基于活性中间产物的中药肝毒性成分早期发现系列关键技术

Xenobiotics are metabolized by CYP450s to various forms of intermediate products, some of which occasionally end up as reactive metabolites (RMs). Depending on their affinity to nucleophiles (high electron density), RMs could act as toxic agents that induce hepatotoxicity by binding to intra-cellular macromolecules. Other mechanistic pathways include mitochondrial dysfunction, cell stress and specific immune reactions. There is enormous amount of evidence indicating a relationship between RMs) and drug-induced liver injury (DILI. Additionally, it is difficult to predict DILI in humans via the classical in vitro cytotoxicity screening and regulatory animal studies. It is therefore important that improvement be made in the early prediction of DILI in human. The purpose of the present study is to develop a series of techniques for high-throughput screening and evaluation of potential hepatotoxic ingredients in herbal medicine. The findings in this study will essentially help understand the connection between reactive functional structural moiety and the potential hepatotoxicity and, to supply some clinically relevant information for the application of Chinese herbal medicine; the development of these techniques on RMs identification and evaluation of hepatotoxic mechanism obviously would facilitate and promote the screening of potential hepatotoxic ingredients (i.e., the hepatic hepatotoxicity profiles), and serve as valuable tools for evaluating the mechanism of action of generic compounds as well as marketed compounds; for standardization in related studies; and finally, would eventually lead to a generation or build up of database for preclinical prediction of hepatotoxicity-induced ingredients during drug research and development of Chinese herbal therapy.

药物在代谢酶作用下产生活性中间产物（RMs），RMs在药物性肝损伤（DILI）发生过程中起重要作用。导致肝毒性的药物本身可能并不具有结构相似性，但RMs介导的肝毒性产生依赖于特定活性官能团的存在。以此为契合点，研究活性官能团与潜在肝毒性成分结构特征之间的相互关联，采用IC50 Shift方法和GSH捕获技术，发现并鉴定RMs；采用基于细胞影像的HCS技术和基于基因芯片的基因聚类分析技术，确定RMs与肝毒性的关联，形成基于RMs及其毒性终端的中药有毒成分早期发现系列关键技术，探索中药肝毒性成分早期发现的新途径。同时构建基于活性官能团的潜在中药肝毒性成分信息库，发现并预警含有与已知毒性成分相同活性官能团的潜在中药肝毒性成分，通过所建立的系列关键技术，阐明致毒机理及其途径，形成完整的中药肝毒性物质基础早期发现研究链。

药物在代谢酶作用下产生活性中间产物（RMs），RMs在药物性肝损伤（DILI）发生过程中起重要作用。导致肝毒性的药物本身可能并不具有结构相似性，但RMs介导的肝毒性产生依赖于特定活性官能团的存在。本课题以此为契合点，按照国家自然科学基金委资助项目计划书要求，针对中药所致肝损伤发现较晚、缺乏早期发现技术及评价指标的现状，以《中华人民共和国药典》收载的中药补骨脂、何首乌、雷公藤、细辛、黄药子、吴茱萸、延胡索、川楝子中含有活性官能团、具有肝毒性表现的中药成分为研究载体，重点针对含有活性官能团的有毒中药质控成分（补骨脂素/异补骨脂素/补骨脂黄酮类/吴茱萸次碱/吴茱萸碱/肉豆蔻醚/大黄酸/大黄素/延胡索乙素/雷公藤生物碱类/黄药子二萜内酯类成分等）为研究对象，（1）开展了活性代谢产物发现、结构鉴定及肝毒性作用及其机制研究，阐明了活性官能团与潜在肝毒性成分结构特征之间的相互关联，形成了基于RMs的中药肝毒性成分早期发现系列关键技术的集成与实施规范，为阐明中药及其活性成分的致毒机理及其途径提供了新的思路和方法；（2）构建了基于活性官能团的潜在中药肝毒性成分信息库，发现并预警含有与已知毒性成分相同活性官能团的潜在中药肝毒性成分，揭示活性官能团与潜在肝毒性成分结构特征之间的相互关联；（3）开展基于中药药代标志物PK-marker和中药质量标志物Q-marker的研究，阐明以黄毒素B为PK-marker，反映和预测黄药子体内物质暴露以及与其疗效和安全性关联；以雷公藤生物碱为Q-marker，构建雷公藤多苷的质控方法，制定符合中药特色的质量控制标准。.本项目在执行期内圆满、超额完成了计划任务，达到了预期研究结果及考核指标，取得一系列重要成果。（1）荣获教育部科技进步奖二等奖1项，中华中医药学会科技进步奖二等奖1项；（2）培养博士研究生3名，硕士研究生11名；（3）申请及授权专利3项，获得软件著作权1项；（4）发表论文23篇，其中SCI收录15篇。