in annual influenza vaccines in 2012, leading to the development and licensure of the inactivated quadrivalent influenza vaccine (QIV). Several published clinical trials have compared the immunogenicity and safety of quadrivalent versus trivalent inactivated influenza vaccines, but to date, no comprehensive and systematic analysis conducted to address the safety profile of quadrivalent compared to trivalent inactivated influenza vaccine has been published. We performed a meta-analysis of adverse events data of clinical trials obtained from the Food and Drug Administration’s database. The data used were from biologics license applications for approved quadrivalent inactivated influenza vaccines only. In pediatric trials, we found significantly increased frequencies of erythema and swelling as well as a statistically decreased occurrence of adverse events in the renal and urinary system organ class in the quadrivalent group versus those in the trivalent group. However, in adult trials, there were no significant differences observed between these two groups. Overall, our findings demonstrated that QIV and TIV have comparable safety profiles from a clinical perspective. This is consistent with the findings in multiple published studies in which pairs of QIV and TIV were compared. Our study confirms that meta-analysis of clinical trials conducted for similar vaccines supports the safety evaluation of these vaccines when viewed individually. This project can provide additional evidence of safety of vaccines to strengthen public trust and could lead to additional areas for research in vaccine development. The findings were presented at both internal and external meetings. We are currently preparing a manuscript to publish the results of these efforts in a peer reviewed scientific journal.

Class of 2012

Michael Bazaco, Ph.D., M.S. (Debra Street, Ph.D., M.P.H., Preceptor) CFSAN Fellow; FDA project: Temporal Trends in Outbreak Attribution of Produce and Juices Compared to Other Food


The Interagency Food Safety Analytics Collaboration (IFSAC) is a federal initiative involving the FDA, CDC and USDA-FSIS. This proposed project will evaluate temporal trends in foodborne outbreak attribution using 14 years of data from the CDC's Foodborne Disease Outbreak Surveillance System (FDOSS). The evaluation will focus on the four IFSAC priority pathogens (Listeria monocyotgenes, Salmonella, Campylobacter and entero-hemorrhagic Escherichia coli). This proposed research has been approved, and project development is currently well under way. This is a new priority project that builds on previous IFSAC work presented publicly through webinars, public meetings, conference presentations, and peer-reviewed manuscripts. The development, preparation, presentation, and prioritization of the project shows the usefulness of IFSAC as a triagency tool for uniform research, decision-making, and communication. This specific project utilizes technical expertise from all three agencies, as well as active involvement of leadership from all three agencies.

Narendranath Reddy Chintagari, B.V.Sc & A.H; M.V.Sc; Ph.D. (Abdu I. Alayash, Ph.D; D.Sc., Preceptor) CBER Fellow; FDA project : Hemoglobin-induced lung injury.

Lung alveoli are the functional units of respiration. They are fluid-free air sacs involved in gas exchange. Acute lung injury and pulmonary vascular damage leads to accumulation, lysis of red blood cells and possible accumulation of acellular hemoglobin in the alveoli. Additional oxidative stress might oxidize hemoglobin to potentially toxic higher oxidation species. Thus, lung alveolar epithelial cells are prone hemoglobin-induced toxicity. Lung alveolar epithelial cells might also be susceptible to hemoglobin-induced toxicity following massive blood transfusion, administration of hemoglobin-based oxygen carriers and in hemolytic diseases.

Lung alveoli are lined by two distinct epithelial cell types, the squamous type I cells (AT1) and cuboidal type II cells. The AT1 cells might be highly susceptible to hemoglobin-induced injury due to their sensitivity and close apposition with pulmonary vasculature. The present study was designed to investigate the effects of hemoglobin exposure on AT1 cells. Several oxidation byproduct of hemoglobin were also used to explore their role in lung injury. The AT1 cells were exposed to 50 μM ferrous hemoglobin [HbFe2+] and 25 μM methemoglobin [HbFe3+] for 24 hrs. The exposure increased the expression of heme oxygenase-1 (HO-1), H- and L-ferritin proteins. The effects of methemoglobin were more pronounced when compared to ferrous hemoglobin. Hemoglobin and methemoglobin activated the NF-κB and ERK signal transduction pathways. Haptoglobin, a natural scavenger of hemoglobin attenuated the ferrous hemoglobin and methemoglobin-induced expression in HO-1, H- and L-ferritin. Further, increased HO-1 expression was associated with its translocation to the mitochondria. Haptoglobin reduced the translocation of HO-1 into the mitochondria. Exposure to ferrous hemoglobin and methemoglobin caused mitochondrial dysfunction as shown by reduced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Methemoglobin-induced a significant mitochondrial stress as shown by decreased OCR and ECAR when compared to ferrous hemoglobin. Similarly, methemoglobin decreased mitochondrial transmembrane potential (ψ) to a greater extent when compared to ferrous hemoglobin. Haptoglobin prevented the decrease in transmembrane potential. On the other hand the highly oxidized ferrylhemoglobin [HbFe4+] induced cell death, cessation of OCR and ECAR within 8 hrs of exposure. Haptoglobin restored the decrease in OCR and ECAR. In summary, our results indicate that oxidized hemoglobin species are more toxic when compared to ferrous hemoglobin. The mitochondrial dysfunction might be due to HO-1 translocation into mitochondria. The study for the first time uncovered a novel pathway of hemoglobin and methemoglobin-induced mitochondrial dysfunction in alveolar epithelial cells.

Srinivasulu Chigurupati, DVM, Ph.D. (Serguei Liachenko, M.D., Ph.D., Preceptor) NCTR Fellow; FDA project: Confirmative Neuropathology studies with MRI Imaging and Informatics.

Toxicology accounts for approximately 33% of failure in new drug development and is a big concern in the drug development to the industry. Impact of imaging in clinical sciences for the past 30 years in diagnosis and treatment is unquestionable. However the use of imaging techniques in preclinical toxicology is negligible. High resolution Magnetic Resonance Imaging (MRI) with whole brain scanning has a potential to identify regions of the laboratory animal brain with evidence of neuro-abnormalities (presumed toxicities) that would precisely identify and direct the plane and loci of slices or sections subsequently taken for confirmatory classical neuropathology with positive lesions. Studies were conducted with ten known neuroxicants which affects different regions of the brain. Tissue were processed and a modified silver staining technique (de Olmos stain) method was performed as per the standard histology and staining method with neutral red counter staining to identify pathological lesions. Stained brain sections were scanned with ultra-high resolution slide scanner (Aperio ScanscopeXT) at 20x magnification. Pathology damage can be quantified with custom made macro software from aperio imagescope and Indicalab, wherever necessary. Also, brain mapping on MRI images (obtained from kanic acid treated rats) with representative positive lesions based on independent pathology report was performed. Based on our preliminary studies with high resolution MRI scanners, MRI has a potential to deliver a qualified non-invasive biomarker of neurotoxicity. After completion of all the studies the data will be submitted for biomarker qualification at CDER.

Peng Duan, Ph.D. (Lei Zhang, Ph.D., Preceptor) CDER Fellow; FDA project: Construction of a Drug Transporter Database and Utilization of the Physiologically - Based Pharmacokinetic (PBPK) Modeling and Simulation to Predict and Analyze Transportermediated Drug - drug Interactions (DDIs).


Drug transporters play an important role in drug disposition, affecting a drug's therapeutic and adverse drug effects. Importantly, transporters may represent the rate-determining step of drug absorption, distribution, and elimination in the intestine, liver, kidney, and blood-brain barrier (BBB), and they are often the sites of drug-drug interactions (DDIs). An FDA's internal transporter database was developed with the collection of transporter-related information such as key transporter parameters, transporter in vitro assays, and clinical DDI data, etc. from new drug submissions including new molecular entity (NME) drugs approved by the FDA since 2012. The transporter database can serve as a knowledge management and review tool, and help reviewers evaluate transporter-related issues including DDI evaluation during the review process. Satins are one of the most prescribed drugs in the United States and are commonly co-administrated with other drugs. Although statins are generally well tolerated, an increase in statin exposure as a result of DDI can lead to exposure-related side effects such as myopathy or even rhabdomyolysis. Depending on the metabolism and transport pathways of each statin, the NME's interaction effects on each statin are not the same. We used in vitro information collected in the database coupled with literature data to build mechanistic static models on seven marketed statin drugs for predicting an NME's interaction effect on various statin drugs. In addition, dynamic mechanistic models (e.g., physiologically-based pharmacokinetic models, PBPK models) on two statin drugs with no published PBPK models, pitavastatin and atorvastatin, were successfully developed and qualified with available clinical data. The availability of both mechanistic static and dynamic PBPK models for statins will provide a powerful modeling tool to evaluate DDIs on statins, and contribute to the agency's mission of ensuring safety of drug products and protecting the public health.

Trisha Eustaquio, Ph.D. (Angel M. Paredes, Ph.D., Preceptor) NCTR Fellow; FDA project: Assessment of Mitochondrial Ultrastructure After Ketamine Treatment In The Developing Rat Brain.


Ketamine, an FDA-approved N-methyl-D-aspartate (NMDA) receptor antagonist, is commonly used for general pediatric anesthesia. Accumulating evidence has indicated that prolonged exposure to ketamine induces widespread apoptotic cell death in the developing brains of experimental animals. Although the mitochondria are known to play a pivotal role in cell death, little is known about the alterations in mitochondrial ultrastructure that occur during ketamine's mechanism of neurotoxicity. The objective of this study was to utilize complementary methods of electron microscopy (EM) to study the mitochondrial ultrastructure in developing rat brains after ketamine treatment: (1) transmission EM (TEM) to study mitochondrial inner membrane topology in two-dimensions (2D) and (2) serial block-face scanning EM (SBF-SEM) to study overall mitochondrial morphology in three-dimensions (3D). The latter method conducts serial imaging and sectioning of a bulk sample, building a Z-stack of TEM-quality images.

