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目的探讨氨氯地平对重组人细胞色素P450 3A5(CYP3A5)酶代谢他克莫司的影响及其机制。方法用重组人CYP3A5酶作为体外代谢体系,以他克莫司作为CYP3A5的底物,建立LC-MS/MS法测定重组酶CYP3A5代谢他克莫司的剩余量。体外给予氨氯地平测定其半数抑制浓度(IC_(50))值,并比较预孵育及共孵育时IC_(50)值的平移,进一步通过酶失活动力学实验来测定氨氯地平介导的灭活CYP3A5酶的失活动力学常数K_(inact)和K_I值。结果孵育体系内源性物质不干扰测定,方法快捷、稳定、灵敏度高。CYP3A5代谢他克莫司的最大反应速率常数为(23.80±2.90)pmol·min~(-1),米氏常数为(0.85±0.28)μmol·L~(-1),清除率为28μL·min~(-1)。共孵育实验及预孵育实验的IC_(50)值分别为18.66,8.95μmol·L~(-1),氨氯地平灭活CYP3A5的灭活动力学参数Kinact和KI值分别为0.03min~(-1),18.87μmol·L~(-1)。结论氨氯地平对CYP3A5代谢他克莫司有抑制作用,其抑制作用是通过机制性灭活来介导。
Objective To investigate the effect of amlodipine on the metabolism of tacrolimus by recombinant human cytochrome P450 3A5 (CYP3A5) and its mechanism. Methods Recombinant human CYP3A5 enzyme was used as in vitro metabolic system, and tacrolimus was used as the substrate of CYP3A5. LC-MS / MS method was used to determine the residual amount of the recombinant enzyme CYP3A5 metabolizing tacrolimus. Amlodipine was used to determine the half-value concentration (IC50), and compared with the pre-incubation IC50 value of the translational shift, and further determined by enzyme inactivation kinetics of amlodipine mediation Kinetic constants of inactivation and K_I of inactivated CYP3A5 enzymes. Results Incubation system endogenous substances do not interfere with the determination, the method is fast, stable, high sensitivity. The maximum rate constant of CYP3A5 metabolism tacrolimus was (23.80 ± 2.90) pmol · min -1, the Mie constant was (0.85 ± 0.28) μmol·L -1 and the clearance rate was 28 μL · min ~ (-1). The IC 50 values of co-incubation and pre-incubation experiments were 18.66 and 8.95 μmol·L -1, respectively. The inactivation kinetic parameters Kinact and KI of amlodipine inactivated CYP3A5 were 0.03 min -1 ), 18.87μmol·L -1. Conclusion Amlodipine can inhibit the metabolism of tacrolimus in CYP3A5, and its inhibitory effect is mediated through mechanism of inactivation.