David Jacobson-Kram

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortiums (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Health Effects of Depleted Uranium on Exposed Gulf War Veterans: A 10-Year Follow-Up

Medical surveillance of a group of U.S. Gulf War veterans who were victims of depleted uranium (DU) “friendly fire” has been carried out since the early 1990s. Findings to date reveal a persistent elevation of urine uranium, more than 10 yr after exposure, in those veterans with retained shrapnel fragments. The excretion is presumably from ongoing mobilization of DU from fragments oxidizing in situ. Other clinical outcomes related to urine uranium measures have revealed few abnormalities. Renal function is normal despite the kidneys expected involvement as the “critical” target organ of uranium toxicity. Subtle perturbations in some proximal tubular parameters may suggest early although not clinically significant effects of uranium exposure. A mixed picture of genotoxic outcomes is also observed, including an association of hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutation frequency with high urine uranium levels. Findings observed in this chronically exposed cohort offer guidance for predicting future health effects in other potentially exposed populations and provide helpful data for hazard communication for future deployed personnel.

Towards consensus practices to qualify safety biomarkers for use in early drug development

Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.

Surveillance of depleted uranium exposed Gulf War veterans: Health effects observed in an enlarged friendly fire cohort

To determine clinical health effects in a small group of US Gulf War veterans (n = 50) who were victims of depleted uranium (DU) “friendly fire,” we performed periodic medical surveillance examinations. We obtained urine uranium determinations, clinical laboratory values, reproductive health measures, neurocognitive assessments, and genotoxicity measures. DU-exposed Gulf War veterans with retained metal shrapnel fragments were excreting elevated levels of urine uranium 8 years after their first exposure (range, 0.018 to 39.1 &mgr;g/g creatinine for DU-exposed Gulf War veterans with retained fragments vs 0.002 to 0.231 &mgr;g/g creatinine in DU exposed but without fragments). The persistence of the elevated urine uranium suggests ongoing mobilization from the DU fragments and results in chronic systemic exposure. Clinical laboratory outcomes, including renal functioning, were essentially normal. Neurocognitive measures showing subtle differences between high and low uranium exposure groups, seen previously, have since diminished. Sister chromatid exchange frequency, a measure of mutation in peripheral lymphocytes, was related to urine uranium level (6.35 sister chromatid exchanges/cell in the high uranium exposure group vs 5.52 sister chromatid exchanges/cell in the low uranium exposure group;P = 0.03). Observed health effects were related to subtle but biologically plausible perturbations in central nervous system function and a general measure of mutagen exposure. The findings related to uranium’s chemical rather than radiologic toxicity. Observations in this group of veterans prompt speculation about the health effects of DU in other exposure scenarios.

Interlaboratory Evaluation of Genomic Signatures for Predicting Carcinogenicity in the Rat

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.

Use of Transgenic Mice in Carcinogenicity Hazard Assessment

Determining the carcinogenic potential of materials to which humans have significant exposure is an important, complex and imperfect exercise. Not only are the methods for such determinations protracted, expensive and utilize large numbers of animals, extrapolation of data from such studies to human risk is imprecise. Toxicologists have long recognized these shortcomings but the 2-year chronic rodent study has remained the gold standard. Recent developments in the field of molecular oncology and development of methods to insert or inactivate specific genes in animals have provided the tools with which to develop the next generation of carcinogenicity assays. With improved understanding of oncogene activation and tumor suppressor gene inactivation a number of animal models have been developed to dramatically reduce latency for chemically induced cancers. This has led to the development of shorter carcinogenicity assays. Also, because the spontaneous tumor frequencies in these animals are low during the in-life portion of the study, and studies are terminated well before the health complications of advanced aging are observed, it has been possible to reduce the group sizes and reduce animal usage. FDAs adoption of ICH S1B in 1997, (ICH, 1997) “Testing for the Carcinogenicity of Pharmaceuticals,” opened the door for the use of such transgenic models in regulatory toxicology. This presentation reviews the current state of the science and its application to regulatory issues.