As in previous studies performed at the NCTR, postnatal day 7 (PND-7) Sprague-Dawley rats were treated with ketamine (6 subcutaneous (s.c.) injections × 20 mg/kg at 2-hr intervals with 6-h withdrawal period after last injection, n=6) or saline (6 s.c. injections × 10 mL/kg at 2-h intervals with 6-h withdrawal period after last injection, n=6). Samples from the frontal cortex were harvested and prepared for TEM or SBF-SEM. TEM and SBF-SEM revealed mitochondrial defects that support ketamine's proposed mechanism of neurotoxicity in the developing rat brain. Conclusions and observations from the study include: (1) examination of mitochondrial inner membrane topology by TEM and mitochondrial surface area and volume measurements by SBF-SEM revealed a significantly higher percentage of apoptotic mitochondria in ketamine-treated samples (p < 0.05); (2) ketamine induced mitochondrial fission indicative of apoptosis as observed in high-resolution SBF-SEM datasets (~5-nm pixel size); and (3) SBF-SEM will allow ultrastructural insight into toxicological processes.

Carmen Gacchina Johnson, Ph.D. (Kenneth J. Cavanaugh Jr., Ph.D. (CDRH) Charles Durfor, Ph.D. (CDRH), and Steven S. Oh, Ph.D. (CBER) Preceptors) CDRH/CBER Fellow; FDA project: Opportunities and challenges in applying existing biocompatibility standards for the safety evaluation of cardiovascular

Device - biologic combination products.

Introduction: Regenerative medicine combination products intended to treat cardiovascular pathologies face particular safety challenges due to complexity in areas such as material composition, cell infiltration, extracellular matrix deposition, and resorption kinetics. These complexities also yield challenges in obtaining quality submissions and executing uniform reviews at the FDA. The objective of this project was to enhance consistency in the review process for cardiovascular combination products. Methods and Results: A Cardiovascular Combination Product Database was developed as a resource for regulatory reviewers to increase reviewer awareness of the cardiovascular combination products currently undergoing clinical investigation and aids in an efficient and consistent review of similar products. Additionally, the Fellow served as a liaison between CDRH and CBER to facilitate communication and understanding of technological development and regulation of regenerative medicine products. This has been achieved through the Fellow's regulatory review work, the Cross-Center Regenerative Medicine Seminar Series, scientific talks being given by the Fellow to CDRH and CBER reviewers, and contributions by the Fellow to scientific publications. Conclusion: The Fellow recommends continual updating of the Cardiovascular Combination Product Database and CBER/CDRH collaboration on regulatory science related to cardiovascular combination product. This will contribute to efficient and consistent science-based regulatory review practices and assist in meeting challenges posed by advances in sciences, evolving technologies, and new clinical indications.

Krystl Haerian, M.D. (Li-Lun Chen, M.D. and Wendy Aaronson, Preceptors) CTP Fellow; FDA project: Informatics methods to streamline tobacco industry submissions to the FDA Center for Tobacco Products.

This project is centered on utilizing informatics methods to streamline tobacco industry submissions, both at the point of submission and data already archived at the FDA Center for Tobacco Products. Streamlining is used to make an organization or system more efficient and effective by employing faster or simpler working methods. The two main methods advanced were: electronic submission standards and application of data mining methods such as topic modeling.

Standardization of the electronic submissions, including applications, is important for: 1) facilitating a timely review process, 2) providing the ability to query across applications, and 3) creating an infrastructure for identification of relevant documents within an application. The fellowship project included: a gap analysis of the electronic common technical document (eCTD) and HL7 Regulated Product Submissions (RPS), hosting a public workshop to obtain stakeholder input on electronic submission standards, and drafting a guidance document on electronic submissions. Standardization is effective at the point of submission, but for data already archived at CTP, topic modeling and other automated methods were researched. Topic modeling allows for automated identification of documents related to certain themes of interest, such as addiction. These informatics approaches will help determine our next steps for organizing, structuring, and accessing health documents submitted by industry in response to sec. 904(b) of the Tobacco Control Act that will help CTP inform and protect public health.

Kirsten Hirneisen, Ph.D. (Donna Williams-Hill, Ph.D., Preceptor) ORA Fellow; FDA project: Developing a Universal Enrichment Broth for Foodborne Bacterial Pathogens.


The Food Safety Modernization Act's aim is to shift the focus of the FDA to a system of prevention. For successful prevention of illness due to foodborne pathogens, rapid and reliable detection methods need to be developed for testing of foods before release to the consumer. The regulatory challenge of the FDA field labs is to complete an accurate detection assay in the shortest time possible. Therefore, it is important to screen foods to find potential pathogens and to eliminate negative samples as quickly as possible so food products, particularly fresh produce and other perishables, can reach the consumer in a timely fashion. As per the Bacteriological Analytical Manual (BAM), depending on the food matrix and target pathogen, different pre-enrichment media are used for sample preparation prior to conventional culturing detection methods. The objective of this Commissioner's Fellowship Project was to develop a universal enrichment broth to simultaneously propagate multiple foodborne bacterial pathogens in food matrices. This project was executed by analyzing growth curves of target pathogens (Salmonella spp., E. coli O157:H7 and Listeria monocytogenes) in candidate enrichment broths using the Bioscreen C, an automated growth curve analyzer. After 24h of enrichment, samples were plated to determine the number of viable cells by assaying for Colony Forming Units (CFU/mL) and DNA was extracted to detect the target pathogens using real-time Polymerase Chain Reaction (qPCR) analysis. Candidate broths analyzed included BAM broths, research broths and modifications of these current broths. Individual growth of each of the three pathogens and simultaneous growth of all three pathogens were characterized. These results indicate that use of a universal enrichment broth could be coupled with a multiplex qPCR detection assay for rapid screening of multiple bacterial pathogens in foods by regulatory field labs.

This approach will reduce media requirements and sample analysis time in addition to resulting in a high-throughput, stream-lined process that increases the capacity to detect multiple pathogens which is critical to the efficiency of food sample analysis by public health laboratories. Due to the complexity of bacterial growth needs under the various environmental conditions encountered by pathogens and the variety of food matrices, there is a need for continuation of this research project to determine the best universal broth for the simultaneous enrichment of these targeted foodborne pathogens in the wide array of food matrices the FDA regulates.

Paul W. Keller, Ph.D. (Ira Berkower, M.D., Ph.D.) CBER Fellow; FDA project: Designing HIV - 1 Vaccine Immunogens by Stabilizing gp120 with an Exposed CD4 Receptor Binding Site.

The key to a successful HIV-1 vaccine is eliciting gp120(Env)-specific broadly neutralizing antibodies (bnAbs). HIV-1 is susceptible to antibody neutralization, but the virus utilizes several strategies to evade immune recognition of Env. The goal of this project was to design a gp120 protein stabilized in an antigenic form, exposing the conserved CD4 receptor binding site and therefore eliciting a strong humoral immune response. Candidate peptides were tested in multiple vaccine vectors, including a live viral and a virus-like particle (VLP) vector. We identified multiple candidate sites for stabilizing mutagenesis within gp120 by structural modeling, and generated mutant proteins for evaluation of expression, immunogenic properties, and structure. These were evaluated along with a selection of externally-created gp120 constructs with immunogenic potential identified through collaboration and literature review. Several constructs showed promising expression in a live rubella viral vector, and are under continued evaluation for immunogenicity in rhesus monkeys. We also developed a novel purification protocol for His-tagged VLPs, allowing rapid purification of material suitable for many downstream applications. This method is antigen-independent, and may therefore have broad applicability to VLP-based vectors. The research conducted during this project contributes to our knowledge of what form an effective HIV-1 immunogen may take, and what vectors will be most suited to delivering active immunogens.

Anita Khatiwara, D.V.M, M.S., Ph.D. (Thomas S. Hammack, M.S., and Yi Chen, Ph.D., Preceptors) NCTR Fellow; FDA project: Development and Validation of Environmental Testing Procedure for Listeria monocytogenes.

Effective environmental testing procedures are critical for identifying source of contamination for surveillance and outbreak preventions. Three years ago, L. monocytogenes was implicated in a major multi-state out-break associated with contaminated cantaloupes and it was the deadliest foodborne outbreak in more than a decade. The investigation of this outbreak identified the processing environment as a source of contamination. The current FDA Bacteriological Analytical Manual (BAM) describes analytical methods for the detection of L.monocytogenes in various food matrices. However, FDA does not have validated methods for environment testing of L. monocytogenes. This research project was focus on the development and validation of testing procedures including sample collection, handling, enrichment, screening, isolation and confirmation of L. monocytogenes in food processing environment, especially food contact surfaces. Various sampling devices were tested for their ability to recover L. monocytogenes from contaminated stainless steel surface. We also tested several commonly used enrichment schemes for L.monocytogenes such as USDA MLG, ISO, and Universal enrichment broth. A meta-genomics study was conducted to study the background micro-flora on cantaloupes and to evaluate the efficacy of Listeria enrichment broths. The results from sampling evaluation indicated the type of moistened swab materials tested did not show statistical significance in the pathogen recovery. The meta-genomics data identified cantaloupe background micro-flora and demonstrated how different enrichment broths inhibited these background floras. Evaluation of enrichment broth shows that broths used by USDA MLG and ISO are more selective than that by FDA BAM, while the broth used by FDA BAM promotes the growth of L. monocytogenes better. Further studies to determine the best enrichment schemes could be performed using the background micro-flora identified in the cantaloupe meta-genomic study. This study provides a framework upon which future studies and regulatory reviews could be based.

Bernard S. Marasa, Ph.D. (Saeed Khan, Ph.D., Preceptor) NCTR Fellow; FDA Project: Biomarker Screening from Post-transcriptional Gene Expression Profiles of Bacillus anthracis Sterne Infected Human Primary Cells.

