Lauren A. Trepanier - US grants

Sulfamethoxazole (SMX), sulfadiazine, dapsone, pentamidine, and procainamide are clinically important drugs, the use of which is limited by the occurrence of hypersensitivity reactions in some patients. These reactions are thought to be related to the presence of a toxic hydroxylamine metabolite and its spontaneous byproduct, the nitroso metabolite. Unlike the parent compounds, these metabolites are both cytotoxic and immunogenic. Hypersensitivity reactions to SMX and related drugs occur in a high percentage (25-60 percent) of patients with AIDS. The reason for this high incidence is unclear, but data from in vitro cytotoxicity assays in peripheral blood mononuclear cells (PBMC's) suggest that both HIV-positive and HIV-negative patients with such hypersensitivity have a defect in hydroxylamine or nitroso detoxification. We hypothesize that detoxification of hydroxylamine and nitroso metabolites by reduction is an important determinant of hypersensitivity to sulfonamide and related drugs. We further hypothesize that NADH cytochrome b5 reductase, which is involved in hydroxylamine reduction in some species, is important for hydroxylamine detoxification in humans. In addition, we have novel data to suggest that flavins (FAD and FMN); in the presence of glutathione, are capable of non- enzymatic reduction of hydroxylamines. We therefore propose that defects in either NADH- or flavin-dependent reduction of hydroxylamines and nitroso metabolites are associated with the outcome of sulfonamide hypersensitivity. To address these hypotheses, we will examine the role of NADH cytochrome b5 reductase (b5R) in hydroxylamine and nitroso reduction in both liver and PBMC's, using expressed human recombinant b5R and antibodies to human b5R. We will evaluate the role of flavins in hydroxylamine and nitroso reduction using in vitro stoichiometric assays in a cell-free system, and correlation of flavin and glutathione content with activity in hepatic microsomes and PBMC's. Finally, we will correlate hydroxylamine and nitroso reduction, b5R expression, and flavin content with the outcome of SMX hypersensitivity in patients with HIV infection. The ultimate goal of these studies is to better understand the pathogenesis of toxicity to sulfonamides and a related group of clinically important drugs which generate hydroxylamine and nitroso metabolites. The results of these experiments, which will identify the pathways involved in the detoxification of these metabolites, will suggest better strategies for the prevention of hypersensitivity and other hydroxylamine-related toxicity.

Project summary: Hydroxylamine and nitroso metabolites have been implicated in the pathogenesis of adverse reactions to sulfamethoxazole (SMX) and other arylamine drugs. Hydroxylamines spontaneously oxidize to reactive nitroso metabolites, which, in the case of SMX, can trigger delayed-type hypersensitivity reactions. These hypersensitivity reactions interfere with the effective use of SMX, which is the drug of choice for the prevention of opportunistic infections in immunocompromised patients. Studies of metabolic risk factors for SMX hypersensitivity have largely yielded negative results;however, these studies have not considered the reductive metabolism of hydroxylamine and nitroso metabolites. We have recently shown that hydroxylamines are detoxified by the flavoprotein NADH cytochrome b5 reductase (b5R) and the hemeprotein cytochrome b5 (cyt b5), through a novel, direct pathway of xenobiotic reduction. We have also shown that ascorbate, in addition to thiols, provides a major pathway of nitroso reduction. Our overall hypothesis is that impaired hydroxylamine or nitroso reduction predisposes patients to adverse reactions to arylamine compounds, such as hypersensitivity to SMX. We will address this hypothesis by first characterizing variability in hydroxylamine reduction and its relationship to genetic polymorphisms in b5R or cyt b5 in humans. We will next determine whether alterations in hydroxylamine or nitroso reduction influence the immunogenic response to SMX metabolites, using ascorbate, thiol, or flavin restriction in a guinea pig model. Finally, we will determine whether impaired hydroxylamine or nitroso reduction is a risk factor for SMX hypersensitivity, in a prospective study of immunocompromised patients with lymphoid malignancies. These studies will advance our understanding of the mechanisms underlying SMX hypersensitivity, and will characterize individual variability in a novel direct pathway of xenobiotic reduction, with clinical implications for responses to many compounds, including amidoxime pro-drugs and arylamine carcinogens, in addition to SMX. Relevance: These studies will help us to better understand individual risk factors that lead to "sulfa drug" allergies in people, by learning about differences in the ways that sulfa drugs are metabolized by different people. The ultimate goal of these studies is to find better ways to prevent these adverse reactions.

