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目的鉴别参与盐酸双苯氟嗪(Dip)代谢的大鼠肝微粒体细胞色素P450酶(CYP)亚型。方法制备SD大鼠肝微粒体并与Dip孵育,产生的Dip代谢产物(M1,M2,M4和M5)采用LC-MS/MS鉴别,通过细胞色素P450酶选择性抑制剂实验、相关性分析实验和重组酶实验确定参与盐酸双苯氟嗪代谢的CYP酶亚型。结果抑制剂研究、相关性分析实验和重组酶实验3种分析方法测定结果均显示,大鼠肝微粒体中CYP2A1、CYP3A和CYP2C11是催化生成M1和M5的酶亚型;代谢盐酸双苯氟嗪生成M2的CYP酶按作用大小依次为CYP3A、CYP2A1、CYP1A2、CYP2C11和CYP2E1;转化盐酸双苯氟嗪生成M4的酶亚型活性高低依次为CYP3A、CYP2A1、CYP2E1和CYP2C11,重组酶实验结果还进一步表明CYP3A2较CYP3A1具有更强的活性。结论 CYP3A和CYP2A1均参与了Dip 4种代谢物的生成反应。
Objective To identify the rat liver microsomal cytochrome P450 enzymes (CYP) subtypes involved in the metabolism of Diphenhydramine hydrochloride (Dip). Methods SD rat liver microsomes were prepared and incubated with Dip. The produced Dip Metabolites (M1, M2, M4 and M5) were identified by LC-MS / MS. Cytochrome P450 selective inhibitor experiments and correlation analysis And recombinase experiments to determine CYP enzyme subtypes involved in the metabolism of dipfluzine hydrochloride. Results Inhibitor studies, correlation analysis experiments and recombinant enzyme assays showed that CYP2A1, CYP3A and CYP2C11 in rat liver microsomes were enzyme subtypes that catalyzed the formation of M1 and M5. Metabolism of dipfluzine hydrochloride CYP3A, CYP2A1, CYP1A2, CYP2C11 and CYP2E1 were formed in order of their effect on the activity of CYP2A1. CYP3A, CYP2A1, CYP2E1 and CYP2C11 were the most active enzymes of the M4 generation transformed by dipfluzine hydrochloride. The results of the recombinase assay were further analyzed This indicates that CYP3A2 is more active than CYP3A1. Conclusion Both CYP3A and CYP2A1 are involved in the production of Dip 4 metabolites.