Bacillus anthracis is a gram positive spore forming bacteria that causes life threatening disease (anthrax). It affects humans and many species of animals and is classified as a bioterrorism agent. The spores cause three types of anthrax infections with vastly different lethality outcomes namely; cutaneous (<1%), gastrointestinal (25-60%) and pulmonary anthrax (>90%). Critically, the molecular mechanisms by which B. anthracis pathogenic factors influence host immunological responses at different sites of infection are poorly understood. We hypothesize that exposure to infectious Bacillus anthracis spores generates a post-transcriptional gene expression (GE) profile that can serve as biomarkers for developing medical counter measures against anthrax exposure.

The cells were infected with the spores of B. anthracis Sterne at a multiplicity of infection of 10. At 1H, 3H, 5H, and 24H post infection (P.I) cells were processed for electron microscopy, and RNA isolation. GE in human epidermal keratinocytes (NHEK), Intestinal myofibroblasts (InMyoFibs) and Small airway epithelial cells (SAECs) were evaluated using Agilent 4x44k whole genome arrays. MicroRNA profiling was performed using the SBI's MirnomeRT system. The data was imported to Partek Genomics Suite and normalized using 75% scaling. One-way ANOVA was used to calculate the significant gene list with cutoff at FDR< 0.05 and fold change > 1.5.

Electron microscopy revealed that the spores were captured by microvilli on the cell surface and internalized. Microarray analysis revealed an up-regulation of 1729, 4981, 1539 genes and down-regulation of 2178, 5558, 698 genes at 24hr P.I for NHEK, InMyoFib and SAEC cells respectively. Biomarker filtering using Ingenuity Pathway Analysis tools showed that there were 36, 489 and 52 unique biomarker genes among NHEK, InMyoFib and SAEC cells respectively compared to 9 common genes and 68 common miRNA biomarkers. These biomarkers included cellular genes exploited by pathogens (CGEPs) such as ITGB1, SGK1, DAB2 and ARAP3 found to significantly enriched in lung cells; while immunological mediators such as CCL3/5/7, IL1A, IL6, IL8, and LIF and transcriptional regulators including c-FOS, c-JUN and NFκB were differentially expressed amongst the three primary cells. These biomarkers could either be used as diagnostic tools for anthrax or for the development of novel medical counter-measure targets to control exposure of B. anthracis.

Niharika Mishra, Ph.D. (John Larkin, Ph.D., Preceptor) CFSAN Fellow; FDA Project: Validation of Extrusion Processing for Inactivation of Salmonella in Low Moisture Model Foods.

The numbers of outbreaks of salmonellosis that have been linked to low-moisture foods such as peanut butter, tree nuts, dry seasoning, peanut butter, powered infant formula, cereal, pet food, and cookie dough are of growing concern to public health. In the US, approximately 1.2 million cases of salmonellosis occur annually. Little research exists on Salmonella inactivation during extrusion processing, yet nearly 50% of outbreaks associated with low-moisture foods since 2006 are linked to extruded foods. Literature reported extrusion studies describing microbial inactivation lack comprehensive data with respect to varying moisture content and temperature. Also, little information is available on heat resistance of Salmonella in flour, a common ingredient in low moisture extruded foods. Therefore, validating extrusion processing with worst product and process parameters and more information on Salmonella heat resistance at different water activities are an urgent need that will aid FDA in developing guidance and regulatory decisions making for this group of low moisture food products.

The goals of this project were to study: (a) Salmonella inactivation under worst-case product and process criteria during extrusion processing; and, (b) heat resistance of Salmonella in different flour matrices at different water activities. For extrusion, oat flour (with ~8.5 indigenous fat) and low temperature (<100 °C), low water activity, low pressure, low shear and single screw extruder were used to meet the worst worst-case product and process criteria. Results of the study indicate that extrusion is an effective method to minimize or eliminate Salmonella under the typical extrusion processing conditions currently employed in industry (14-28% moisture, >100°C). These conditions are sufficient to reduce a population of Salmonella by 5-log10 CFU/g. Consequently, Salmonella contamination in extruded foods is most likely the result of improper processing, processing failures or post-processing contamination. The study also revealed that Salmonella is more resistant to heat at low water activity and that matrix may also affect heat resistance of Salmonella and should be considered when implementing preventive controls such as extrusion.

Yuzhuo Pan, Ph.D. (Ping Zhao, Ph.D., Preceptor) CDER Fellow; FDA Project: Application of the FDA PBPK Knowledgebase in Evaluating Model Predictability for Drug-drug Interactions.

Drug developers have increasingly used physiologically-based pharmacokinetic (PBPK) models to assess drug-drug interactions (DDIs) potential. Using established an FDA PBPK knowledgebase in the OCP/FDA to evaluate the capability of a substrate PBPK model in predicting DDI potential in the presence of enzyme/transporter inhibitors. Substrates were selected based on the following criteria :(1) having clinical DDI data with inhibitors and (2) having PBPK models developed by the sponsors using in vitro ADME and/or human PK data, and not informed by DDI outcome. Predictive performance was evaluated using R =predicted /observed PK metric (AUC or Cmax ratios with/without inhibitor: AUCR and CmaxR), with a predefined success measure of 0.8≤ R≤1.25. The selected substrates from sponsors met selection criteria, with a variety of elimination pathways. The clinical DDI studies using different enzyme/transporter inhibitors were available for these substrates. The observed AUCR and CmaxR values covered wide range. Predictive accuracies for AUCR and CmaxR were 93% and 87%, respectively. These findings increase our confidence in predicting enzyme/transporter inhibition for substrate drugs using PBPK.

Javier Revollo, Ph.D. (Vasily Dobrovolsky, Ph.D., Preceptor) NCTR Fellow; FDA project: Characterization of the TK6 Exome by 454 Next Generation.

TK6 cells are heterozygous for the thymidine kinase gene (TK+/-) but wild-type for the p53 gene. They are routinely used in genetic toxicology to assess the mutagenicity of chemicals by using loss of function of the remaining wild-type TK allele as a reporter. To determine if TK6 cells carry other genetic changes that may affect their response to DNA damaging agents, we used 454 Next Generation Sequencing to characterize their normalized transcriptome. We identified 1928 genetic variations from a total of 7471 detected genes. 1120 of these variants are known single nucleotide polymorphisms (SNPs), while 808 constitute variants not found in normal human populations. Importantly, we detected several genetic changes that could affect the response of these cells to genotoxins, such as clinically relevant SNPs at the thiopurine S-methyltransferase (TPMT) and NADPH quinine oxidoreductase 1 (NQO1) loci, as well as a novel frame-shift mutation in the heavy subunit of ferritin (FTH1). Our data provide a catalog of genetic anomalies in the transcriptome of the TK6 cell line.

Justina Tam, Ph.D. (Charles Clavet, Ph.D., Preceptor) ORA Fellow; FDA project: Rapid Detection of Pathogens using Nanoparticles functionalized with Aptamers, with Realtime Analysis.

Rapid diagnostic devices are urgently needed to rapidly detect pathogens that threaten the health of Americans. Despite large initial advances, there are still significant areas of improvement that must be urgently addressed to protect the American public from the variety of pathogens that are constantly introduced to the country via evolutionary mutations, climate change, urbanization, and increased global travel. Current rapid diagnostic tests are severely limited in the areas of: multi-analyte detection, sensitivity, specificity, and real-time monitoring. We have addressed these limitations by developing a multiplexed lateral flow-based point-of-care (POC) platform device that is used with a mobile phone application, allowing the user to send results from their device online. We have validated our prototype on a model pathogen, dengue virus, which has recently caused outbreaks in the U.S., and for which there is no rapid test commercially available in the country. Additionally, we developed methods to increase the sensitivity and lower the manufacturing cost of our device by optimizing the visual markers on the lateral flow test. These visual markers are gold nanoparticles that were conjugated to anti-dengue nonstructural protein 1 (NS1) antibodies via specific linker to orient the antibodies. Antibody-conjugated nanoparticles were characterized by UV Vis, dynamic light scattering, gel electrophoresis, and lateral flow tests. Intensity of signal from the test area of the lateral flow tests was analyzed using ImageJ software. The larger-scale impact of developing this rapid test is the ability of public health officials to monitor the spread of a disease in real-time on a global scale, leading to more rapid implementation of prevention and control programs on identified infected and at-risk populations.

Carolyn Yong, Ph.D. (Stephen S. Oh, PH.D. (CBER), Charles M. Durfor, Ph.D. (CDRH), Kenneth J. Cavanaugh, Jr. Ph.D. (CDRH) Preceptors) CBER/CDRH Fellow; FDA project: Evaluation of regenerative medicine devices that produce a biological product at point-of-care: Assessment of device and output controls and enhancement of FDA review.

Due to the rapidly evolving regenerative medicine technology published in scientific literature and/ or submitted to the FDA, the purpose of this project has been to advance the assurance of the safety, efficacy, and security of biological products through enhancing the Agency's ability to regulate devices used at the patient "point-of-care" (POC) that process autologous biological material. As the FDA relies on available scientific knowledge to set product standards to ensure the quality of the biological output of such devices, a comprehensive retrospective analysis of regulatory submissions of these products to CBER and CDRH was conducted along with current scientific literature reviews to evaluate regulatory science tools and update the FDA knowledge base, respectively, through database and discussion paper development. In addition, by engaging in regulatory review in both Centers, the project evolved to address the latest technologies, challenges, and deficiencies in the field. Moreover, this project focused on identifying special controls for POC devices to guide FDA policies with regards to product standards and ultimately enhance review practices. Finally, inter-center activities were undertaken to support cross-center communication and proactively promote interactions and knowledge synthesis in relevant areas of regenerative medicine.

