Develop predictive human cardiomyocyte-based all optical assay for cardiotoxicity

 描述(由申请人提供)：开发基于人类心肌细胞的心脏毒性全光测定法。心脏毒性是药物化合物安全驱使停药的主要原因.目前的心脏安全范例非临床指南ICH S7B专注于体外HERG检测，事实证明，在预测化合物的人类心脏毒性方面，HERG检测是次优的。在新药开发成本高、生产率低的背景下，更好的心脏安全性评估成为一个重要的公共卫生问题。CSRC、HESI和FDA正在开发一种新的心脏安全范例：全面体外心律失常分析(CIPA)，其中包括表征化合物在IPSC来源的人心肌细胞中的电生理效应。Q-State的平台能够以比目前的膜片钳分析更高的吞吐量、更低的成本和更高的信息量(多模式：电压、钙、pH和ATP)，以及比其他光学筛选工具更好的时间和空间分辨率以及更低的光毒性来探测这类细胞的电生理。在68万美元的NINDS SBIR第一阶段奖励和天使基金的支持下，Q-State Biosciences正在开发一种专有的交钥匙仪器和软件系统(Optopatch)，用于同时进行光学微扰和神经元膜电压的光学测量。我们建议将Optopach用于心肌细胞的测量，并开发和验证一种检测药物对人IPSC来源的心肌细胞的心脏毒性的Optopatch分析方法。目标1.分子工具。通过比较启动子、运输基序和基因传递方法，选择最有效的执行器(将蓝色光转换为起搏的电刺激)和报告器(将AP波形转换为近红外荧光信号)，并彻底表征分子工具的灵敏度、速度、光稳定性和可重复性。目标2.仪器。采用Optopatch硬件和软件，可同时对1,000个心肌细胞进行光学刺激和记录。开发流体和环境控制；修改软件以支持光学起搏、图像分割和心脏相关AP参数的计算。目的3.检测方法的建立和检测。优化急性和慢性心脏毒性的检测方法。通过量化AP在一组具有已知机制和心脏安全概况的药物下的变化来验证该分析。我们将在领先识别和优化阶段向药物发现公司提供非GLP心脏毒性筛查服务，补充并最终有可能取代HERG检测。我们还将向学术界提供Optopatch-心脏仪器和试剂；使机械化研究成为可能，并扩大电生理平台的应用。

 DESCRIPTION (provided by applicant): Develop predictive human cardiomyocyte-based all optical assay for cardiotoxicity. Cardiotoxicity is the leading cause of safety-driven withdrawal a all stages and limitation of drug compounds. The current cardiac safety paradigm nonclinical guidance ICH S7B focuses on in vitro hERG assays, which prove to be suboptimal in predicting a compound's cardiotoxicity in human. In the context of high cost and low productivity for new drug discovery, better cardiac safety evaluation becomes a significant public health issue. CSRC, HESI, and FDA are developing a new cardio safety paradigm: Comprehensive In vitro Proarrhythmia Assay (CiPA), which comprises characterizing electrophysiological effects of compounds in iPSC-derived human cardiomyocytes. Q-State's platform can probe electrophysiology of such cells with higher throughput, lower cost, and higher information content (multi-modalities: voltage, Ca2+, pH, and ATP) than current patch clamp assays, and with better temporal and spatial resolution and lower phototoxicity than other optical screening tools. Supported by a $680K NINDS SBIR Phase I award and angel funding, Q-State Biosciences is developing a proprietary turnkey instrument and software system (Optopatch) for simultaneous optical perturbation and optical measurement of membrane voltage in neurons. We propose to adapt Optopach for cardiomyocyte measurements and to develop and validate an Optopatch assay for drug-induced cardiotoxicity in human iPSC- derived cardiomyocytes. Aim 1. Molecular tools. Select the most effective actuators (converting blue light into electrical stimuli for pacing) and reporters (converting AP waveforms into near infrared fluorescence signals) by comparing promoters, trafficking motifs and gene delivery methods, and to thoroughly characterize the sensitivity, speed, photostability, and repeatability of the molecular tools. Aim 2. Instrumentation. Adapt Optopatch hardware and software to optically stimulate and record from > 1,000 cardiomyocytes simultaneously. Develop fluidics and environmental controls; modify software to support optical pacing, image segmentation, and calculation of cardiac-relevant AP parameters. Aim 3. Assay development and testing. Optimize assays for acute and chronic cardiotoxicity. Validate the assay by quantifying AP changes under a panel of drugs with known mechanisms and cardiac safety profiles. We will provide non-GLP cardiotoxicity screening services to drug discovery companies at the lead identification and optimization stages, supplementing, with the potential to eventually replace, hERG assays. We will also make Optopatch-cardio instrumentation and reagents available to the academic community; to enable mechanistic studies and broaden the applications of electrophysiology platform.