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

描述（由申请方提供）：需要对大鼠心肌肌钙蛋白（cTnI）、快收缩骨骼肌肌钙蛋白-I（fsTnI）和慢收缩骨骼肌肌钙蛋白-I（ssTnI）进行高特异性、超灵敏度、低容量测定，以评估和区分临床前研究中的心脏和肌肉毒性。这项工作的主要目的是开发一种多面板的超灵敏度，单分子计数肌钙蛋白-I测定。这些检测试剂盒将对肌钙蛋白-I亚型（肌钙蛋白-快型、肌钙蛋白-慢型和心脏型）具有特异性，但在大鼠和人同源物之间具有交叉反应性，用于大鼠临床前毒性试验和随后的人体临床研究。该建议的意义在于开发一种新的工具，以在临床前研究期间更好地筛选候选药物的心肌和心脏毒性。鉴于最近FDA对他汀类药物（辛伐他汀）和罗格列酮（文迪雅）这两种主要处方药的安全警报，这一点尤其重要。在高剂量下，这些药物已被证明会增加不良肌肉毒性和心脏毒性作用的风险，尽管这两种药物以前被认为是安全的，并被FDA批准。自批准以来，它们已被广泛销售和广泛处方，因此引起了深切关注。提出的创新将创建用于肌钙蛋白亚型的超灵敏单分子免疫测定，用作肌毒性和心脏毒性的替代生物标志物，从而能够更早地检测药物诱导的损伤。目前市售的肌钙蛋白测定无法在健康动物中进行基线定量，因此通过心脏和肌肉组织的组织病理学检查来评价传统药物诱导的心脏和肌肉毒性，以确定是否存在指示药物诱导损伤的硬化和坏死病变。然而，这些方法只能用于识别晚期毒性，在大量器官损伤已经发生之后。使用肌钙蛋白作为替代标志物早期检测毒性将是当前临床前安全性评价的显著改进。本提案的主要假设是，区分心肌和骨骼肌钙蛋白-I（快和慢）形式的高度特异性测定，具有定量和监测基线动物循环浓度的灵敏度，将能够在早期临床前研究中检测和区分毒性药物化合物引起的心肌和肌肉特异性损伤。该提案的具体目标包括：（1）开发快速和慢速收缩肌肉中骨骼肌钙蛋白-I的新检测方法，并显示初步分析验证，包括灵敏度、定量限、范围、亚型特异性、人-大鼠交叉反应性和精确度。(2)结合cTnI测定正常大鼠和人血清样本中快肌和慢肌骨骼肌钙蛋白I的基线水平。(3)使用开发的试验组检测大鼠的存档血清样本，与具有已知肌肉毒性的化合物（即他汀类药物）相比，这些大鼠接受了已知心脏毒性化合物（即异丙肾上腺素）给药，作为相应生理毒性终点检测特异性的试验。在完成本提案的I期后，我们将准备使用我们的多试验肌钙蛋白-I毒性组在给药前和给药后大鼠的纵向时间过程研究中筛选其他化合物（即受体酪氨酸激酶抑制剂）用于心脏和/或肌肉特异性损伤的适应症。这些研究将大大有利于开发对心脏和肌肉损伤具有高安全性的药物，即使是目前无法研究的低剂量。 公共卫生相关性：我们建议开发一种多组超灵敏、高特异性肌钙蛋白-I检测试剂盒，以检测和区分临床前模型中的心脏和肌肉损伤，以检测也可用于人体的毒性。这些试验将对肌钙蛋白-I亚型（肌钙蛋白-快型、肌钙蛋白-慢型和心脏型）具有特异性，但在大鼠和人同系物之间具有交叉反应性，用于大鼠心脏毒性临床前模型和后续的人体临床研究。该建议的意义在于开发一种新的工具，以在临床前研究期间更好地筛选候选药物的心肌和心脏毒性。鉴于最近FDA对他汀类药物（辛伐他汀）和罗格列酮（文迪雅）这两种主要处方药的安全警报，这一点尤其重要。

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 differentiate between 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 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). At high doses these 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 surrogate markers 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 to develop 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).