Yueqin Zhao, Ph.D. (Ram Tiwari, Ph.D., Preceptor) CDER Fellow; FDA project: Likelihood Ratio Test-Based Methods for Analysis of Clinical Trial Safety Data with Applications to the Rivaroxaban Drug.

The adverse events data of randomized clinical trials are often analyzed in an unstructured way, where hundreds of adverse events are analyzed independently. These methods do not adequately control the type-I error rate, and inflate the risk of making false positive errors. A likelihood ratio test (LRT), recently developed for the detection of signals of adverse events (AEs) for a drug of interest in the FDA Adverse Events Reporting System (FAERS) database and that controls the type-I error rate, is now extended to detect signals of AEs simultaneously in randomized clinical trials. The proposed method is applied to a clinical trial dataset for Rivaroxaban.

Class of 2011

Barbara Abrams, M.D., J.D. (Elizabeth Mansfield, Ph.D. and Katherine Serrano, B.S., Preceptors) CDRH Fellow; FDA project: Developing Guidance Documents forResearch Use Only/investigational Use Only Componentsin in vitro Devices andRisk-basedClassification of Novel in vitro Devices.

FDA's Commissioner's Fellowship Program allowed me to gain an appreciation of the complexity of FDA's regulatory environment and how science, laws, regulations, and politics all combine to influence the development of public health policy. The majority of my time in the Fellowship program was spent in the Office of In Vitro Diagnostics where I was able to further the Agency's mission by working on a variety projects including taking a guidance document from draft to final stage, developing a program to decrease recall classification times for in vitro diagnostic devices, and leading the effort to create product codes to allow for the importation of investigational use only in vitro diagnostic devices. Two developmental assignments outside the Office of In Vitro Diagnostics allowed me to research a method to capture adverse events associated with the off-label use of certain drugs in the pediatric population and to assist with FDA's interactions with Congress.

Kimberly M. Anderson, Ph.D. (Sunee Himathongkham, Ph.D., D.V.M., M.P.V.M., Preceptor) ORA Fellow; FDA project: Rapid Molecular Typing of Shiga Toxin-Producing Escherichia coli Using the Automated DiversiLab Repetitive-Sequence-Based PCR System.

Shiga toxin-producing E. coli (STEC) are of major public health concern due to their frequent contamination of food products and ability to trigger foodborne illnesses such as diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. In this study, the DiversiLab rep-PCR system was used to generate a DNA fingerprint profile for (60) O157 and (100) non-O157 STEC, and later results were compared to those obtained with the "gold-standard" PFGE molecular typing method. STEC isolates from human, food, and animal sources were collected from Michigan State University (n=112), as well as the Center for Veterinary Medicine (n=48) within the FDA. Among the 160 isolates tested, all were typeable by rep-PCR. Twenty serogroups, including the "Big 6" E. coli serogroups, were analyzed using the Extended Jaccard statistical method to determine distance matrices and the unweighted pair group method of averages algorithm (UPGMA) to create dendrograms. Excellent reproducibility of rep-PCR profiles was observed with % values ranging from 98.4%-99.7% and 96.3%-99.8% for intra-run and inter-run studies, respectively. A comparison of non-O157 isolates revealed that DL clustered 79/89 (88.8%) isolates according to serogroup status with peak differences ranging from 2 peak differences (94.3% similarity) up to 12 peak differences (59.6% similarity). Furthermore, the genetic relatedness among the non-O157 serogroups mirrored the branching of distinct clonal groups elucidated by other investigators. Among each set of O157 and non-O157 isolates, DL was able to distinguish isolates with a Simpson's Index of Diversity greater than 0.92. Even though the discriminatory power of DL was less than PFGE (Simpson's Index of Diversity >0.99), DL was able to identify 24 pattern types for O157 isolates and 32 pattern types for non-O157 isolates, suggesting it has the ability for strain typing. Rep-PCR profiles for STEC isolates were also distinct from 12 other bacterial species that included: S. flexneri, L. monocytogenes, V. cholera, K. pneumoniae, and S. enterica. These results indicate the ability of DL to distinguish between and within O157 and non-O157 STEC groups, as well as other foodborne pathogens. Therefore, PCR-based typing methods, like rep-PCR, may facilitate the identification of STEC by excluding unrelated STEC isolates within 24 hrs during outbreak investigations.

Olgica Ceric, Ph.D. (Renate Reimschuessel, Ph.D., Preceptor) CVM Fellow; FDA project: Investigation of etiology of Jerky Pet Treat related renal failure and Fanconi syndrome in dogs.

In September of 2007, FDA issued a cautionary warning to consumers regarding jerky pet treat products imported from China, and a Preliminary Animal Health Notification in December of 2008. Since 2008, FDA issued several consumer updates advising pet owners about complaints associated with jerky treats. The number of complaints dropped off during 2009 and 2010, but in 2011 and 2012 there was an increase in the number of complaints of dog illnesses associated with consumption of jerky pet treats (JPT). As of March 31, 2013, FDA received more than 2800 consumer complaints involving more than 3400 dogs and more than 550 deaths. Signs reported in the complaints include decreased appetite; decreased activity; vomiting; diarrhea, sometimes with blood; increased water consumption and/or increased urination. In some cases, blood tests indicate kidney failure (increased urea nitrogen and creatinine), occasionally coupled with Fanconi syndrome (increased urinary glucose with normal blood glucose). FDA, in collaboration with several veterinary diagnostic laboratories in the U.S., is working to determine why these products are associated with illness in dogs. The objective of our project is to test jerky pet treat products focusing on agents known to cause kidney failure/Fanconi syndrome. In addition to testing jerky pet treats, we are reaching out to owners of affected dogs in order to obtain diagnostic samples from the affected animals. To date, testing for contaminants in jerky pet treats has not revealed a cause for the symptoms that have been reported to us. Since 2011,we have collected approximately 250 jerky treat samples from more than 165 consumer related complaints, and more than 200 "retail samples" (unopened bags obtained from a store or shipment), and performed more than 1000 tests on these samples. We also ran more than 240 tests on "historical" samples (samples received in 2007-2011).

Cynthia J. Chang, D. Phil. (Jiyoung Dang, Ph.D. (CDRH), Mark H. Lee, PH.D (CBER), Elias Mallis, B.S. (CDRH) Preceptors) CDRH/CBER Fellow; FDA project: Cross-center project to enhance the premarket review process for scaffold-based products, including surgical mesh devices, cell-scaffold engineered combination products, and unclassified wound dressing products.

The purpose of this project has been to advance the regulatory process for premarket review of scaffold-based combination products in the regenerative medicine (RM) arena. The project has focused on two product areas: surgical mesh devices and cell-scaffold engineered combination products, and unclassified wound dressing products. Both sub-projects relied on the assessment of the content of historical submissions to develop and improve the review process. The surgical mesh/cell-scaffold product project focused on the assessment and promotion of standards useful to evaluating the performance of these products. The wound dressing project focused on the assessment of previously cleared unclassified wound dressing products, with the goal of identifying special controls as part of regulation development as well as publishing guidance. Fundamental to the project, and to the RM Fellowship, is the importance of cross-center communication and interaction; hence, formal and informal cross-center activities have been undertaken throughout the course of the Fellowship to support the project. Ample progress in all parts of the project has been accomplished. The project is intended to standardize the review process to facilitate premarket evaluation of scaffold-based products, to benefit industry and FDA by providing clear guidelines and a streamlined process, and to benefit public health by advancing the regulatory science of RM products.

Laurie M. Clotilde, Ph.D. (Andrew Lin, Ph.D., Preceptor) ORA Fellow; FDA project: Detection of Shiga Toxin-Producing Escherichia coli.

Because of the increase of outbreaks caused by non-O157 Shiga toxin-producing Escherichia coli (STEC), facilitating and speeding the detection of those organisms in contaminated foods is becoming extremely important. Therefore, we proposed to compare and improve the efficiency of a 10-Plex PCR-based Luminex assay (E. coli serogroups O26, O45, O91, O103, O111, O113, O121, O128, O145, and O157), a 9-Plex antibody-based Luminex assay (Shiga toxins 1 and 2 [Stx1 and Stx2], E. coli serogroups O26, O45, O103, O111, O121, O145, and O157), and a newly developed agar (SHIBAM) to the methods used in the FDA-Bacteriological Analytical Manual (BAM; Chapter 4a). These different assays have been previously developed by the Principal Investigator and/or her preceptor and were further tested. Using the Luminex technology present many advantages: 1) The multiplexed format (up to 100 analytes) will save time, reagents, and test sample; and 2) Many Food and Drug Administration (FDA), Food Safety and Inspection Service (FSIS), and Food Emergency Response Network (FERN) laboratories currently utilize the Luminex platform for other assays. Therefore our assay was directly transferable and implemented by many laboratories.

My work has extreme relevance to public health as it provides meaningful data for releasing safer foods, thus minimizing annual cost and numbers of recalls, enhancing public health, and allowing utilization of the same standard protocol throughout regulatory agencies in a more user-friendly manner. In fact our newly developed SHIBAM agar was recently added into the BAM (Chapter 4a).

Brian E. Erkkila Ph.D. (Raymond Yeager, Ph.D., Preceptor) CTP Fellow; FDA project: An Assessment of the Risks Associated with Childhood Exposure to Environmental Tobacco Smoke.