Health surveillance of Gulf War I veterans exposed to depleted uranium: Updating the cohort

A cohort of seventy-four 1991 Gulf War soldiers with known exposure to depleted uranium (DU) resulting from their involvement in friendly-fire incidents with DU munitions is being followed by the Baltimore Veterans Affairs Medical Center. Biennial medical surveillance visits designed to identify uranium-related changes in health have been conducted since 1993. On-going systemic exposure to DU in veterans with embedded metal fragments is indicated by elevated urine uranium (U) excretion at concentrations up to 1,000-fold higher than that seen in the normal population. Health outcome results from the subcohort of this group of veterans attending the 2005 surveillance visit were examined based on two measures of U exposure. As in previous years, current U exposure is measured by determining urine U concentration at the time of their surveillance visit. A cumulative measure of U exposure was also calculated based on each veterans past urine U concentrations since first exposure in 1991. Using either exposure metric, results continued to show no evidence of clinically significant DU-related health effects. Urine concentrations of retinol binding protein (RBP), a biomarker of renal proximal tubule function, were not significantly different between the low vs. high U groups based on either the current or cumulative exposure metric. Continued evidence of a weak genotoxic effect from the on-going DU exposure as measured at the HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus and suggested by the fluorescent in-situ hybridization (FISH) results in peripheral blood recommends the need for continued surveillance of this population.

Re-evaluation of the mutagenic potential of quinacrine dihydrochloride dihydrate.

Quinacrine has been used for voluntary female non-surgical sterilization for its ability to produce tubal occlusion. Safety issues regarding quinacrine have been raised because it has been shown to intercalate with DNA. Therefore, safety issues need to be resolved by appropriate toxicology studies to support a review for human transcervical use. Such toxicology studies include mutagenicity assays. Here we report an evaluation of the genotoxicity of quinacrine dihydrochloride dihydrate (QH) using a battery of assays. In the bacterial mutagenicity assay, QH was strongly positive in Salmonella typhimurium tester strain TA1537 with and without S9-activation and in S. typhimurium tester strain TA98 with S9-activation; QH was also strongly positive in Escherichia coli WP2 uvrA without S9-activation. QH was not mutagenic in S. typhimurium tester strains TA100 and TA1535 with and without S9-activation. QH was mutagenic in the mouse lymphoma assay in the absence of S9-activation. QH was clastogenic in Chinese hamster ovary (CHO) cells, with and without S9-activation. QH was negative for polyploidy in the same chromosome aberration test. Using a triple intraperitoneal injection treatment protocol in both male and female mice, QH was negative in the in vivo mouse micronucleated erythrocyte (micronucleus) assay. These results confirm that QH is mutagenic and clastogenic in vitro and suggest a potential risk to human health due to QH exposure after intrauterine exposure.

Cancer risk assessment approaches at the FDA/CDER: Is the era of the 2-year bioassay drawing to a close?

Determining the carcinogenic potential of materials to which humans have significant exposures is an important, complex, and imperfect exercise. Not only are the methods for such determinations protracted and expensive and use large numbers of animals, extrapolation of data from such studies to human risk is imprecise. With improved understanding of oncogene activation and tumor suppressor gene inactivation, a number of animal models have been developed to dramatically reduce latency for chemically induced cancers and has led to the development and use of shorter carcinogenicity assays. Recent studies by a number of investigators suggest that specific gene signature patterns seen after short-term exposure of rats to test chemicals can predict long-term outcomes in cancer bioassays with relatively high accuracy. In addition, a recent survey performed by PhRMA member companies examined two hundred drug years to determine whether histological biomarkers seen at the end of a six- or twelve-month toxicology study in rats can predict the outcome of a two-year carcinogenicity study. With only a handful of exceptions, chronic studies appear capable of predicting effects at the end of two years with good accuracy. It is hoped that the combination of results from transgenic mouse assays and six-month rat studies will soon supplant the need for most two-year bioassays.

Pharmacogenomic data submissions to the FDA: non-clinical case studies

John K Leighton†1, Joseph DeGeorge2, David Jacobson-Kram3, James MacGregor4, Donna Mendrick5 & Alexandra Worobec6 †Author for correspondence 1Supervisory Pharmacologist, Division of Oncology Drug Products, CDER, FDA, 5600 Fishers Lane, Rockville, MD 20850, USA E-mail: leightonj@ cder.fda.gov 2Vice President, Research and Development Safety Assessment, Novartis Pharmaceuticals, USA 3Associate Director for Pharmacology and Toxicology, CDER, Office of New Drugs, FDA, USA 4Deputy Director, National Center for Toxicological Research, FDA, USA (retired) 5Vice President, Toxicogenomics, Gene-Logic, USA 6Medical Reviewer, CDER, FDA, USA