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

 描述（由申请人提供）使用人多能干细胞衍生的心肌细胞的代谢组学鉴定心脏毒性的生物标志物项目概述/摘要心脏安全性是制药工业中化合物损耗和FDA批准的药物从市场撤回的主要原因之一。该提案的目的是通过开发体外试验预测化合物的心脏毒性潜力，改善公众健康，并减轻由于心脏毒性引起的化合物消耗的经济负担。为了实现这一点，Stemina Biomarker Discovery（“Stemina”）提出使用暴露于已知的人类心脏毒性和非心脏毒性化合物的人类诱导多能干细胞衍生的心肌细胞（hiPSC-CM）的代谢组学。这些技术将用于发现人类内源性代谢物生物标志物，其预测一般心脏毒性以及特定类型的心脏毒性（例如，功能性、结构性）。使用代谢组学来测量hiPSC-CM响应于化合物暴露而分泌的小分子是一种用于评估心脏毒性的新方法，并且可能为使用人类细胞进行新一代更准确的预测性毒理学筛选铺平道路。Stemina已经使用这种范例完成了I期SBIR申请（1 R43 GM 100640 -01），并开发了预测方法来评估未分化多能干细胞（devTOX（tm））的发育毒性潜力。斯特米娜的 长期目标是开发基于人类细胞的高通量心脏毒性筛选，作为药物、生物技术和农业化学公司在治疗剂和化学品的早期开发期间的有价值的工具。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，以评估从hiPSC-CM收集的废培养基（目的1）。我们将评估各种样品制备方法、LC-MS色谱柱和条件，并进行耐用性检测，以使用我们的系统建立hiPSC-CM中全套分泌代谢物或分泌组的最重现性测量。在目标2中，我们将使用上述平台来评估来自hiPSC-CM的废培养基对由功能性、结构性、一般性和非心脏毒性物质组成的60种化合物的训练集的响应，以建立预测性代谢组学模型。其丰度取决于细胞是否用心脏毒性诱导剂或非诱导剂处理的小分子将作为心脏毒性的候选生物标志物。这里获得的数据将是 用于建立指示一般心脏毒性和特定类型心脏毒性的预测性代谢特征（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志物特征的预测模型的性能。在目标3中，Stemina将确认预测代谢物的结构特性，并评价其作为确认的生物标志物的生物学意义。最后，在目标4中，将使用靶向LC-MS方法开发靶向生物标志物测定法，该方法测量经确认的生物标志物。此外，我们的生物标志物充分预测心脏毒性的能力将通过使用由从合作公司获得的化合物组成的盲法研究进行测试。这些目标的完成将使得能够开发能够检测心脏毒性的经验证的生物标志物的商业测定法，类似于Stemina目前市场上用于发育毒性的现有测试（devTOX（tm）quickPredict）。Stemina随后将利用该检测方法销售一种能够预测化合物是否会诱导心脏毒性的服务，并为制药公司提供临床前筛选试验。这种服务提供了第一个基于心肌细胞代谢的基于人类细胞的心脏毒性筛查试验。

 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.