瑞格列奈在人肝微粒体中的酶促反应动力学及葛根素对其代谢的影响

Effect of puerarin on the metabolism of repaglinide in human liver microsomes by CYP 450

目的通过体外人肝微粒孵育实验考察瑞格列奈的酶促反应动力学,并研究葛根素对瑞格列奈代谢的影响。方法以阿托伐他汀为内标,建立UPLC法测定人肝微粒体中瑞格列奈的浓度。建立人肝微粒体体外孵育体系,确定最佳蛋白浓度和孵育时间;采用原形药物减少法,通过GraphPad Prism 5.0软件计算酶促反应动力学参数(V_(max)和K_(m));系列浓度葛根素(0~50μmol·L^(-1))与瑞格列奈(5.5μmol·L^(-1))在肝微粒体孵育体系中于37℃水浴共同孵育,测定肝微粒体中瑞格列奈的浓度,并计算减少量,从而考察葛根素对瑞格列奈代谢的影响。结果瑞格列奈在人肝微粒体的最佳蛋白质量浓度为0.5 mg·mL^(-1),最佳孵育时间为40 min;酶促反应动力学参数V_(max)=1.71μmol·min^(-1)·(mg·protein)^(-1),K_(m)=5.66μmol·L^(-1)。结论UPLC法测定人肝微粒体中瑞格列奈的浓度,灵敏度高、操作简便。通过人混合肝微粒体孵育实验得到瑞格列奈的酶促反应动力学参数,且进一步研究显示葛根素对瑞格列奈在人肝微粒体未产生显著的抑制作用,可为两药的联合应用提供参考。

Objective To obtain the enzymatic kinetic parameters of repaglinide by human liver microsome incubation experiment,and determine the effect of puerarin on the metabolism of repaglinide.Methods The UPLC method was established to determine the concentration of repaglinide in human liver microsomes with atorvastatin as the internal standard.The human liver microsomes incubation system was established and the optimal protein concentration and incubation time were determined.The enzymatic kinetic parameters of V_(max) and K_(m) were calculated by GraphPad Prism 5.0 software based on the reduction of repaglinide.Repaglinide(5.5μmol·L^(-1))was co-incubated with puerarin(0~50μmol·L^(-1))at 37℃water bath.The concentrations of repaglinide were detected.The effect of puerarin on the metabolism of repaglinide was measured by calculating the reduction of repaglinide.Results The optimal incubation time and protein concentration in human liver microsomes were 40 min and 0.5 mg·mL^(-1),respectively.The enzyme kinetics of repaglinide were Vmax=1.71μmol·min^(-1)·(mg·protein)^(-1),and K_(m)=5.66μmol·L^(-1).Conclusion The UPLC method can determine the concentration of repaglinide in human liver microsomes,which is simple and sensitive,and the enzyme kinetic parameters of repaglinide are obtained.Puerarin dose not obviously inhibit repaglinide in human liver microsomes,which may provide support for the combination of the two drugs.