钩吻素子在人和猪、大鼠、猴、犬肝微粒体的酶促动力学及CYP450酶亚型分析

摘要

目的 研究和比较钩吻素子(KM)在人与各种实验动物肝微粒体体外代谢的酶促动力学及选择性CYP450酶抑制剂对其代谢的影响.方法 采用优化的反应体系和UPLC检测方法,测定系列浓度的KM与各种属肝微粒体孵育的降解曲线,以底物消除法计算酶动力学参数;共孵育方法考察选择性CYP450酶抑制剂对KM在各种属肝微粒体代谢的影响.结果 在人肝微粒体,KM的米氏常数(Km)、最大反应速率(Vmax)、半哀期(T1/2)、固有清除率(CLint)分别为(0.135±0.067)mmol/L、(1.89±0.19)μmol·min-1·g-1pro、(712±23)min和(15.7±5.2)mL·min-1·g-1pro.与人相比,犬、猪的Km值较大,大鼠及猴的CLint较高,4种实验动物的Vmax值均较大、T1/2均较短.酮康唑在各种属均可显著抑制KM代谢,在人、猪、犬IC50<1 μmol/L,在大鼠、猴IC50为1~10 μmol/L,可见CYP3A为主要代谢酶.噻氯匹定、奎尼丁和磺胺苯吡唑仅对大鼠,噻氯匹定仅对猴和犬的KM代谢为弱抑制(抑制率20.43%~44.31%),说明在体情况下,CYP2B、CYP2D和CYP2C与KM的代谢可能无关.结论 KM在人与猪、大鼠、犬、猴肝微粒体中的主要代谢途径相同,均由CYP3A酶催化,但酶活性和代谢动力学特征有一定差异.%Objective To study the enzyme kinetics and the effects of selective CYP450 enzyme inhibitors on the koumine metabolism in liver microsomes of human,pig,rat,monkey and dog.Methods Koumine in liver microsome was quantified through UPLC.Based on substrate deletion approach,various concentrations of koumine were incubated with liver microsomes under optimized condition,and residual substrate concentrations were measured at different incubating time.Enzymatic kinetic parameters were calculated by reciprocal plot.Influence of four selective inhibitors on koumine metabolism was investigated to identify the enzyme subtypes of the metabolism.Results Km,Vmax,T1/2 and CLint of koumine in human liver microsomes were (0.135±0.067) mmol/L,(1.89±0.19)μmol·min-1·g-1pro,(712±23)min and (15.7±5.2)mL·min-1·g-1pro,respectively.The parameters in liver microsomes of the four specie animals were observed differences from human in varying degrees,such as: Km were higher in dog and pig;CLint were higher in rat and monkey;Vmax were higher and T1/2 were smaller among four specie animals.Ketoconazole significantly inhibited the koumine metabolism in liver microsomes from human,pig,dog(IC50<1 μmol/L)and rat,monkey (IC50 between 1 and 10 μmol/L),indicating that CYP3A is the major enzyme of koumine metabolism in these species in vitro.The inhibitory ratio of ticlopidine,quinidine and sulfaphenazole to the koumine metabolism in rat,ticlopidine in monkey or dog,were 20.43%~44.31% (IC50>10 μmol/L),and no other inhibitory effects were observed by ticlopidine,quinidine and sulfaphenazole.Thus CYP2B,CYP2D and CYP2C are unlikely to participate koumine metabolism in vivo.Conclusion The major metabolic pathway of koumine is similar in the liver microsomes among five species,and CYP3A is the major metabolic enzymes in vitro.However,there are some differences among the various species in enzyme kinetic characters and enzyme activity of koumine metabolism.