建立人肝微粒体―小鼠3T3成纤维细胞模型评价5―羟基雷公藤内酯醇代谢产物的细胞毒性.docVIP

建立人肝微粒体―小鼠3T3成纤维细胞模型评价5―羟基雷公藤内酯醇代谢产物的细胞毒性 　　摘 要 目的：以环磷酰胺（cyclophosphamide，CPA）和他莫昔芬（tamoxifen，TMX）为模型药物，建立人肝微粒体-小鼠3T3成纤维细胞模型，并用该模型评价5-羟基雷公藤内酯醇（T8）代谢产物的细胞毒性。方法：采用人肝微粒体（加或不加NADPH）-小鼠3T3成纤维细胞模型，测定吸光度（A值）并计算细胞孵育液的EC50值和EC50 shift，以EC50 shift评价代谢产物毒性。结果：模型药物CPA和TMX及创新药物T8的EC50 shift分别为0.34、4.7和1.2，表明CPA和TMX的代谢物会分别增加和降低细胞毒性，而T8代谢产物不影响或稍许降低对细胞的毒性。结论：本实验成功建立人肝微粒体-小鼠3T3成纤维细胞模型，这为T8进入体内的安全性提供了一定依据，也为候选化合物开发初期在不合成代谢物的情况下初步评价代谢物的细胞毒性提供了一种体外方法。 　　关键词 人肝微粒体 3T3成纤维细胞 细胞毒性 环磷酰胺 他莫昔芬 5-羟基雷公藤内酯醇 　　中图分类号：R992； R965.3 文献标识码：A 文章编号：1006-1533（2014）19-0074-05 　　Evaluation of the cytotoxicity of 5-hydroxytriptolide metabolites 　　with human liver microsome and mouse 3T3 fibroblast experimental system 　　LI Hua， XU Jia， LIU Haiyan* 　　（Central Research Institute， Shanghai Pharmaceuticals Holding Co.， Ltd.， Shanghai 201203， China） 　　ABSTRACT Objective： To evaluate the cytotoxicity of 5-hydroxytriptolide （T8） metabolites by establishing human liver microsome （HLM） and mouse 3T3 fibroblast experimental system with cyclophosphamide （CPA） and tamoxifen （TMX） as model toxicants. Methods： Absorbance （A value） was determined based on HLM incubation systems （with or without NADPH） and the 3T3 fibroblast experimental system and the EC50 value and EC50 shift were calculated. The cytotoxicity of metabolites was evaluated by EC50 shift. Results： The EC50 shift for model toxicant CPA and TMX and novel compound T8 were 0.34， 4.7 and 1.2， respectively， which demonstrated that the cytotoxicity could be increased or reduced by the metabolites of CPA and TMX， respectively while could not be affected or slightly reduced by the metabolites of T8. Conclusion： A human liver microsome and mouse 3T3 fibroblast experimental system was successfully established， which can provide a certain basis for the safety of T8 in human body as well as an in vitro method for the preliminary evaluation of the cytotoxicity of metabolites without synthesis of possible metabolites in the early development phase of drug candi