Study On The Drug Metabolic Properties Of Cytochrome P450 2C9 R150H And R150L Variants

Cytochrome P450 2C9(CYP2C9)is one of the most important drug-metabolizing enzymes(phase I)in liver,which catalyzes about 16%of the commonly used clinical drugs.CYP2C9 has high polymorphism(SNP),which is of great significance for clinical use.Both CYP2C9*8(449G>A,R150H)and CYP2C9*27(G449>T,R150L)have mutations in the 449th base,and the 150th arginine in the protein sequence has been mutated to histidine and leucine,respectively.However,it has been reported that the catalytic activity of CYP2C9*8 and CYP2C9*27 is quite different.For studying the metabolic mechanism of CYP2C9*8 and CYP2C9*27,a baculovirus insect cell expression system with clear composition and high expression was constructed.An electro-spray ionisation ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method was constructed to determin CYP2C9 SNPs enzyme kinetics.The results showed that the expression system had high bioactive CYP450 content and porase content.It was used to determine P450 content by CO spectrophotometry,and the interference of CYP content in P420 form on drug metabolism was eliminated.The system method was established correctly.Characteristics of UPLC-MS/MS were good.There was no endogenous interference,no tail and bifurcation between substrate,metabolite and um in CYP2C9incubation system.Compared with wild type,Michaelis constant(K_m)of CYP2C9*8 for warfarin,phenytoin and diclofenac were 1.241,35.87 and 3.59μmol/L,respectively,the relative clearances were 128.86%,97.22%and 21.62%.The K_m of CYP2C9*27 for warfarin,phenytoin and diclofenac metabolism were 3.807,30.93 and 1.286μmol/L,respectively,and relative clearances were 43.07%,136.55%and 79.13%.In this study,we established a good expression system model of CYP2C9 and POR in vitro through molecular biology and cytology,established an efficient method for the determination of metabolites by precise analysis instruments,and studied the enzyme dynamics of CYP2C9 polymorphism and the metabolism of drugs.This study provides experimental basis for the study of the mechanism of CYP2C9 polymorphism enzyme catalysis drugs.