川芎嗪的肝微粒体代谢动力学及代谢表型研究

Investigation of metabolic kinetics and reaction phenotyping of ligustrazin by using liver microsomes and recombinant human enzymes

应用体外人和大鼠肝微粒体以及重组人源代谢酶孵育体系,研究了中药有效成分川芎嗪(TMPz)的肝代谢动力学特征和代谢酶表型。将TMPz与加入不同辅酶的人(HLM)和大鼠肝微粒体(RLM)孵育,应用LC-MS/MS法测定其剩余浓度,考察TMPz的肝微粒体代谢稳定性和酶动力学。在+NADPH和+NADPH+UDPGA的肝微粒体中,TMPz发生代谢转化。在同时加入NADPH和UDPGA的HLM和RLM中,TMPz的t1/2、Km和Vmax分别为(94.24±4.53)和(105.07±9.44)min、(22.74±1.89)和(33.09±2.74)μmol·L-1、以及(253.50±10.06)和(190.40±8.35)nmol·min-1·mg-1(protein)。TMPz在HLM的代谢消除略快于RLM,其氧化生成活性产物2-羟甲基-3,5,6-三甲基吡嗪(HTMP)后可进一步与葡糖醛酸结合。应用重组人源CYP酶及特异化学抑制剂法确定CYP1A2、2C9和3A4是参与川芎嗪Ⅰ相代谢的CYP同工酶,并介导了HTMP的生成。经整体归一化法得到CYP1A2、2C9和3A4的贡献率分别是19.32%、27.79%和52.90%。应用LC-MS/MS峰面积相对定量法,观察到UGT1A1、1A4和1A6是介导HTMP葡糖醛酸结合反应的主要尿苷二磷酸葡糖醛酸转移酶(UGT)亚型。上述结果表明,川芎嗪的肝脏代谢涉及多酶介导的Ⅰ相和Ⅱ相代谢。

The metabolic characteristics of ligustrazin （TMPz） in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes （CYP） and UDP glucuronosyltransferases （UGT）. TMPz was incubated at 37 ℃ with human （HLM） and rat liver microsomes （RLM） in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH＋UDPGA. In the HLM and RLM with NADPH＋UDPGA, t1/2, gm and Vmax of TMPz were 94.24±4.53 and 105.07±9.44 min, 22.74±1.89 and 33.09 ±2.74 μmol·L^-1, 253.50 ±10.06 and 190.40 ±8.35 nmol·min^-1·mg^-1（protein）, respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine （HTMP）, could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms （rCYP） and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed by using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms （rUGT）. UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.