采用特异性探针药物法对双峰驼CYP1A酶体外活性的测定

Determination of in vitro activities of bactrian camel CYP1A enzyme by specific probe drug

为探究双峰驼CYP1A酶体外活性及其对外源性药物代谢的影响,首先采用差速离心法制备双峰驼肝微粒体,并优化其体外孵育体系,然后采用高效液相色谱紫外检测法测定双峰驼肝微粒体孵育体系中CYP1A酶特异性底物非那西丁的代谢产物对乙酰氨基酚的动态含量,并借助Origin Pro8.6软件计算CYP1A酶的动力学参数。结果表明,双峰驼肝微粒体具有良好的酶活性,其蛋白质含量为1.652mg/g±0.341mg/g。经优化后孵育体系的最适宜底物的质量浓度为200μg/mL,最佳肝微粒体的质量浓度为4.95mg/mL,最适宜孵育时间为30min。双峰驼CYP1A酶体外孵育体系的最大反应速度为0.224 6nmol/(min·mg)±0.041 8nmol/(min·mg),米氏常数为5.577 9μmol/L±0.634 2μmol/L,内在代谢清除率为0.080 4(mL·min)/mg±0.023 1(mL·min)/mg。结果表明,本试验通过借助特异性探针药物动力学特征的研究,成功检测了双峰驼CYP1A酶的体外代谢活性。

In order to explore the in vitro activities of bactrian camel CYP1 Aon drug metabolism,firstly the bactrian camel hepatic microsome was prepared by using differential centrifugation,and the incubation system was optimized.And then the specific substrate of CYP1 Aenzyme and its metabolites-phenacetin and 4-Acetamidophenol concentration in bactrian camel liver microsomal incubation system were determined by high performance liquid chromatography with UV method（HPLC-UV）.Finally the kinetic parameters of CYP1 A were calculated by using Origin Pro8.6software.The results showed that bactrian camel liver microsome possess good activities,and their protein content is 1.652mg/g±0.341mg/g.The most suitable substrate concentration was 200μg/mL,the best liver microsome concentration was 4.95mg/mL,and the optimum incubation time was 30 min,respectively.Maximum velocity of bactrian camel CYP1 Aenzymes was 0.224 6nmol/（min·mg）±0.041 8nmol/（min·mg）in vitro incubation system,the Michaelis constant was 5.577 9μmol/L±0.634 2μmol/L,and the intrinsic metabolic clearance rate was0.080 4（mL·min）/mg±0.023 1（mL·min）/mg.In conclusion,the in vitro metabolic activities of bactrian camel CYP1 Aenzymes were successfully detected by studying the pharmacokinetic characteristics of specific probe drug.