芦荟大黄素的体外肝微粒体代谢动力学和代谢酶表型研究

目的 研究芦荟大黄素在人肝微粒体(HLM)和大鼠肝微粒体(RLM)的代谢特征,并确定其代谢酶表型.方法 将芦荟大黄素分别与加入不同辅酶因子的人和大鼠肝微粒体孵育,LC-MS/MS法定量分析芦荟大黄素的剩余含量,考察芦荟大黄素的代谢稳定性和酶动力学.将芦荟大黄素分别与一组重组人源CYP同工酶(CYP1A2、2B6、2C8、2C9、2C19、2D6、3A4)孵育,或在HLM中加入各同工酶的特异性抑制剂,确定其CYP酶代谢表型.结果 在HLM和RLM中,芦荟大黄素均可发生依赖于NADPH的Ⅰ相代谢消除.经加热法确定,Ⅰ相代谢主要由CYP酶介导,30 min的代谢百分率分别为85.8%和81.7%,消除半衰期t1/2分别为(10.3±0.3)min和(11.5±3.3)min;内在清除率CLint分别为(420.1±10.9)和(573.4±188.2)ml/(min·kg);表观酶动力学参数Km分别为(2.4±0.9)和(3.9±1.4)μmol/L,Vmax分别为(1492±170.5)和(2783±595.8)nmol/(min·g protein).在RLM中还观察到芦荟大黄素的葡糖醛酸结合反应,30 min代谢转化率为38.5%,消除半衰期t1/2为31.6 min,CLint为(197.1±15.5)ml/(min·kg).CYP酶代谢表型研究表明,芦荟大黄素的肝微粒体代谢由多个CYP同工酶介导,其中CYP1A2、3A4、2B6和2C9的整体归一化贡献率分别为35.4%、21.9%、6.6%和6.8%.结论 芦荟大黄素在HLM和RLM中,主要发生多个CYP同工酶介导的Ⅰ相代谢消除,其中CYP1A2和3A4的代谢贡献率>20%;HLM和RLM代谢存在一定的种属差异,RLM还存在葡糖醛酸结合反应.

Characterization of metabolic kinetics and CYP phenotyping of aloe emodin in liver microsomes

Objective To characterize the metabolic kinetics of aloe emodin in human liver microsomes(HLM)and rat liver microsomes(RLM)and identify the CYP phenotyping of phaseⅠmetabolism. Methods Aloe emodin was incubated at 37℃ with HLM and RLM in the presence or absence of NADPH,UDGPA or NADPH+UDGPA. The remaining aloe emodin was determined with a validated LC-MS/MS method to assess the metabolic stability and enzymatic kinetics. A panel of rCYP isoforms(CYP1A2,2B6,2C8, 2C9,2C19,2D6 and 3A4)and HLM with specific inhibitors of CYP isoforms were used to identify the CYP phenotyping of aloe emo?din. Results In HLM and RLM,aloe emodin was metabolically eliminated in the presence of NADPH,with 85.8%and 81.7%of the parent compounds eliminated in 30 min,respectively. The t1/2 were(10.3±0.3)and(11.5±3.3)min,and the CLint were(420.1±10.9) and(573.4±188.2)ml/(min·kg),respectively. The apparent Km and Vmax for HLM and RLM were obtained and found to be(2.4±0.9) and(3.9±1.4)μmol/L,(1492±170.5)and(2783±595.8)nmol/(min·g protein),respectively. In RLM with UDPGA,38.5%of aloe emodin was metabolized in 30 min with t1/2 of 31.6 min and CLint of(197.1±15.5)ml/(min·kg). The results of CYP phenotyping indi?cated that CYP1A2,2B6,2C19 and 3A4 were the major enzymes involved in the metabolism of aloe emodin. By using the method of total normalized rate,the contributions of the major enzymes were assessed to be 35.4%,6.6%,2.2%and 21.9%,respectively. Con?clusion Aloe emodin is mainly eliminated by CYP mediated metabolism in HLM and RLM. CYP1A2 and 3A4 are the major responsi?ble enzymes of aloe emodin,and the contributions are above 20%. Species differences in liver metabolism of aloe emodin are observed. It undergoes notable glucuronidation in RLM only.