DESCRIPTION (provided by applicant): Veterinarians obtain broad comparative training in the pathophysiology of diseases across many species, and are therefore well positioned to conduct translational research that benefits human as well as veterinary health. However, most veterinarians do not obtain advanced training after obtaining their DVM degrees. In order to spark interest in formal research training programs, we propose the development of a Clinician Scientist Training Workshop that would provide entry-level research training to veterinary clinicians. The goals of this workshop are to enhance interest and applicable skills in applied research among veterinary residents and new veterinary faculty, to expose trainees in the human medical fields to spontaneous animal models of disease, and to promote collaborations with veterinary schools for bench-to-bedside research. The School of Veterinary Medicine, UW-Madison, will organize the two-day workshop, in collaboration with the University of Wisconsin-Madison Institute for Clinical and Translational Research. We will involve veterinary clinicians and other interested CTSA center members by remote conferencing to UC Davis, the University of Pennsylvania, Tufts University, and Ohio State, which are sites with both CTSAs and veterinary schools. The workshop is scheduled for the first or second weekend in October, and will be held at the Health Sciences Learning Center at UW-Madison. We are seeking support for the first year, but our goal is to offer this workshop biennially. Project PUBLIC HEALTH RELEVANCE (provided by applicant): Most veterinary clinical trainees do not obtain adequate research training, yet these are the same clinicians that will become faculty members at veterinary schools across the country, and will be expected to mentor the next generation of veterinary clinician scientists. Many young clinical veterinary faculty, while confident and talented as clinicians and teachers, are ill-prepared to conduct their own independent, quality research. These faculty members have enormous untapped potential to contribute to collaborative and translational research initiatives. One of the NIH Roadmap for Medical Research initiatives asserts that " we must cultivate and train a cadre of clinical researchers with skills that match the increasing complexity and needs of the research enterprise...The clinical research workforce must be large enough to facilitate bench-to-bedside research... Clinicians must be trained to work in the interdisciplinary, team-oriented environments that characterize today's emerging research efforts." This Workshop is the first national meeting to provide entry-level research training to veterinary clinicians from various specialties. It is unique in its collaboration with the UW Institute for Clinical and Translational Research, and is therefore well situated to promote research collaborations between veterinarians and medical, nursing, pharmacy, and engineering professionals. In addition, this is the first meeting to bring together veterinary members of CTSA-funded centers across the country, and should promote creative discussion of various collaboration models. This short workshop is by its nature and overview to start. We hope to interest participants in enrolling in ICTR's Capstone Certificate in the Fundamentals of Clinical Research or the Graduate Program in Clinical Investigation. We also hope to promote ongoing exploration of collaborations that can be developed among veterinary members of CTSA-funded units across the country.

DESCRIPTION (provided by applicant): Mechanisms of risk for sulfonamide hypersensitivity Hypersensitivity (HS) to potentiated sulfonamide antibiotics is one of the most common idiosyncratic adverse drug reactions, affecting about 3% of the general population, and up to 50-60% of HIV-infected patients. Sulfamethoxazole (SMX), in combination with trimethoprim (TMP), can lead to fever, skin rash and multi-organ toxicity, and is the leading cause of life-threatening bullous skin eruptions in human patients. A better understanding of the mechanisms of risk in both immunocompetent and HIV-infected patients is needed to better predict and prevent these reactions. This is particularly important in light of the widespread use of these antimicrobials fo infection prophylaxis in immunocompromised patients, as well as their renewed use for methicillin-resistant Staph. aureus infections. Specific aim 1a will focus on genetic risk of sulfonamide HS in immunocompetent patients, which appears to be familial, using a two-stage GWAS design with tolerant patients as controls. Specific aim 1b will focus on the observation that peripheral blood mononuclear leukocytes (PBMCs) from HS patients are more susceptible to toxicity from sulfonamide metabolites compared to drug tolerant patients. Understanding the mechanism(s) for this surrogate marker may provide insight into the mechanisms of risk for systemic drug hypersensitivity. This subaim will identify differentially expressed transcripts in PBMCs from carefully phenotyped sulfonamide HS versus tolerant patients, and confirm the mechanistic significance of candidate transcripts by determining the effects of knock-down or over-expression on the cytotoxicity from sulfonamide metabolites in lymphoid cells. Together, these studies will characterize genetic risk for sulfonamide HS patients on both the genomic and transcriptional levels. In Aim 2, we will explore acquired risk factors for sulfonamide HS in HIV infection, using an SIV infection model in rhesus macaques. Baseline data, to include antioxidant and cytokine profiles, in vitro cytotoxicity assays, and liver and leukocyte expression profiles, will be obtained, followed by oral administration of a therapeutic dosage of TMP-SMX. Primary outcomes will include development of serum drug adducts, drug specific T cells, and toxicologic signs consistent with sulfonamide HS, along with baseline predictors for these outcomes. The goal of these aims is to better understand the genetic and acquired risk factors for sulfonamide drug hypersensitivity, so that better predictive and preventative measures can be developed. The ultimate goal is to improve the safety of this inexpensive antimicrobial for both the general population and for HIV-infected patients.

