体内、体外相结合评价CYP2E1酶活性的方法学研究

Method for in vivo and in vitro Evaluation of CYP2E1 Activities

摘要:

目的: 以氯唑沙宗(chlorzoxazone,CZX)为"探针药物",建立一种快速、准确的体内、体外相结合评价细胞色素P450 2E1(cytochrome P450 2E1, CYP2E1)酶活性的HPLC-DAD检测方法。 方法: 体内实验为大鼠静脉注射CZX,剂量20 mg·kg^-1,按时间点眼眶取血,检测CZX与其代谢产物6-羟基氯唑沙宗(6-hydroxy chlorzoxazone, 6-OHCZX)的血浆药物浓度。体外实验为不同浓度CZX与大鼠肝微粒体温孵后,检测温孵体系中6-OHCZX的浓度,计算酶动力学参数来评价CYP2E1酶的活性。采用Agilent Extend-C₁₈色谱柱(6 mm×100 mm, 5 μm),以非那西丁为内标,流动相为乙腈-0.6%乙酸水溶液,流速1.0 mL·min^-1,紫外检测波长290 nm,柱温30 ℃。 结果: 体内、体外实验结果发现CZX,6-OHCZX的线性范围、日内和日间精密度、相对回收率、稳定性均符合生物样品的检测要求。 结论: 大鼠血浆及肝微粒体温孵体系中的其他内源性物质不干扰待测物的测定。该方法快速、稳定、灵敏度高,适合CZX及其代谢产物6-OHCZX的测定。

Abstract:

Objective:Using chlorzoxazone (CZX) as the 'probe drug’,to establish a rapid and accurate method for evaluating the activities of cytochrome P4502E1(CYP2E1) by the high-performance liquid chromatography-UV detection. Method: In vivo study: 20 mg·kg^-1 of CZX was given to the rats via the tail vein. Blood samples were collected at designed time points post dose and plasma samples were obtained. CZX and 6-hydroxy chlorzoxazone (6-OHCZX) were determined by an HPLC method. In vitro study:After incubation, the formation rate of 6-OHCZX was used to evaluate the function of CYP2E1.The analysis was performed on the Agilent Extend-C₁₈ column (4.6 mm×100 mm, 5 μm), with a mobile phase consisting of acetonitrile-0.6% acetic acid in water at a flow rate of 1.0 mL·min^-1. Phenacetin was used as the internal standard. The detection wavelength was 290 nm, and column temperature was set at 30 ℃. Result: In vivo and in vitro experiments revealed that the methods had good linear ranges and their intra-and inter-day relative standard deviations, relative recoveries, stabilities met the requirement of biological estimation. Conclusion: Other endogenous substances in the plasma and incubation system did not interfere with the determination. The method is rapid, stable and highly sensitive. It is suitable for determination of CZX and its metabolites 6-OHCZX and can be used for in vivo and in vitro evaluation of CYP2E1 activitise and research of enzyme kinetics.