Identification of Biomarkers of Cardiotoxicity using Metabolomics of Human Pluripotent Stem Cell-Derived Cardiomyocytes

 使用人类多能干细胞衍生的心肌细胞代谢组学鉴定心脏毒性的生物标记物项目摘要/摘要心脏安全性是制药业化合物损耗和FDA批准的药物退出市场的主要原因之一。这项建议的目的是通过开发一种体外试验来预测化合物的心脏毒性潜力，以改善公众健康，并减轻由于心脏毒性而造成的化合物磨损的经济负担。为了实现这一目标，Stemina Biomarker Discovery(“Stemina”)建议使用人类诱导多能干细胞来源的心肌细胞(hiPSC-CMS)暴露于已知的人类心脏毒性和非心脏毒性化合物的代谢组学。这些技术将被用于发现人类内源性代谢物生物标志物，这些生物标志物可以预测一般的心脏毒性以及特定类型的心脏毒性(例如，功能性、结构性)。利用代谢组学来测量HiPSC-CMS对化合物暴露的反应所分泌的小分子是一种评估心脏毒性的新方法，并可能为使用人类细胞进行更准确、更具预测性的新一代毒理学筛查铺平道路。Stemina已经使用这种范例完成了第一阶段SBIR应用(1R43GM100640-01)，并开发了评估未分化多能干细胞发育毒性潜力的预测方法(devTOX(Tm))。Stemina‘s 长期目标是开发一种基于人类细胞的高通量心脏毒性筛查，作为制药、生物技术和农化公司在治疗和化学品早期开发过程中的宝贵工具。为了实现这一目标，Stemina首先提出开发一个优化的和可重复的实验平台来评估从HiPSC-CMS收集的废介质(目标1)。我们将评估各种样品制备方法、LC-MS色谱柱和条件，并进行稳健性测试，以建立使用我们的系统对HiPSC-CMS中完整的分泌型代谢物或分泌组进行最具重复性的测量。在目标2中，我们将使用上述平台评估从HiPSC-CMS对包含功能、结构、一般和非心脏毒物的60种化合物的训练集的耗用介质，以建立一个预测的代谢组学模型。小分子的丰度变化取决于细胞是用心脏毒性诱导剂还是非诱导剂处理，它们将作为心脏毒性的候选生物标记物。此处获取的数据将为 用于建立预测代谢特征，指示一般心脏毒性和特定类型的心脏毒性(例如，功能性、结构性)。然后，Stemina将在20种化合物的测试集上测试生物标记物签名的预测模型(S)的性能。在目标3中，Stemina将确认预测代谢物的结构同一性，并评估它们作为已确认生物标记物的生物学意义。最后，在目标4中，将使用测量已确认生物标记物的靶向LC-MS方法来开发靶向生物标记物分析。此外，我们的生物标记物充分预测心脏毒性的能力将通过使用由合作公司获得的化合物组成的盲目研究来测试。这些目标的完成将使一种能够检测心脏毒性生物标志物的商业测试得以开发，类似于Stemina目前市场上销售的一种开发毒性测试(devTOX(Tm)QuickPredict)。Stemina随后将利用这种测试来营销一种能够预测一种化合物是否会引起心脏毒性的服务，并在临床前筛选试验中为制药公司提供服务。这项服务提供了第一个基于人类细胞的心脏毒性筛选试验，建立在心肌细胞新陈代谢的基础上。

 DESCRIPTION (provided by applicant) Identification of Biomarkers of Cardiotoxicity using Metabolomics of Human Pluripotent Stem Cell- Derived Cardiomyocytes Project Summary/Abstract Cardiac safety is one of the leading causes of compound attrition in the pharmaceutical industry and withdrawal of FDA-approved drugs from the market. The purpose of this proposal is to improve public health, as well as alleviate the financial burden of compound attrition due to cardiotoxicity, through development of an in vitro assay to predict a compound's cardiotoxicity potential. To accomplish this, Stemina Biomarker Discovery ("Stemina") proposes to use metabolomics of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to known human cardiotoxic and non-cardiotoxic compounds. These technologies will be used to discover human, endogenous metabolite biomarkers which predict general cardiotoxicity as well as the specific type of cardiotoxicity (e.g., functional, structural). The use of metabolomics to measure small molecules secreted by hiPSC-CMs in response to compound exposure is a novel approach for evaluating cardiotoxicity and may pave the way for a new generation of more accurate, predictive toxicology screens using human cells. Stemina already used such a paradigm to complete the Phase I SBIR Application (1R43GM100640-01), as well as developed predictive methods to assess developmental toxicity potential in undifferentiated pluripotent stem cells (devTOX(tm)). Stemina's long-term goal is to develop a human cell- based, high-throughput cardiotoxicity screen as a valuable tool to pharmaceutical, biotech, and agrichemical companies during early development of therapeutics and chemicals. In order to achieve this goal, Stemina first proposes to develop an optimized and reproducible experimental platform to evaluate spent media collected from hiPSC-CMs (aim 1). We will evaluate various sample preparation methods, LC-MS columns and conditions, and perform robustness testing in order to establish the most reproducible measurement of the complete set of secreted metabolites, or secretome, in hiPSC-CMs using our system. In aim 2, we will use the above platform to evaluate spent media from hiPSC- CMs response to a training set of 60 compounds consisting of functional-, structural-, general-, and non- cardiotoxicants to establish a predictive metabolomic model. Small molecules whose abundances vary dependent upon whether cells were treated with an inducer or non-inducer of cardiotoxicity will serve as candidate biomarkers of cardiotoxicity. The data acquired here will be used to establish a predictive metabolic signature indicative of general cardiotoxicity and specific type of cardiotoxicity (e.g., functional, structural). Stemina will then test the performace of the predictive model(s) of biomarker signature(s) on a test set of 20 compounds. In aim 3, Stemina will confirm the structural identity of the predictive metabolites and evaluate their biological significance as confirmed biomarkers. Lastly in aim 4, a targeted biomarker assay will be developed using targeted LC-MS methods that measure the confirmed biomarkers. Further, the ability of our biomarkers to adequately predict cardiotoxicity will be tested through the use o a blind study comprised of compounds acquired from partnering companies. Completion of these aims will enable the development of a commercial assay able to detect the validated biomarkers of cardiotoxicity, similar to an existing test Stemina currently markets for developmental toxicity (devTOX(tm) quickPredict). Stemina will subsequently utilize this assay to market a service capable of predicting whether a compound will induce cardiotoxicity and serve pharmaceutical companies in preclinical screening trials. Such a service provides the first human cell-based screening assay for cardiotoxicity founded on cardiomyocyte metabolism.