William Mattes

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.

Strategic paths for biomarker qualification

Biomarkers may be qualified using different qualification processes. A passive approach for qualification has been to accept the end of discussions in the scientific literature as an indication that a biomarker has been accepted. An active approach to qualification requires development of a comprehensive process by which a consensus may be reached about the qualification of a biomarker. Active strategies for qualification include those associated with context-independent as well as context-dependent qualifications.

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.

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.

Research at the interface of industry, academia and regulatory science

volume 28 number 5 mAY 2010 nature biotechnology 1Critical Path Institute, Tucson, Arizona, USA. 2European Medicines Agency, Canary Wharf, London, UK. 3Department of Laboratory Sciences and Investigative Toxicology, Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania, USA. 4Novartis, San Diego, California, USA. 5Food and Drug Administration, Silver Spring, Maryland, USA. e-mail: wbmattes@gmail.com Consortia have played key roles in addressing technological problems common to a competitive industry. For instance, the Sematech consortium, formed in 1987 and comprising 14 leading US semiconductor producers, addressed common issues in semiconductor manufacture and increased R&D efficiency by avoiding duplicative research16. Sematech demonstrates that consortia provide the opportunity for industry scientists to share their experiences in identifying and solving problems, to pool their expertise and to collectively consider mutual questions. To create similar models in drug, diagnostic and device development, the Critical Path Institute (C-Path) was incorporated as a “neutral, third party” to serve as a consortium organizer14 and interface between industry members and the FDA17. One of the first consortia formed by C-Path to address one of the Critical Path gaps was the Predictive Safety Testing Consortium (PSTC)18,19. As noted in the Critical Path Opportunities list, there is a need for “preclinical biomarkers that predict human liver or kidney toxicity” and “collaborations among sponsors to share what is known about existing safety assays”15. Indeed, the preamble to the legal agreement that binds PSTC members notes that “the parties to this Agreement also recognize the importance of validated safety biomarkers to pharmaceutical and biotechnology research and development efforts and wish...to conduct research and development projects, under the coordination of C-Path, to identify and validate such biomarkers to increase drug safety.” Thus, the PSTC is committed to cooperative research resulting in tools beneficial to both pharmaceutical development and regulatory science (termed Critical Path Research). Of course, these tools could be valuable to medical situations where improved monitoring for drug safety would improve outcomes. The PSTC legal agreement furnishes not only a clear set of goals and deliverables that provide guidance for actions and decisions of the consortium, but also a framework to address issues such as antitrust, intellectual property and confidentiality. This assures open data sharing and collaboration in a manner consistent with applicable legal requirements. In particular, the confidentiality provisions also assure that publications (which are encouraged) respect member contributions, again fostering openness and participation. As noted above, C-Path provides executive functions and contributes overall scientific leadership, whereas members lead strategic and technical execution of the scientific working groups pursuing biomarkers of several critical toxicities where understanding of new biomarkers is desired. Members also participate in an advisory committee that, among other functions, reviews new proposals and ongoing projects and guides their scope and growth. A medicine is defined as ‘a substance or preparation used in treating disease’. Society expects that the benefits of medicines should substantially exceed their risks, and this expectation has been translated into governmental policy around the world. Part of the mission of the US Food and Drug Administration (FDA) is to protect the public health by assuring the safety and efficacy of medicines1. The FDA has carried out its mission by relying upon the best current scientific knowledge and practice2. By definition, gaps in current scientific knowledge and practice limit the ability of regulatory agencies, such as the FDA and the European Medicines Agency (EMEA; London), to carry out their mission. Current gaps include a limited ability to extrapolate animal data to humans3–5, the difficulty of evaluating genetic and carcinogenic risks6,7, and our poor understanding of gender-specific responses8. It is hoped that new knowledge, technologies and tools can address these and other gaps and improve the evaluation of new drugs and medicines9–12. In this context, the FDA has advocated a ‘Critical Path Initiative’13,14 to intentionally address gaps in applied and regulatory science. The initial report and subsequent listing of specific opportunities15 called attention to research and tools needed to improve the process of drug development that extends from preclinical testing to ultimate regulatory registration. Although this area is vital for improving the development of new medicines and getting them to the public, it receives little academic, public or legislative attention and, thus, little funding. Rather, the focus of both academic research and news organizations is often on novel discoveries and/or the risks and benefits of drugs after they have reached the marketing phase. Nevertheless, a great deal of essential work must be accomplished between discovery and delivery (that is, in the critical path) to accomplish the delivery of safe and effective medicines to the public. With the goal of improving that process, the FDA has not only identified gaps in ‘Critical Path Research’ but also suggested that an effective approach to address these gaps would be to form consortia of industry, academic and regulatory scientists to share resources, expertise and experience toward accomplishing shared common specific objectives. Research at the interface of industry, academia and regulatory science

