Development of an ultra-sensitive SMC multi-assay toxicity panel for pre-clinical

DESCRIPTION (provided by applicant): There is a need for highly specific, ultra-sensitive, low volume assays for cardiac troponin (cTnI), fast-twitch skeletal troponin-I (fsTnI) and slow-twitch skeletal troponin-I (ssTnI)in rats to assess and differentiatebetween cardiac and muscle toxicity in pre-clinical studies. The primary objective of this work is to develop a multi-panel of ultra-sensitive, single molecule counting troponin-I assays. These assays will be specific for the troponin-I isoform (skeletal-fast, skeletal-slow and cardiac) but will be cross reactive between rat and human homologs, for use in rat pre-clinical toxicity testing and in subsequent human clinical studies. The significance of this proposal is to develop a novel tool to better screenfor myo- and cardio-toxicity of pharmaceutical candidates during pre-clinical studies. This is especially relevant in light of recent FDA safety alerts for two prominently prescribed drugs, statins (simvastatin) and rosiglitazone (avandia). At high dosesthese drugs have been shown to increase risk of adverse myotoxic and cardiotoxic effects, even though both drugs were previously considered safe and were approved by the FDA. Since approval they have been widely marketed and extensively prescribed, thus generating deep concern. The innovation proposed will create ultra- sensitive, single molecule immunoassays for troponin isoforms to be used as surrogate biomarkers for myo- and cardio- toxicity, enabling earlier detection of drug induced injury. Current at-market troponin assays are incapable of baseline quantification in healthy animals, and thus traditional drug induced cardio- and myo-toxicity have been evaluated by histo- pathological examination of heart and muscle tissue for the presence of sclerotic and necrotic lesions indicative of drug induced damage. However these methods can only be used to identify advanced stages of toxicity, after significant amounts of organ damage have already occurred. Early detection of toxicity with troponins as surrogatemarkers will be a significant improvement upon current pre-clinical safety evaluation. The primary hypothesis of this proposal is that highly specific assays that differentiate between cardiac and skeletal (fast and slow) forms of troponin-I, with the sensitivity to quantify and monitor circulating concentrations in baseline animals, will be able to detect and differentiate between cardiac and muscle specific damage caused by toxic pharmaceutical compounds in early stage pre-clinical studies. The specific aims of this proposal include: (1) Develop new assays for skeletal troponin-I in fast- and slow- twitch muscle and show preliminary analytical validation, including sensitivity, limit of quantification, range, isoform specificity, human-rat cross reactivity, and precision. (2) Determine the baseline levels of fast- and slow-twitch skeletal troponin-I in normal rat and human serum samples in conjunction with cTnI. (3) Use the developed panel of assays to test archival serum samples from rats that have been dosed with known cardiotoxic compounds (i.e. Isoproterenol) compared to compounds with known muscle toxicity (i.e. statins) as a test for specificity of detection for corresponding physiological toxicity endpoints. Upon completion of Phase I of this proposal, we will be poised to use our multi-assay troponin-I toxicity panel to screen additional compounds (i.e. receptor tyrosine kinase inhibitors) for indications of cardiac and/or muscle specific damage in longitudinal time-course studies of pre- and post-dose rats. These studies will be of large benefit to the development of pharmaceuticals with a high safety profile for cardiac and muscular damage, even at low doses that are not currently feasible to investigate. PUBLIC HEALTH RELEVANCE: We propose todevelop a multi-panel of ultra-sensitive, highly specific troponin-I assays to detect and differentiate between cardiac and muscle damage in pre-clinical models for toxicity that can also be utilized in humans. These assays will be specific for the troponin-I isoform (skeletal-fast, skeletal-slow and cardiac) but will be cross reactive between rat and human homologs, for use in rat pre-clinical models of cardiotoxicity and in subsequent human clinical studies. The significance of this proposal is to develop a novel tool to better screen for myo- and cardio-toxicity of pharmaceutical candidates during pre-clinical studies. This is especially relevant in light of recent FDA safety alerts for two prominently prescribed drugs, statins (simvastatin) and rosiglitazone (avandia).