The mission of CTP is "to protect Americans from tobacco-related death and disease by regulating the manufacture, distribution, and marketing of tobacco products and by educating the public, especially young people, about tobacco products and the dangers their use poses to themselves and others". In doing so, the Center is charged with protecting the health of both tobacco product users and non-users. The health of millions of Americans is jeopardized due to exposure to environmental tobacco smoke (ETS), more commonly known as secondhand smoke. ETS is composed of thousands of constituents (nitrosamines, polycyclic aromatic hydrocarbons, metals, etc.) and exposure to these constituents has been associated with respiratory infections, asthma, sudden infant death syndrome and cancer. For his FDA Commissioner's Fellowship Dr. Brian E. Erkkila analyzed available research on ETS constituent exposure in order to perform a risk assessment of childhood exposure to ETS. His efforts led to identification of a number of data gaps, development of a model to estimate exposure, and a preliminary quantification of the risks associated with a set of ETS harmful and potentially harmful constituents. This project lays the groundwork for what could potentially be an important factor in regulatory decisions at the Center for Tobacco Products.

Alifiya H. Ghadiali, Ph.D. (Julie A. Kase, Ph.D., Preceptor) CFSAN Fellow; FDA project: Molecular Risk Assessment of non-O157 shiga toxin-producing Escherichia coli isolates.

Enterohemorrhagic E. coli (EHEC) are a highly pathogenic sub-group of shiga toxin-producing E. coli (STEC) that can cause bloody diarrhea as well as potentially fatal diseases including hemolytic uremicsyndrome and hemorrhagic colitis. EHEC O157:H7 was, until recently, the most prevalent serogroup inthe United States and worldwide. However, other EHEC serogroups are emerging as food bornepathogens that pose a serious health risk to humans. While the pathogenesis of STEC infections is notfully comprehended, several important virulence factors have been established. However, there is limitedconsensus on the virulence markers that need to be used to assert human pathogenicity. A pan-genomicmicroarray chip (FDA-ECID) was used to identify genetic signatures that distinguish EHEC strains withhigher virulence capability. The FDA-ECID chip represents ~42,000 non-redundant genes and 10,000SNPs from >300 whole genome alignments of E. coli and Shigella genomes and plasmids. The designand validation of the microarray is presented as part of a separate ASM poster by Jackson et al. and isnot discussed here. The analysis represented a temporally and geographically diverse collection of non-O157 isolates. These isolates included sporadic cases of illness caused by a wide variety of STECserotypes between the years 2000-2011. Also included were STEC strains isolated from food productsbetween the years 2002-2012. The microarray platform allowed us to rapidly determine the virulencegene repertoire in each individual isolate. Our results suggest that the STECs display an extensive pangenomic variability both within and between serogroups. Using a molecular risk assessment strategy, we have defined a set of clinically important genes and determined the prevalence of this subset in non-O157 STEC isolates from food. Furthermore, the microarray based approach is amenable to rapid surveillance, outbreak detection, investigation and evaluation of efficiency of control measures.

Charles Haggart, Ph.D. (Murray Sheldon, M.D. and Day Ranamukhaarachchi, Ph.D., Preceptors) CDRH Fellow; FDA project: U.S. Medical Device Innovation (FY00-12): An Analysis of Total Time to Market for approved Original PMAs.

The FDA's mission of protecting and promoting the public health is advanced by fostering innovation of safe and effective medical devices. While some FDA stakeholders make claims of a regulatory standard that is too high for innovative, high risk devices, there has historically been a dearth of objective data to validate or invalidate such critiques. We have thus defined Total Time to Market (TTM) as the time from the initial device-specific sponsor-FDA interaction to the final market approval of that device. To assess the historical pace of high-risk medical device innovation in the U.S., we constructed a comprehensive TTM database that spans all original PMAs approved between FY2000 and FY2012 and includes both regulatory and clinical development attributes and milestone dates beginning with pivotal IDE submission and ending with PMA approval. We have performed numerous analyses on this TTM data, paying particular attention to variations in TTM across clinical specialties and trends in TTM and sub-intervals timelines in recent compared to earlier years captured within the database. This retrospective database and analysis provides FDA with a proof-of-concept for prospective tracking and analysis of IDE approval and clinical trial timelines that would allow FDA to quantitatively assess the effects of regulatory policy-making on medical device innovation in real-time. By understanding the driving forces behind regulatory and clinical development of innovative, high risk medical devices in the U.S., FDA will further improve its ability to advance its mission and benefit public health in the U.S.

Yanna S. Kang, Ph.D. (Brian Fitzgerald, B.Sc., Preceptor) CDRH Fellow: FDA project: An Automated Text Mining Approach.

Background: Analysts at the CDRH study Medical Device Reports (MDRs), which are adverse event (AE) reports related to medical devices, for identifying safety signals. These signals could be divided into two categories for an arbitrary device D: (1) a deviation relative to the historical baseline behavior between D and a known AE associated with it, e.g., a spike in the number of MDRs for D and a known AE in comparison to the past, and (2) a previously unknown AE associated with D. To identify the two types of safety signals described above, one needs to define known AEs associated with D. These known AEs are manifest in the historical MDRs or in the warnings, contra-indications, and precautions of the pre-market submissions. Currently, there is no tool that is available to help analysts to identify information about known AEs in these documents. This process, if done manually, is labor-intensive and time-consuming because (1) all these documents are presented in narrative form, and (2) the number of documents requiring study and analysis is increasing rapidly. Therefore, there is an urgent need to develop tools for efficient AE recognition.

Methods: We designed a text-mining framework called Medical Device Adverse event Labeling (or MEDAL) and applied to this framework a state-of-the-art probabilistic model, Conditional Random Fields (CRFs), for this AE term recognition task. First, we obtained the parameters of the CRFs model by training the model on gold standard documents that have human-annotated adverse event terms. Then, we applied the trained model to new un-annotated documents and the model automatically predicted the AE label (AE or non-AE) for each word in the documents. For preliminary experiments, we selected 510(k) documents received from 2001 to 2011 that have subject device automated external defibrillators. We finally retained 38 documents that do not contain duplicate text regarding warnings, precautions, or contra-indications. We performed 10-fold cross validation experiments on these 38 documents to evaluate the predication performance of MEDAL.

Results: MEDAL achieved an average precision rate of 97.6% and an average recall rate of 88% over the 10-fold cross validation experiments on these 38 documents. We believe that the relatively lower recall rate was due to the small size of training examples we used for tune the parameters of the CRFs model: if patterns of AE terms were seen in the training examples, CRFs could recognize these AE terms with a high precision rate; otherwise, it would be difficult for it to detect new, unseen patterns of AE terms. We conjectured that the recall performance would be boosted once it was given a large set of training examples.

Conclusions: MEDAL is promising in recognizing device-related adverse event terms in unstructured FDA pre-market submission documents. With this tool, adverse event terms can be automatically labeled/highlighted, which will save FDA analysts huge amounts of time manually screening tons of documents for adverse event signals. *Keywords: adverse medical device event; text mining; probabilistic methods; machine learning.

Jennifer Y. Kelly, Ph.D. (Scott McNamee, Ph.D.) CDRH Fellow; FDA project: Effects of Extreme Weather and Natural Disasters on Medical Device Product Lifecycle.

Extreme weather events such as super storm Hurricane Sandy in October 2012 and other natural disasters can affect the public health in many ways. Resulting conditions including lengthy power and network outages, extensive flooding, extreme temperature ranges, and structural damage can affect medical device manufacturing processes and medical device safety and effectiveness. The purpose of this Commissioner's Fellowship project was to examine the ways that extreme weather events could affect the medical device industry and the public health.

Discussion sessions were held across the Agency and the Department to identify key areas of vulnerability for medical devices affected by extreme weather events. Three areas were identified: 1) medical devices used during or in the aftermath of an extreme weather event (including those requiring electricity or fresh water); 2) medical devices and components caught in the path of extreme weather (as during transport or storage); and 3) medical device manufacturing processes interrupted by extreme weather (power outage, facility flooding).

This Commissioner's Fellowship project also looked at whether the current device regulatory framework addresses risks and vulnerabilities to the medical device industry resulting from extreme weather. The scope of this project included identifying future steps that the Agency and the medical device industry might take to better tolerate or mitigate such risks. A Device Good Manufacturing Practice Advisory Committee meeting was held to discuss this topic. The advisory committee was in unanimous agreement that the Quality System regulation does not need revision. The committee did point out that industry has an opportunity to put robustness into its device design and to evaluate risk management and crisis management procedures as to whether they address risks from extreme weather and natural disasters. The Agency may also consider reviewing reference and guidance documents related to the Quality System regulation since it was written in 1996 to identify where moderate updates may be appropriate.

Gumei Liu, MD, Ph.D. ( Anne Pariser, M.D., Preceptor) CDER Fellow; FDA project: The Role of Natural History Studies in Drug Development for Rare Diseases: A Five-year Analysis.

Objective: An understanding of disease natural history (NH) is critical for clinical trial design and outcome assessment for drug and biologic product ("drug") development. Unlike common diseases, there is little existing knowledge on disease natural history for most rare diseases (RD), which makes NH studies of particular importance for RD drug development. The objectives of this analysis were 1) to assess the role of NH studies in RD drug development, and 2) to identify key factors in NH studies that may facilitate RD drug development.

Method: We analyzed NH studies utilized in marketing applications (New Drug Applications (NDAs) and Biologics License Applications (BLAs)) for new molecular entity (NME) and new biologic (NB) products approved by FDA CDER during the PDUFA IV period (October 1, 2007 to September 30, 2012). Associations between the use of NH studies and characteristics of marketing applications, such as whether the drug was "first in disease", used a "novel endpoint" or underwent "priority review" were investigated.

Results: Our data revealed that a wide variety of NH studies, both implicit and explicit, were integrated in NME/NB marketing applications. NH data sources included disease registries/databases, clinical studies, and literature reviews. While there were few examples of sponsor-conducted NH studies (e.g., prospective longitudinal studies or retrospective chart reviews), when they were performed, those NH studies often provided critical support to the marketing applications. Most commonly, NH studies were used to guide clinical endpoint and patient population selection for clinical trials, as well as to address safety considerations. We found a statistically significantly higher number of NH studies used in support of orphan drug approvals than for common disease approvals. In addition, the use of NH studies was positively associated with the marketing application characteristics of first in disease, novel endpoint, and priority review.

