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

描述(申请人提供)：在临床前研究中，需要对大鼠的心肌肌钙蛋白(CTnI)、快抽动骨骼肌肌钙蛋白I(FsTnI)和慢抽动骨骼肌肌钙蛋白I(SsTnI)进行高特异性、超灵敏、低容量的检测，以评估和区分心肌和肌肉毒性。这项工作的主要目标是开发一种多面板的超灵敏、单分子计数的肌钙蛋白-I检测方法。这些检测将专用于肌钙蛋白-I亚型(骨骼快型、骨骼慢型和心脏型)，但将在大鼠和人类同系物之间产生交叉反应，用于大鼠临床前毒性测试和后续的人类临床研究。这项建议的意义是开发一种新的工具，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FDA最近对他汀类药物(辛伐他汀)和罗格列酮(文迪雅)这两种重要处方药的安全警告，这一点尤其相关。在高剂量下，这些药物被证明增加了不良的肌肉毒性和心脏毒性作用的风险，尽管这两种药物之前都被认为是安全的，并得到了FDA的批准。自批准以来，它们得到了广泛的市场推广和广泛的处方，因此引起了深切的关注。这项拟议的创新将创建超灵敏的单分子免疫分析方法，将肌钙蛋白亚型用作心肌和心脏毒性的替代生物标记物，从而能够更早地检测到药物引起的损伤。目前市场上的肌钙蛋白测定不能对健康动物进行基线定量，因此，传统药物引起的心脏和肌肉毒性已经通过心脏和肌肉组织的组织病理学检查来评估，以确定是否存在表明药物所致损害的硬化性和坏死性损害。然而，这些方法只能在大量器官损伤已经发生后，才能用于识别晚期毒性。使用肌钙蛋白作为替代标记物及早检测毒性将是对目前临床前安全性评估的重大改进。这一建议的主要假设是，区分心肌和骨骼(快和慢)形式的肌钙蛋白-I的高度特异性分析，以及对量化和监测基线动物循环浓度的敏感性，将能够在早期临床前研究中检测和区分有毒药物化合物造成的心肌和肌肉特异性损伤。这项建议的具体目标包括：(1)开发新的快速和慢速抽动肌肉中骨骼肌肌钙蛋白-I的分析方法，并进行初步的分析验证，包括灵敏度、定量限度、范围、异构体特异性、人-大鼠交叉反应和精密度。(2)结合cTnI测定正常大鼠和人血清中快、慢收缩骨骼肌钙蛋白-I的基线水平。(3)使用开发的一组分析方法，测试已服用已知心脏毒性化合物(即异丙肾上腺素)的大鼠的档案血清样本，并与已知的肌肉毒性化合物(即他汀类药物)进行比较，作为检测相应生理毒性终点的特异性测试。在这项建议的第一阶段完成后，我们将准备使用我们的多分析肌钙蛋白I毒性小组来筛选额外的化合物(即受体酪氨酸激酶抑制剂)，用于在给药前和给药后的纵向时间进程研究中显示心脏和/或肌肉的特异性损伤。这些研究将对开发对心脏和肌肉损伤具有高安全性的药物有很大好处，即使是在目前尚不可行的低剂量药物研究中也是如此。 与公共卫生相关：我们建议开发一组超灵敏、高度特异的肌钙蛋白-I检测方法，以检测和区分临床前毒性模型中的心肌和肌肉损伤，这种毒性也可以用于人类。这些检测将是肌钙蛋白-I亚型(骨骼-快、骨骼-慢和心脏)的特异性检测，但将在大鼠和人类同系物之间交叉反应，用于大鼠心脏毒性的临床前模型和后续的人类临床研究。这项建议的意义是开发一种新的工具，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FDA最近对他汀类药物(辛伐他汀)和罗格列酮(文迪雅)这两种重要处方药的安全警告，这一点尤其相关。

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).