中国汉族人群24种CYP2C19新突变体的体外活性研究

发布时间：2018-10-08 09:58

【摘要】：目的以S-美芬妥英为探针药,体外分析24种汉族人群CYP2C19新变异体的酶学活性。方法以野生型CYP2C19 c DNA为模板,通过定点诱变方法获得24种CYP2C19新变异体和典型变异体CYP2C19.3的c DNA,利用Bac-toBac Baculovirus Expression System试剂盒,获得高表达各型CYP2C19变异体的昆虫微粒体。Western blot法进行CYP2C19蛋白定量。以S-美芬妥英为探针底物药,体外测定不同药物浓度下各型CYP2C19变异体的最大反应速率Vmax和米氏常数Km,评价新突变类型对CYP2C19体外代谢活性的影响。结果利用昆虫表达系统,成功获得高表达典型变异体CYP2C19.3和24种CYP2C19新变异体的昆虫微粒体。Western blot结果显示,变异体CYP2C19.3和35FS无正常CYP2C19酶的表达,与野生型相比,变异体CYP2C19.29和T130M表达量明显下降,其余变异体蛋白表达量与野生型接近。体外代谢实验结果显示,变异体L16F针对S-美芬妥英的体外清除率(Vmax/Km)较野生型升高,T130M的活性与野生型相似,其余各突变型的清除率均低于野生型。其中,突变体CYP2C19.3、35FS和R124Q对于该底物药无代谢活性。结论新发现的24种CYP2C19基因变异大多可引起酶体外药代活性显著降低,提示携带此类基因突变的个体服用经由CYP2C19代谢药物时,体内药物代谢速度可能会显著变慢,临床用药时需密切关注。
[Abstract]:Objective to analyze the enzymatic activity of 24 new variants of CYP2C19 in vitro using S-mephenytoin as a probe. Methods using wild type CYP2C19 c DNA as template, the c DNA, of 24 new CYP2C19 variants and typical variants CYP2C19.3 were obtained by site-directed mutagenesis. Insect microsomes with high expression of various CYP2C19 variants were obtained. Western blot assay was used to quantify CYP2C19 protein. Using S-mephenytoin as the probe substrate, the maximum reaction rate (Vmax) and the Michlet constant (Km,) of various CYP2C19 variants at different drug concentrations were determined in vitro to evaluate the effect of new mutation types on the metabolic activity of CYP2C19 in vitro. Results using insect expression system, the insect microsomes with high expression of typical variant CYP2C19.3 and 24 new variants of CYP2C19 were successfully obtained. Western blot results showed that the mutant CYP2C19.3 and 35FS had no normal CYP2C19 enzyme expression, compared with wild type. The expression of CYP2C19.29 and T130M decreased significantly, and the other variants were similar to wild-type. The results of metabolism in vitro showed that the in vitro clearance rate (Vmax/Km) of variant L16F against S-mephenytoin was similar to that of wild type, and the clearance rate of other mutant types was lower than that of wild type. The mutants CYP2C19.3,35FS and R124Q had no metabolic activity against the substrate. Conclusion most of the 24 CYP2C19 gene mutations can cause the decrease of pharmacokinetic activity in vitro, which suggests that the drug metabolism may slow down significantly when the individuals with this mutation take the metabolic drugs via CYP2C19. Close attention should be paid to the clinical use of drugs.
【作者单位】：卫生部北京医院检验科;卫生部北京医院卫生部北京老年医学研究所;温州医科大学药学院药理教研室;浙江省丽水市人民医院临床药学实验室;
【基金】：国家自然科学基金资助项目(31371280) 卫生部行业科研专项经费资助项目(201302008)
【分类号】：R394

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