Conclusion: In the 5-year period between 2007 and 2012, NH studies were shown to provide a scientific foundation for drug development, especially for rare diseases. A clear understanding of disease natural history is essential, and well-conducted NH studies were shown to facilitate rare disease drug development and approval.

Gajendiran Mahadevan, Ph.D (Oscar A. Chiesa, D.V.M., M.S., Ph.D., Preceptor) CVM Fellow; FDA project: Determination of Ceftiofur Metabolite Desfuroylceftiofur Cysteine Disulfide in Bovine Small Intestine by LC-MS/MS.

Antimicrobial drugs are used in both humans and animals for the treatment and prevention of diseases caused by variety of microorganisms, such as bacteria, fungi, and parasites. If bacteria or other microbes are exposed to antimicrobial drugs inappropriately they could become resistant to that drug or class of drugs. If resistant bacteria enter the food supply, drugs normally used to treat people infected with those bacteria may not work effectively. In order to understand antimicrobial resistance development in food-producing animals it is necessary to determine the concentration of antimicrobial drugs to which the intestinal floras are exposed. Ceftiofur is a cephalosporin �-lactam antibiotic that is approved in the United States for treating respiratory diseases in cattle and mastitis in lactating dairy cattle. The objective of the present study is to develop and validate a fast, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the concentration of desfuroylceftiofur cysteine disulfide (DCCD), an active metabolite of ceftiofur in duodenum, jejunum, and ileum of the bovine small intestine. An accurate assessment of the concentration of an antimicrobial agent in the small intestine may be useful in predicting the potential emergence of antimicrobial-resistant bacterial pathogens from food-producing animals. Eight steers were dosed with ceftiofur sodium at 2.2 mg/kg, intramuscularly at 24 hour intervals for 5 days. Different sections of the small intestine were collected from steers harvested at 12, 24, 30, 36, 48, 60, 64, and 76 hrs after the last dose, respectively. Stable isotope labeled d3-DCCD was used as an internal standard (IS). The analyte of interest and IS were extracted from sample tissues by precipitating proteins using 1% formic acid in acetonitrile. The sample extract was cleaned up by solid phase extraction cartridges. The sample extracts were centrifuged and the supernatants were chromatographically separated with C18 chromatography and analyzed by triple quadrupole tandem mass spectrometry. The calibration curves were linear over a wide range of concentrations. Independent sources of control small intestinal tissues were used for the determination of accuracy and extraction efficiency. The average inter-day accuracy met the acceptance criteria of the FDA guidance (GFI#145). The detectable concentrations of DCCD were observed only from ileum tissues. The concentrations of DCCD in the ileum tissue were relatively higher than the plasma concentration at the same harvest time point. This finding necessitates considering a potential risk evaluation procedure for the possible emergence of antimicrobial resistant bacteria due to exposure in the small intestine of food-producing animals.

Michael Mendicino, Ph.D. (Frank Weichold, M.D., Ph.D. and Vicki Seyfert-Margolis, Ph.D., Preceptors) CBER Fellow; FDA project: Advancing Regulatory Science and Innovation in Stem Cells and Regenerative Medicine.

'MSCs', commonly referred to as Mesenchymal Stem Cells or Mesenchymal Stromal Cells, are a diverse population of cells with a wide range of potential therapeutic applications. There has been an explosion of interest in the production and utilization of MSCs, as evidenced by the number of scientific publications. The number of publications investigating MSCs submitted per year increased at least 20-fold from 2000 to 2012 (PubMed search for 'Mesenchymal Stem Cell OR Mesenchymal Stromal Cell'). Academic clinical investigators and commercial clinical trial sponsors have begun to develop MSC-based products for numerous clinical indications in recent years. However, there is no consistent nomenclature or definition of MSCs. There have been efforts by leaders in the field to propose minimal criteria to characterize MSCs, such as cell surface marker expression. One major question is whether commonly described markers in the MSC literature are sufficient in terms of defining what an MSC-based product contains or the function(s) that can be performed. In order to better understand what an MSC population contains and how the cells exert biological activity, it would be useful to be able to correlate MSC markers with MSC bioactivity in vitro and in vivo. This is especially difficult due to incomplete knowledge about MSC mechanisms-of-action and how these may relate to outcomes for different clinical indications. To facilitate development of cell therapies through better understanding of the biology of MSCs, FDA's Center for Biologics Evaluation's (CBER) Office of Cellular, Tissue and Gene Therapies (OCTGT) has developed a number of initiatives including undertaking a MSC Consortium project to identify and correlate multi-parametric product characteristics, such as product marker-based expression and bioactivity, with in vitro and in vivo assays, and animal models of human injury or disease.

This project focused on MSC product characterization and analysis of peer-reviewed scientific literature and clinical trials that are evaluating MSC-based products. In addition, the MSC Consortium's approach was emphasized in relation to the scientific literature and regulatory perspectives, regarding both MSC-based product characterization and parameters that have been shown to influence product characteristics.

Clark Meyer, Ph.D. (Nandini Duraiswamy, Ph.D., Preceptor) CDRH Fellow; FDA project:Computational modeling of Atrial Septal Defects and Occluders.

A project centered on computational solid mechanical modeling and analysis of atrial septal defects (ASD) and occluders investigated the etiology of treatment failure modes for the two devices currently approved. Atrial septal defects (of secundum type II) are congenital heart defects that are either treated surgically or percutaneously with transcatheter devices called occluders. Failure modes in this class of device are rare but can be life threatening and include erosion, fracture, and embolization. The patient population is primarily young pediatric cases with the device intended to remain in place for the life of the patient.

Finite element models and simulations of solid mechanical equilibrium were developed using devices designed and made by the two companies currently marketing devices in the US. These models and simulations were supported by a combination of bench top testing and imaging studies including: microCT, extension testing, pull-through testing, differential scanning calorimetry, and porcine (or clinical) model imaging (from external collaborators). These bench top studies and further processing of the porcine imaging studies provided essential data to support the verification and validation of the computational modeling approaches for the device under development. These approaches will help determine our next steps in modeling towards device-tissue interaction using clinical imaging data.

Anh Nguyen, M.D., MBA (Markham Luke, M.D., Ph.D., Preceptor) CDRH Fellow; FDA project: Guidance Development for Future Medical Devices.

Dr. Nguyen's CFP project was the completion of a Special Controls Document for a future medical design. Special controls are regulatory requirements for class II devices. FDA classifies into class II devices for which general controls alone are insufficient to provide reasonable assurance of the safety and effectiveness of the device, and for which there is sufficient information to establish special controls to provide such assurance. Special controls are usually device-specific and include:

  • Performance standards
  • Postmarket surveillance
  • Patient registries
  • Special labeling requirements
  • Premarket data requirements
  • Guidelines

Dr. Nguyen's manuscript is completed and currently under internal review at FDA.

Li Pang, M.D. (Beverly Lyn-Cook, Ph.D., Preceptor) NCTR Fellow; FDA project: Role of ABC Transporters in Pancreatic Cancer Chemoresistance.

Polymorphisms in ATP-binding cassette (ABC) transporter genes have been implicated in different responses to chemotherapy and disease susceptibility in various cancers. In this study, we investigated whether single nucleotide polymorphisms (SNPs) in ABC transporter genes are associated with the abnormal expression of the efflux pumps in normal and pancreatic cancer specimens. Four SNPs were selected from the ABCB1/MDR1, ABCC1/MRP1, and ABCG2/BCRP genes according to the following criteria: 1) Minor allele frequency of the SNP is greater than 10%; 2) The SNP has been reported to affect expression or function of the ABC transporter and has been associated with cancer risk or clinical outcome in prior studies. We found that the T/T genotypes in exon 21 (G2677T) and exon 26 (C3435T) of ABCB1/MDR1 gene were significantly associated with a lower risk of developing pancreatic cancer (p = 0.013, OR = 0.35 and p = 0.015, OR = 0.29, respectively). Moreover, the expression levels of MDR1, MRP1 and BCRP were increased in the pancreatic cancer specimens (-2.0, -5.1, and -1.4 folds, p<0.01, respectively), compared to normal pancreas. But the expression of MDR1 in the 2677TT and 3435TT carriers was lower compared to the wild type homozygotes and heterozygotes. Interestingly, the pancreatic cancer cell lines, MIA1 and BXPC3, are MDR1 2677TT and 3435TT homozygotes and cell viability assays showed that these two cell lines were much more sensitive to gemcitabine than PANC1, SU86.86, PL-45, and ASPC1, which are either wild type homozygotes or heterozygotes for MDR1 G2677T and C3435T. This result is consistent with the clinical observation that for patients receiving post-operative gemcitabine chemotherapy, the overall survival is tended to be longer in the mutant type homozygotes of MDR1 G2677T and C3435T. In conclusion: Polymorphisms in MDR1 G2677T and G3435T are associated with different responses to chemotherapy and disease susceptibility in pancreatic cancer.

Ji-Young Park, Ph.D. (Sean W. Linder, Ph.D..Preceptor) ORA Fellow; FDA project: Development of Methodologies for the Characterization of FDA Regulated Liposomal Products.

Nanotechnology is commonly defined as the control and manipulation of materials in which one size dimension (length or width) is between approximately 1-100 nm. One example of a commercially available nanomaterial is the liposome, which is typically used as a delivery carrier for therapeutic agents or dietary supplements. It has been well documented that the utilization of liposomal formulations can enhance the efficacy of such agents and/or supplements within biological systems. Methodologies to characterize the physicochemical properties of the commercialized products have not been well documented in the scientific literature. Therefore the main goal of this study is to address this knowledge gap by developing appropriate methods, which could ultimately be used in repose to a regulatory need or adverse public health event.