The Predictive Safety Testing Consortium: A synthesis of the goals, challenges and accomplishments of the Critical Path

The qualification of biomarkers of drug safety requires data on many compounds and nonclinical and clinical studies. The cost and effort associated with these qualifications cannot be easily covered by a single pharmaceutical company. Intellectual property associated with safety biomarkers is also held by many different companies. Consortia between different pharmaceutical companies can overcome cost and intellectual property hurdles to biomarker qualification. The Predictive Safety Testing Consortium (PSTC) is a collaborative effort between 16 different pharmaceutical companies to generate data supporting biomarker qualification. This Consortium is coordinated through the C-Path Institute, and currently has five biomarker qualification working groups engaged in this collaboration: nephrotoxicity, hepatotoxicity, vascular injury, myopathy, and non-genotoxic carcinogenicity. These working groups are aided by a data management team and a translational strategy team. Qualification studies of promising biomarkers are already progressing in several of the working groups, and results in the nephrotoxicity working group warranted a data submission to the FDA and EMEA for regulatory qualification of new nephrotoxicity biomarkers.:

Regorafenib impairs mitochondrial functions, activates AMP-activated protein kinase, induces autophagy, and causes rat hepatocyte necrosis.

The tyrosine kinase inhibitor regorafenib was approved by regulatory agencies for cancer treatment, albeit with strong warnings of severe hepatotoxicity included in the product label. The basis of this toxicity is unknown; one possible mechanism, that of mitochondrial damage, was tested. In isolated rat liver mitochondria, regorafenib directly uncoupled oxidative phosphorylation (OXPHOS) and promoted calcium overload-induced swelling, which were respectively prevented by the recoupler 6-ketocholestanol (KC) and the mitochondrial permeability transition (MPT) pore blocker cyclosporine A (CsA). In primary hepatocytes, regorafenib uncoupled OXPHOS, disrupted mitochondrial inner membrane potential (MMP), and decreased cellular ATP at 1h, and triggered MPT at 3h, which was followed by necrosis but not apoptosis at 7h and 24h, all of which were abrogated by KC. The combination of the glycolysis enhancer fructose plus the mitochondrial ATPase synthase inhibitor oligomycin A abolished regorafenib induced necrosis at 7h. This effect was not seen at 24h nor with the fructose or oligomycin A separately. CsA in combination with trifluoperazine, both MPT blockers, showed similar effects. Two compensatory mechanisms, activation of AMP-activated protein kinase (AMPK) to ameliorate ATP shortage and induction of autophagy to remove dysfunctional mitochondria, were found to be mobilized. Hepatocyte necrosis was enhanced either by the AMPK inhibitor Compound C or the autophagy inhibitor chloroquine, while autophagy inducer rapamycin was strongly cytoprotective. Remarkably, all toxic effects were observed at clinically-relevant concentrations of 2.5-15μM. These data suggest that uncoupling of OXPHOS and the resulting ATP shortage and MPT induction are the key mechanisms for regorafenib induced hepatocyte injury, and AMPK activation and autophagy induction serve as pro-survival pathways against such toxicity.

Public Consortium Efforts in Toxicogenomics

Public consortia provide a forum for addressing questions requiring more resources than one organization alone could bring to bear and engaging many sectors of the scientific community. They are particular well suited for tackling some of the questions encountered in the field of toxicogenomics, where the number of studies and microarray analyses would be prohibitively expensive for a single organization to carry out. Five consortia that stand out in the field of toxicogenomics are the Institutional Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Committee on the Application of Genomics to Mechanism Based Risk Assessment, the Toxicogenomics Research Consortium, the MicroArray Quality Control (MAQC) Consortium, the InnoMed PredTox effort, and the Predictive Safety Testing Consortium. Collectively, these consortia efforts have addressed issues such as reproducibility of microarray results, standard practice for assays and analysis, relevance of microarray results to conventional end points, and robustness of statistical models on diverse data sets. Their results demonstrate the impact that the pooling of resources, experience, expertise, and insight found in consortia can have.

Microarray Probe Mapping and Annotation in Cross-Species Comparative Toxicogenomics

Genomics-based tools, such as microarrays, do appear to offer promise in evaluating the relevance of one species to another in terms of molecular and cellular response to a given treatment. However, to fulfill this promise the individual end points (i.e., the genes, proteins, or mRNAs) measured in one species must be mapped to corresponding end points in another species. Several approaches, along with their strengths and weaknesses, are described in this chapter. A sequential approach is described that first makes use of a Genome To Genome Through Orthology method, where probe sequences for a given species are mapped into full-length sequences for that species, associated with the locus for those sequences and then into a second species by consulting orthology resources. The second step supplements these results by mapping the probe sequences for the given species into the best matching transcript from any organism, which then are mapped into the appropriate native locus and finally into the second species via an orthology resource. The results of this method are given for an experiment comparing the transcriptional response of canine liver to phenobarbital with that of rat liver.