黄独素B的体外代谢通路及其代谢产物研究

目的基于体外代谢模型对黄独素B的代谢稳定性、主要CYP450代谢酶表型及其代谢产物进行研究。方法黄独素B分别在人肝微粒体(HLM)和大鼠肝微粒体(RLM)中共同孵育,采用UPLC-MS/MS检测孵育液中剩余的黄独素B含量,分析其在HLM和RLM中的代谢稳定性。利用10种重组人CYP450酶(1A1、1A2、1B1、2A13、2A6、2B6、2D6、2C9、2C19、3A4)分别与黄独素B共同孵育,确定其代谢酶表型,结合大鼠离体肝灌流模型对黄独素B的主要代谢酶表型进行确认。此外,分别对HLM和RLM孵育体系中的黄独素B代谢产物进行定性分析,考察黄独素B在HLM和RLM中代谢产物的区别。结果在HLM和RLM中,黄独素B的转化率分别为37%、59%,经HLM和RLM代谢的体外半衰期(t1/2)为97.4、52.3 min,推算得到的HLM和RLM中的固有清除率(CLint, in vivo)为8.23、23.9 mL/(min·kg),肝清除率(CLh)为5.89、16.8 mL/(min·kg),由此可知黄独素B在RLM体系中的代谢转化速率较HLM中快。黄独素B体外代谢酶表型结果可知其I相代谢是由多个CYP同工酶介导的,包括3A4、2C19、2C9、1A13及1A1,其中CYP3A4对黄独素B的代谢起主导作用;肝灌流实验结果显示,随着酮康唑给药剂量的增加,对肝脏中CYP3A4的抑制作用增强,黄独素B在肝脏中的代谢减少,在灌流液中的剩余量增加,印证了CYP3A4对黄独素B的代谢作用。此外,2种肝微粒体孵育后的黄独素B都只产生了1个代谢产物(M1),为黄独素B去甲基化产物。结论黄独素B在RLM中的代谢转化速率较HLM中快。黄独素B的主要代谢酶表型为CYP3A4,其在HLM和RLM中产生的代谢物均为去甲基化产物。

Study on metabolic pathways and metabolites of Diosbulbin B in vitro

Objective To investigate the metabolic stability, the main CYP450 enzymes phenotypes and metabolites of Diosbulbin B based on in vitro metabolism model. Methods For metabolic stability study, UPLC-MS/MS was used to detect the remaining Diosbulbin B content in the incubation solution after being incubated with human and rat liver microsomes, respectively. Ten recombinant human CYP450 enzymes (1A1, 1A2, 1B1, 2A13, 2A6, 2B6, 2D6, 2C9, 2C19, 3A4) were used for identifying the metabolic enzyme phenotypes of Diosbulbin B. Moreover, the major metabolic enzyme phenotype for the metabolism of Diosbulbin B was confirmed and verified by the rat isolated hepatic perfusion model. The metabolites of Diosbulbin B in human and rat liver microsomes were determined by LC-MS/MS. Results The metabolic percentage of Diosbulbin B in human and rat liver microsomes were 37% and 59%, respectively. Its half-lives t_1/2 in human and rat liver microsomes were 97.4 and 52.3 min, respectively. The intrinsic clearance rates CL_int in human and rat livers were 8.23 and 23.9 mL/(min·kg), and liver clearance CL_h in human and rat livers were 5.89 and 16.8 mL/(min·kg). It can be found that the metabolic rate of Diosbulbin B in rat liver microsomes was faster than in human liver microsomes. There were five CYP enzymes, including 3A4, 2C19, 2C9, 1A13 and 1A1, related to the metabolism of Diosbulbin B, especially CYP3A4. The hepatic perfusion experimental results showed that the metabolism of Diosbulbin B was inhibited by ketoconazole, and the inhibitory effect was enhanced along with the increasing dosage of ketoconazole, which confirmed that CYP3A4 played an important role in metabolism of Diosbulbin B. There was one metabolite (M1) of Diosbulbin B has been found in both human and rat liver microsomes incubation. Conclusion The metabolic rate of Diosbulbin B in rat liver microsomes was faster than human liver microsomes. The CYP3A4 plays a leading role in the metabolism of Diosbulbin B. And a demethylated metabolite of Diosbulbin B was appeared in both human and rat liver microsomes incubation.