Katya Petrova, Ph.D. (Laura Schnackenberg, Ph.D., Preceptor) NCTR Fellow; FDA project: Development and Application of LC-SPE-NMR Methods to Evaluate Biomarkers of Hepatotoxicity.

Drug-induced hepatotoxicity is one of the major reasons for withdrawal of FDA-approved drugs from the market and hence is of major concern to the FDA and to consumers. The currently used clinical measures of hepatotoxicity, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are sensitive but not necessarily liver specific. This research project aimed to 1) develop hyphenated liquid chromatography nuclear magnetic resonance (LC-NMR) spectroscopy methods to identify metabolites of interest in liver tissue and 2) identify metabolite biomarkers of liver damage due to drug toxicity and drug-dietary supplement interactions. While NMR data is highly reproducible and directly related to concentration, sensitivity issues prevent metabolites such as bile acids (BAs), present in biofluids and tissue at sub-micromolar concentrations from being measured. Perturbations in the BAs pathway have been reported in previous studies of model hepatotoxicants. Hyphenated methods such as LC-NMR coupled with solid phase extraction (SPE) provide a means to evaluate less abundant metabolites. An LC-time slice-SPE-NMR method was developed for detection of free and conjugated BAs. The LC-time slice-SPE approach coupled with NMR detection can potentially be used for systematic investigation of low abundant analytes with weak UV chromophores, such as bile acids and oxidative stress-related metabolites.

NMR methods were also employed to evaluate the effects of co-exposure to acetaminophen (APAP) and green tea extract (GTE). Increased use of dietary and herbal supplements necessitates the application of tools such as metabolomics to understand the synergistic effects of the supplement on prescription or over-the counter drugs. The effect of exposure to GTE on APAP-induced hepatotoxicity was evaluated for three different dosing scenarios. The changes in the metabolite profiles of mouse liver tissue were different depending on whether GTE was given prior to or after a single dose of APAP. GTE prior to APAP decreased severity of APAP-induced hepatotoxicity, while GTE post-APAP potentiated hepatotoxicity in a dose dependent manner. Metabolites involved in glutathione metabolism and energy pathways were significantly altered and had strong correlations with liver necrosis and serum ALT. The results demonstrate the potential for drug dietary supplements interactions and the need to understand such interactions with increased individual consumption of dietary supplement in the US and worldwide.

Prakash Rath, Ph.D. (Anita Richardson, M.A.S. B.S., M.T., Preceptor) CBER Fellow; FDA project: Science Policy and Compliance Program Risk-Modeling Related to the Regulation of Human Cells, Tissues and Cellular and Tissue-Based Products (HCT/Ps).

This Commissioner's Fellowship project was designed to assist the Office of Compliance and Biologics Quality (OCBQ), Center for Biologics Evaluation and Research (CBER), in its role of ensuring the safety of human cells, tissues, and cellular and tissue-based products (HCT/Ps). HCT/Ps are articles containing or consisting of human cells or tissues that are intended for implantation, transplantation, infusion, or transfer into a human recipient. Examples of HCT/Ps include bone, ligaments, cartilage, skin, dura mater, hematopoietic stem/progenitor cells, oocytes, embryos, cultured cartilage and nerve cells, gene therapy products, and human cells using somatic cell nuclear transfer techniques.

The goal was to understand and contribute to the policy development and compliance programs related to HCT/Ps, designed to impact public health by increasing the overall safety of HCT/Ps. Projects included developing a draft guidance for industry on HCT/P deviation reporting, developing a risk-based assessment program that can be used as tool to help prioritize inspections of registered establishments that manufacture HCT/Ps, and participating in working groups that involve developing and analyzing HCT/P policies.

Subrat N. Rout, Ph.D. (Gary L. Hartman, Preceptor) CBER Fellow; FDA Fellow; FDA project: Development of a liquid mid-density micro array assay for the detection of food-borne enteric viruses using Luminex® xMAPTM technology.

In the current climate of globalization, food borne hepatitis E outbreak can occur due to importation of contaminated food from endemic countries. Contamination of growing beds with Hepatitis E Virus (HEV) from human sewage, animal waste, and drinking contaminated water is a major source for food-borne infection. Diagnostic tests that are rapid and have the ability to identify multiple pathogens in a single tube hold a great promise to minimize food borne infection and reduce cost. Developing a molecular method targeting HEV nucleic acid and multiplexing the method along with other food-borne hepatitis viruses will give a proven advantage over conventional tests reducing time and resources. In this study, we designed and developed a real time quantitative RT-PCR based assay to detect RNA of multiple HEV genotypes and subsequently developed a bead based liquid mid-density microarray assay using Luminex® xMAP technology on Bioplex® platform. The ORF1 regions of three different HEV genotypes (HEV1, HEV2 and HEV3) were targeted for this assay development. The minigenomes, primers and probes of three each HEV genotype ORF1 region were synthesized. The viral sub-genomic RNA was transcribed from their respective linear genomic plasmid using T7 RNA polymerase kit. The integrity and purity of the RNA samples were checked using Agilent 2100 Bioanalyzer. RNA samples with >90% purity were stored at -80º C for future use. Transcribed RNA samples of each HEV genotype was used as template to develop real time quantitative RT-PCR (RT-qPCR) using Cepheid SmartCycler®.

The 3 HEV assays were optimized individually and as a multiplex reaction over a broad range of RNA primers and probe concentration. In addition, an internal control was incorporated into reactions to determine assay condition. Subsequently primers and probes of three HEV genotypes were used to develop a single tube multiplex qRT -PCR reaction. For development of bead based assay, paramagnetic polystyrene microspheres containing unique fluorescent signature conjugated with probe complementary to each HEV genome were used to hybridize with amplified RT-PCR products. PCR products were amplified using a biotin labeled primer and subsequently detected by the use of a reporter dye, Streptavidin-phycoerythrin. This developed assay can be supported by a systematic study of matrix and platform extension, pathogen multiplexing and high throughput capabilities to detect multiple viral pathogens in food samples.

Kristin Schultz-Kuszak, Ph.D. (Michail Alterman, Ph.D., Preceptor) CBER Fellow; FDA project: Proteomic Characterization of Multipotent Stromal Cells Seeded on Different Scaffolds to Uncover

Osteogenic Differentiation Biomarkers.

Multipotent stromal cells (MSCs) have garnered a lot of attention in the field of regenerative medicine due to their pluripotency, low immunogenicity, and immunomodulatory properties. Acquiring FDA approval of these MSC therapies is complex because there are few established characterization methodologies. The goal of this research project was to characterize MSCs undergoing osteogenesis to find biomarkers indicative of MSC cell fate. Two pathways for inducing osteogenesis were explored: 1) the classical route using chemical supplements in the culture media and 2) culturing the cells on a polymeric scaffold (aka spontaneous osteogenesis). Mass spectrometry based proteomics analysis of MSCs cultured on flat surfaces or scaffolds reveals differences in the mechanisms behind these osteogenesis pathways. Initially both Electrospray (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry methods were included in the characterization of the MSC proteome. Data analysis was scaled back to only include ESI mass spectrometry when the MSCs provided by NIST yielded low levels of protein. To address this unforeseen issue, biological replicates were then pooled and prepared solely for electrospray analysis because this method offers faster data collection, ease of operation, and the enhanced on-line separation capabilities.

ESI analysis showed that culture conditions greatly affected the measured MSC proteome. The differences in protein expression measured in these experiments were due to the culture environment and not heterogeneous protein expression amongst donors because control MSCs from NIST showed similar expression profiles to what was measured with MSCs cultured in-house. Based on the collaborator's published microarray data, it was expected the MSCs cultured on the 3D scaffold would exhibit a similar phenotype to those cultured with osteogenic supplements. Hierarchical cluster analysis of the proteomics data indicated a different trend: the 3D scaffold data clustered with the 2D data while the supplemented cells clustered with the control cells. When the micro-array experiments were repeated using replicate MSCs from the same donor, similar clustering patterns were observed, indicating the strong influence of donor to donor heterogeneity in comparing MSC phenotypes and activity. Though the microarray data and the proteomics data revealed similar phenotypic clustering, the measured magnitude of the response was quite large for several proteins. This discrepancy mirrors literature reports showing that mRNA expression is not directly correlated to changes in protein expression. Overall these findings indicate the critical importance of culture conditions in MSC preparations and protein expression. Therefore differences in culturing conditions may alter the therapeutic potential of MSCs by altering the expression of key proteins and modulating important signaling pathways.

Jonathan Swoboda, Ph.D. (Karen Elkins, Ph.D., Preceptor) CBER Fellow; FDA project: The Biological Role of the Cytokine p40 in Clearing Chronic Infections Caused by Vaccination with Francisella

Tularensis Live Vaccine Strain (LVS).

Francisella tularensis is a Gram-negative facultative intracellular pathogen and is the causative agent of tularemia. Infection studies using Francisella can provide a model system for studying other intracellular pathogens such as Mycobacteria, Chlamydia, Salmonella, and Leishmania. Francisella has always been a public health concern since inhalation of as few as 10 bacteria can result in death. Further, the CDC has designated Francisella as a Category A pathogen and a potential biological threat. Due to a limited supply of antibiotics, prophylactic protection using a vaccine against intracellular pathogens would be the ideal prevention.

