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

开发用于临床前的超灵敏 SMC 多重检测毒性面板

• 批准号: 8058915
• 负责人: John Allan Todd
• 金额: $ 9.94万
• 依托单位: SINGULEX, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-15 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8058915
• 关键词: Animals; Biological Assay; Biological Markers; Cardiac; Cardiotoxicity; Clinical Research; Detection; Development; Dose; Drug Prescriptions; Early Diagnosis; Evaluation; FDA approved; Homologous Gene; Human; Immunoassay; Injury; Isoproterenol; Light; Marketing; Methods; Monitor; Muscle; Myocardial tissue; Myocardium; Necrotic Lesion; Organ; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physiological; Pre-Clinical Model; Protein Isoforms; Rattus; Risk; Safety; Sampling; Serum; Simvastatin; Specificity; Staging; Surrogate Markers; Testing; Time; Toxic effect; Toxicity Tests; Troponin; Troponin I; Tyrosine Kinase Inhibitor; Validation; Work; cross reactivity; innovation; novel; pre-clinical; preclinical study; receptor; rosiglitazone; single molecule; skeletal; tool

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).

描述（由申请人提供）：在临床前研究中，需要对大鼠心肌肌钙蛋白（cTnI）、快抽搐骨骼肌钙蛋白- i （fsTnI）和慢抽搐骨骼肌钙蛋白- i （ssTnI）进行高特异性、超灵敏、低容量的检测，以评估和区分心脏和肌肉毒性。这项工作的主要目的是开发一个多面板的超敏感，单分子计数肌钙蛋白- 1测定。这些检测将针对肌钙蛋白- 1亚型（快骨型、慢骨型和心脏型），但将在大鼠和人类同源物之间进行交叉反应，用于大鼠临床前毒性测试和随后的人类临床研究。该建议的意义在于开发一种新的工具，以便在临床前研究中更好地筛选候选药物的肌和心脏毒性。鉴于最近FDA对两种重要处方药——他汀类药物（辛伐他汀）和罗格列酮（文迪雅）的安全警告，这一点尤为重要。尽管这两种药物之前被认为是安全的，并得到了FDA的批准，但高剂量的这两种药物已被证明会增加不良肌毒和心脏毒性作用的风险。自批准以来，它们已广泛销售和广泛开处方，因此引起深切关注。提出的创新将为肌钙蛋白异构体创造超敏感的单分子免疫测定方法，作为肌和心脏毒性的替代生物标志物，使药物诱导损伤的早期检测成为可能。目前市场上的肌钙蛋白测定法无法在健康动物中进行基线定量，因此传统药物引起的心脏和肌肉毒性是通过心脏和肌肉组织的组织病理学检查来评估的，以确定是否存在表明药物引起的损伤的硬化和坏死病变。然而，这些方法只能用于识别毒性的晚期阶段，在大量器官损害已经发生之后。用肌钙蛋白作为替代标记物的早期毒性检测将是目前临床前安全性评估的重大改进。该提案的主要假设是，区分心脏和骨骼（快速和缓慢）形式的肌钙蛋白- 1的高度特异性分析，具有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由有毒药物化合物引起的心脏和肌肉特异性损伤。本提案的具体目标包括：(1)开发快速和缓慢抽搐肌中骨骼肌钙蛋白- 1的新检测方法，并显示初步的分析验证，包括灵敏度，定量限制，范围，同型特异性，人鼠交叉反应性和精度。(2)结合cTnI测定正常大鼠和人血清样品中快速和慢速抽搐骨骼肌钙蛋白- i的基线水平。(3)使用已开发的检测方法，将使用已知心脏毒性化合物（即异丙肾上腺素）与已知肌肉毒性化合物（即他汀类药物）的大鼠档案血清样本进行测试，以检测相应生理毒性终点的特异性。在该提案的I期完成后，我们将准备使用我们的多测定肌钙蛋白- 1毒性小组来筛选其他化合物（即受体酪氨酸激酶抑制剂），用于在给药前和给药后大鼠的纵向时间过程研究中用于心脏和/或肌肉特异性损伤的指征。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的好处，即使是目前无法进行研究的低剂量药物。