【摘要】 目的测定大鼠肠微粒体中细胞色素P4503A(CYP3A)的活性,并对其体外孵育条件进行优化。方法采用1′-羟基咪哒唑仑的生成量表示肠中CYP3A的活性,反相高效液相色谱法测定肠微粒体中1′-羟基咪哒唑仑的浓度,用单因素实验法对其体外孵育条件进行优化,用线性Lineweaver-Burk双倒数作图法研究肠CYP3A酶促动力学。结果1′-羟基咪哒唑仑在9.10～910.00ng.mL-1内线性关系良好;体外优化的孵育条件为10μmol.L-1咪哒唑仑、4μg肠微粒体、孵育20min;肠CYP3A酶促动力学参数Km为7.61μmol.L-1,Vmax为1.26nmol.min-1.mg-1。结论HPLC操作简便、灵敏、快速,适用于肠微粒体中1′-羟基咪哒唑仑的浓度测定,肠CYP3A孵育条件及其酶促动力学研究为研究经CYP3A代谢的药物相互作用及其他物质对CYP3A酶的影响提供理论依据。更多还原

【Abstract】 OBJECTIVE To determine the activity of CYP3A and optimize the incubation conditions in the rat intestinal microsomes.METHODS The formation of 1′-hydroxymidazolam determined by RP-HPLC was used to indicate the activity of CYP3A.Single effect experimental design was used to optimize the incubation conditions and the enzyme kinetics was evaluated by graphical analysis with Lineweaver-Burk double reciprocal plots (1/velocity vs.1/[substrate]).RESULTS The calibration curves of 1′-hydroxymidazolam were linear in the range of 9.10-910.00 ng·mL-1 ( r2= 0.999 9).The optimal incubation conditions were 10 μmol·L-1,4 μg and 20 min for midazolam concentration,intestinal microsomes and incubation time,respectively.The parameters of CYP3A in the intestinal microsomes for Km and Vmax were 7.61 μmol·L-1 and 1.26 nmol·min-1·mg-1,respectively.CONCLUSION The HPLC method is simple,accurate,rapid,and suitable for the determination of 1′-hydroxymidazolam in the intestinal microsomes.The incubation conditions of CYP3A and its kinetics can be applied for study on the interaction between drugs metabolized by CYP3A.更多还原