Various attenuated strains of Francisella have been used with some apparent success in the former U.S.S.R. as vaccines against Francisella infections. However, the FDA has not yet approved a vaccine for Francisella. Currently, a variant denoted Live Vaccine Strain (LVS) is the only vaccine in clinical development for the treatment of tularemia in the U.S. Designing field trials to evaluate vaccines for tularemia, which is rare in nature, would be challenging. However, successful immunity to Francisella using LVS and clinical strains has been mimicked to some degree in several animal models including mice, providing a potential strategy to advance vaccine development in a preclinical setting and to support licensing via the FDA's "Animal Rule." Most vaccines targeting infectious diseases today rely on antibody responses that are simple to measure, and that in some cases correlate well with protection. However, vaccines targeting intracellular pathogens such as Francisella more like provoke cell-based immunity mediated by T lymphocytes. Measurements of T cell responses have not yet been associated with efficacy. Therefore, a major challenge to utilizing the "Animal Rule" and creating an approved vaccine for this class of pathogens lies in understanding the immune mechanisms, including effector molecules, that are important during efficacious T cell-based immunity and that thus might be measured to bridge between animals and in people.

The purpose of this project was to evaluate one such effector, p40, and better understand its required role in clearing a vaccinating dose of LVS. Information from this project could potentially be applied to other pathogens that live within cells, thereby aiding the development of vaccines and medical countermeasures for emerging biological threats caused by these types of bacteria. These results directly contribute to the FDA's mission of using research-based science to protect the public health, enabling safety evaluations as well as the use of the FDA's "Animal Rule" in approving new vaccines targeting intracellular pathogens such as Francisella.

Wendy Tan, Ph.D., (Marian E., Major, Ph.D., Preceptor) CBER Fellow; FDA project: Improvement of hepatitis C virus (HCV) vaccines through T-cell phenotypic analysis and potency assay development.

Hepatitis C virus (HCV) infection is a global health threat. According to WHO, about 170 million people worldwide are chronically infected, with about 3-4 million new cases each year. Current therapeutics available to treat this infection include ribavirin, pegylated interferon alpha and protease inhibitors. However, no prophylactic vaccine is available to prevent HCV infections. T cell-based vaccines represent an important and rapidly developing class of vaccines as prophylaxis for prevention and control of chronic diseases such as HIV, tuberculosis and Malaria. The correlates of protection after T-cell based vaccinations are complicated and differ with each pathogenic antigen. However, certain canonical biomarkers and functional qualities expressed by antigen-experienced T cells at the memory stage present some clues to the qualitative differences among various vaccine platforms. The purpose of this project is to determine the qualitative and quantitative differences among HCV-specific T cells induced post vaccination with various commonly used vaccine vectors and to understand if these differences play a role in the protection following exposure to the pathogen. Antigen-specific CD8+ T cell responses to HCV-NS3 vaccination were successfully tracked in the B6 mouse model. The phenotypes and functional qualities of CD8+ T cells post vaccination were compared among three different viral vectored vaccines.

The ability of memory T cells generated from the three different vaccination regimens to respond to subsequent antigen exposure using a surrogate virus challenge model was also compared.

Immune correlates of protection post vaccination involve very complex immunological and molecular events in vivo, for which it is difficult to set criteria. For some vaccines no true correlates can be determined, but only useful surrogates for an unknown protective response. This presents a great challenge for the regulation of T cell-based vaccines. In this study, the phenotypic and functional data generated may provide some insights into the types of memory T cells that are protective versus non- or less protective memory T cells. Further studies to elucidate other aspects of the immune responses post vaccination can be performed using the B6 mouse system to characterize the immune responses post HCV-NS3 vaccination. I hope that this study will provide a framework upon which future studies and regulatory reviews of T-cell based vaccines can be based.

Jacquline Yancy, Ph.D. (Elizabeth Mansfield, Ph.D. and Katherine Serranco, B.S., Preceptors) CDRH Fellow; FDA project: Development of guidance for Direct-to-Consumer (DTC) genetic testing: A Rapid and Systematic approach for determining the clinical significance and validity of adding new intended uses to already approved/cleared DTC genetic tests.

Direct to consumer (DTC) tests, now redefined as Direct Access Genetic Tests (DAGTs), are a type of test that allows patients (mostly healthy) to order a test and receive results directly from the company providing the test rather than through a more traditional means of having a physician order the test with a prescription, receive and deliver the results. In recent years, tests for numerous genetic variations with clinical implications have been introduced to the market and have been sold without FDA oversight as Laboratory Developed Tests (LDTs) and offered and marketed directly to consumers. Although FDA has cleared a number of genetic tests, none of the genetic tests being marketed and offered directly to consumers has undergone premarket review by the FDA, which would provide for an independent assessment of the diagnostic test's ability to generate results that are safe and effective. This offering of DAGTs without premarket clearance or approval stems from an interpretation of FDA's current policy of enforcement discretion for LDTs, in which no FDA oversight is required.

The DAGT issue has spurred a heated public health, social, legal and ethical debate in the recent years. Some of the outcomes of these debates have included public meetings, advisory panel meetings, several "It has come to our attention" letters issued by FDA to DAGT offerers and a GAO investigation. DAGT offerers believe the ability for a consumer to select and purchase a genetic test without the requirement for involvement of a physician empowers them to know their own genetic code, and be more active in the management of their health care. On the other hand, FDA believes that direct access to genetic tests raises considerable health and safety concerns that that need to be addressed to apply the proper safeguards to ensure safety and effectiveness of the tests. While there are no DAGTs have been approved or cleared currently, the FDA believes that these tests, when offered without a prescription, do not fit the model of an LDT, and should be actively regulated as medical devices according to the standard regulatory scheme. Given that these tests are already on the market, FDA has chosen to implement a reasonable, risk based and effective regulatory framework for reviewing and approving/clearing DTC genetic test claims while they are still on the market.

This project was designed to provide guidance on the definition of a DAGT and the criteria required for sponsors to obtain clearance or approval of such devices. As a result, draft guidance was developed to define the scope and regulatory status of in vitro diagnostic devices (IVDs), to address the types of genetic tests that FDA believes may be appropriately marketed through a direct access model and the regulatory issues for DAGTs evaluated via the premarket review process is under review at the center level.

Xiaoxia Yang, Ph.D. (Jeffrey Fisher, Ph.D., Preceptor) NCTR Fellow; FDA Project: Pharmacokinetic Modeling: Predication and Evaluation of Route Dependent Dosimetry of Bisphenol A in Rats at Different Development Stages.

Bisphenol A (BPA), a synthetic chemical widely used in consumer products, has received considerable attention throughout the last decade. A physiologically based pharmacokinetic (PBPK) model was developed in adult and neonatal rats to quantitatively evaluate age-dependent pharmacokinetic behavior of BPA and its phase II metabolites. The PBPK model was calibrated in adult rats using studies on BPA metabolism and excretion in the liver and gastrointestinal tract, and pharmacokinetic data with BPA in adult rats. For immature rats the hepatic and gastrointestinal metabolism of BPA was inferred from studies on the maturation of phase II enzymes coupled with serum time course data in pups. Metabolism of BPA in the small intestine and liver was responsible for the low oral bioavailability of BPA and enterohepatic recirculation was predicted to prolong the systemic levels of both BPA and its metabolites. The observed age dependence of internal BPA exposure could be partially explained by the immature metabolic capacity of pups. The assessment of BPA dosimetry across rats, non-human primates, and humans suggests that dose adjustments would be necessary to extrapolate toxicity studies from neonatal rats to infant humans.

Diana M. Yoon, Ph.D. (Jiyoung Dang, Ph.D., Mark Lee, Ph.D., Elias Mallis, B.S., Preceptors) CBER/CDRH Fellow; FDA project: Opportunities and challenges of using standards for premarket review of bone regenerative medicine products at CBER and CDRH.

The purpose of this project was to identify how standards can be utilized to assess the safety, efficacy, and performance of FDA-regulated bone tissue engineering/regenerative medicine (TERM) products which are composed of biologics, devices, drugs, tissues and/or a combination of these components. A critical retrospective review of regulatory submissions of these products to CBER and CDRH was conducted to create a database focused on assessing the pre-clinical testing (i.e., performance assays and animal studies) performed on TERM bone products. A complementary analysis of available FDA guidance documents and voluntary consensus standards that describe the pre-clinical test methods for bone TERM products developed by Standard Development Organizations (SDOs) was also conducted in order to assess the information that is publically available to stakeholders. The results from this project identified differences in performance tests used by sponsors to assess bone TERM product properties.

Standards were identified as a potential solution to allow for consistency in assessing the properties of similar products across different submissions. Standards can be complementary to FDA regulations and guidance and can be a potential resource to help meet regulatory requirements of this specific product class. The current challenges identified for standards, such as timely development and consistent use, were addressed through efforts to increase the knowledge base and awareness, both internally at the FDA through generation of useful databases and staff training, and externally in the form of future publications and public workshops.

Shifu Zhao, Ph.D. (Frank Weichold, M.D., Ph.D.) OC Fellow; FDA project: FDA Patient-Centered Outcomes Research Promoting Personalized Medicine.

Funded by the American Recovery and Reinvestment Act (ARRA), the FDA Patient-Centered Outcomes Research (PCOR) Project addresses the Agency's critical needs in upgrading its data infrastructure and building its PCOR research capacity toward the goal of supporting personalized medicine. This fellowship research work directly supports the FDA PCOR project and contributes to the project's initiatives in legacy data conversion of 101 selected clinical trial studies relevant to the FDA's current PCOR efforts, development of innovative trial design and data analysis methods, and development of an FDA streamlined product review environment based on standardized data and advanced analytical tools.

This multi-team project has produced a large number of deliverables including standardized study data, research methods and data systems, which will continue to play important roles in supporting the FDA's regulatory review and research related to clinical data. In addition to supporting various day-to-day activities of the FDA PCOR project, this fellowship work included in-depth analyses of the CDISC SDTM data standard as it was applied to the FDA PCOR project. As a result, an innovative clinical data standardization guide is proposed to provide simple and clear guidance to achieve data standardization goals - enabling standardized study data submission by sponsors and efficient and consistent data review at the FDA as well as streamlined data life cycle management.

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