建立一种测定细胞色素P450 3A4活性的方法

 目的建立一种细胞色素P450 3A4(cytochrome P450 3A4,CYP3A4)活性定量的新方法。方法将CYP3A4底物硝苯地平与人肝微粒体进行体外温孵,以高效液相色谱法测定硝苯地平及其氧化产物,以甲醇-水(64∶36)为流动相,流速为0.5 mL·min^-1,检测波长为254 nm。结果在所建立的HPLC条件下,硝苯地平及其氧化产物的出峰时间分别在8.44和6.15 min,能够完全分离,且无其他生物基质峰干扰;氧化硝苯地平在人肝微粒体的最低检测限为25μg·L^-1(S/N=3),线性范围是0.25～32 mg·L^-1(r=0.999 4),符合检测要求;在0.5,4.0,20.0 mg·L^-1时的萃取率分别为(67.05±3.49)%,(66.63±3.60)%,(67.08±2.78)%(n=5);方法回收率分别为(100.17±6.97)%,(91.96±3.71)%,(96.55±3.98)%(n=5);日内及日间RSD均小于7%。结论本实验所建立的HPLC能够准确、快速检测出硝苯地平及其在人肝微粒体中的氧化产物,能够对CYP 3A4活性进行快速评价,适合于体外药物代谢动力学研究和第三代多药抗药性逆转剂的筛选。

Determination of CYP 3A4 Activity in Vitro

OBJECTIVE To determine CYP3A4 activity in vitro.METHODS Nifedipine,a substrate of CYP3A4,was incubated with human liver microsomes in vitro.Nifedipine and oxidized nifedipine were determined by HPLC.The mobile phase was composed of CH₃OH-H₂O(64∶36) at a flow rate of 0.5 mL·min^-1,and the detection was 254 nm.RESULTS The retention times of nifedipine and nifedipine appeared at 8.44 and 6.15 min under the condition of HPLC,respectively,while there were not interference peaks.The lowest concentration of detection in human liver microsomes was 25 μg·L^-1 for oxidized nifedipine(S/N=3),and its suitable linear range was 0.25～32.0(mg·L^-1)(r=0.999 4),which met the requirement of the whole examination.At the concentration of 0.5,4.0,20.0 mg·L^-1,the recoverys of extraction were(67.05±3.49)%,(66.63±3.60)% and(67.08±2.78)%(n=5),respectively,and the recoveries of method were(100.17±6.97)%,(91.96±3.71)% and(96.55±3.98)%(n=5),respectively.The RSDs of the within-day and between-day validation were less than 7%.CONCLUSION Nifedipine and oxidized nifedipine be detected accurately and rapidly by the proposed method,which can be applied to evaluate CYP3A4 activity and screen the third generation multidrug resistance modulators.