抗癫痫新药氯桂丁胺在大鼠肝微粒体中的酶促反应动力学研究

Enzyme kinetics of novel antiepileptic drug 3,4-dichlorophenyl-propenoyl-sec.-butylamine in rat liver microsomes in vitro

目的观察肝微粒体药物代谢酶系对抗癫痫新药氯桂丁胺生物转化的影响及酶促动力学特征。方法差速离心法制备大鼠肝微粒体,高效液相色谱法检测氯桂丁胺及其主要代谢物的浓度,双倒数作图法计算酶促动力学参数。结果原形药的Km为13.46μg.mL-1,Vmax为14.88 nmol.min-1.mg-1蛋白,t1/2为48.53 min。代谢物M1,M2和M3的Km分别为8.82,0.26和43.37μg.mL-1,Vmax分别为0.17,4.44×10-2和3.24 nmol.min-1.mg-1蛋白。结论氯桂丁胺经肝微粒体代谢是其主要的消除形式,并呈反应时间、酶蛋白浓度和底物依赖性酶促动力学特征。

Objective To evaluate the enzyme catalyzed kinetic characteristics of 3,4-dichlorophenyl-propenoyl-sec.-butylamine（3,4-DCPB）,an anti-epileptic ramification of cinnamamides,with hepatic microsomal cytochrome P450 in rats in vitro.Methods Liver microsomes were obtained by differential centrifugation.Quantitation of 3,4-DCPB and its metabolites using HPLC-UV detection.The enzyme kinetic characteristics were calculated by Lineweaver-Burke method.Results In an optimized HPLC-UV method,3,4-DCPB 0.05~16.00 μg·mL-1 was incubated with microsomal protein 0.4 mg·mL-1 for 5 min.The michaelis constants of 3,4-DCPB were evaluated as Km 13.46 μg·mL-1,Vmax 14.88 nmol·min-1·mg-1 protein,and the elimination half-life was 48.53 min.The enzyme kinetic constants of three major metabolites M1,M2 and M3 were as follows： Km was 8.82,0.26 and 43.37 μg·mL-1,Vmax was 0.17,4.44×10-2 and 3.24 nmol·min-1·mg-1 protein respectively.Conclusion These results suggest that the biotransformation of 3,4-DCPB in liver was important in its elimination pathway.