! Project summary Advances in human health rely on valid animal models of disease. Inbred or genetically engineered rodent models can answer important questions about disease pathogenesis and test promising therapies, but can sometimes fail to predict disease responses seen in humans. Spontaneously occurring diseases in companion and other domestic animals can complement the use of engineered laboratory models to understand human diseases, especially those that involve complex genetic traits, disease-modifying gene loci, environmental-gene interactions, or chronic disease progression. Veterinary clinician-scientists have expertise in these naturally occurring animal diseases, and have much to contribute to interdisciplinary research teams that are working to solve human health problems. The purpose of this proposal is to support the development of veterinary specialists into clinician-scientists capable of being productive contributors to translational research. This proposal outlines 3 initiatives: 1) targeted post-residency Translational Research Fellowships for veterinary specialists, to perform research with inter-disciplinary teams on spontaneous diseases shared by humans and animals; 2) a Translational Research Immersion Program for early career clinical faculty, to provide key training in grant writing and mentorship, and showcase successful models of interdisciplinary research collaborations; and 3) convene Translational Research Summits for established veterinary and human medical researchers working on the same diseases, to accelerate the use of spontaneous animal models to understand human disease. The long-term goals of this Translational Research Workforce Training proposal are to catalyze interdisciplinary translational research among veterinarians, basic scientists, physicians and other human health professionals. These three initiatives will provide a generalizable model to recruit clinical specialists of all backgrounds to centers of excellence within the CTSA network, provide immersion research training for clinical faculty followed by evidence-based mentoring support, and bring together clinicians and scientists from different walks of life to collaborate around shared disease interests.

Project Summary: The University of Wisconsin (UW) Graduate Program in Clinical Investigation (GPCI) was developed by the UW Institute for Clinical and Translational Research (ICTR) and continues to be administered by the ICTR Workforce Development group. GPCI houses multiple educational options for trainees in the translational research continuum, but all are based on a common didactic foundation encompassing biostatistics, epidemiology, clinical study design, clinical trials conduct, and the ethical and responsible conduct of research. These essential curriculum elements are shared by the Certificate in the Fundamentals of Clinical Research, the MS in Clinical Investigation, and two PhD programs. The PhD in Clinical Investigation is an applied degree in which trainees focus on the creation of novel methodologies and tools for translational science within the context of a specific biomedical discipline. The PhD in Clinical and Translational Science leverages the core curriculum to give trainees the skills and tools necessary to move their biomedical research along the translational pathway. Since its inception in 2008, GPCI has created a robust infrastructure for training future members of the translational workforce that has allowed students in the program to accumulate an impressive record of publications and research awards. For this NRSA application, new leadership has been appointed distinct from the parental UL1. David DeMets, PhD, and Elizabeth Meyerand, PhD, both have outstanding training records with mentees at all levels and are exemplary investigators in translational sciences. Specific aims include maintaining both existing, high-performing PhD programs; applying an integrated evaluation approach for continuous program improvement and longitudinal outcomes tracking; and developing recruitment strategies to foster trainee diversity and success. In addition, a new postdoctoral training program will be created for individuals with professional degrees (MD, DVM, PharmD, Nursing) to enhance the translational potential of their prior research and provide a bridge to future independent careers in translational sciences. All trainees will benefit from the existing comprehensive mentoring approach, development of individualized career development plans (ICDP), and integrated program activities including federally-mandated training (HIPAA, CITI, GCP, RCR, etc.) and additional training in team science, scientific leadership, mentor-mentee relationships, and writing manuscripts and grants. GPCI also provides career development seminars and workshops in innovation and entrepreneurship. The expanded program will support 15 two-year appointments combined in all pre and postdoctoral categories.