Aim: We aimed to systematically examine the effects of enzymatic activity of 38 human CYP2C9  alleles and 21 human CYP3A4  alleles, including wild-type CYP2C9.1  and CYP3A4.1 , which contain the 24 CYP2C9  novel alleles (*36–*60)  and 6 CYP3A4  novel alleles (*28–*34 ) newly found in the Chinese population, on sildenafil metabolism through in vitro experiment.
Methods: The recombinant cytochrome P450 alleles protein of CYP2C9  and CYP3A4  expressed in insect baculovirus expression system were reacted with 10– 500 μM sildenafil for 30 minutes at 37°C, and the reaction was terminated by cooling to − 80°C immediately. Next, we used ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection system to detect sildenafil and its active metabolite N-desmethyl sildenafil.
Results: The intrinsic clearance (Vmax/Km) values of most CYP2C9  variants were significantly altered when compared with the wild-type CYP2C9*1 , with most of these variants exhibiting either reduced Vmax and/or increased Km values. Four alleles (CYP2C9*11, *14, *31, *49 ) exhibited no markedly decreased relative clearance (1-fold). The relative clearance of the remaining thirty-three variants exhibited decrease in different levels, ranging from 1.81% to 88.42%. For the CYP3A4  metabolic pathway, when compared with the wild-type CYP3A4*1 , the relative clearance values of four variants (CYP3A4*3, *10, *14  and *I335T ) showed significantly higher relative clearance (130.7– 134.9%), while five variants (CYP3A4*2, *5, *24, *L22V  and *F113I ) exhibited sharply reduced relative clearance values (1.80– 74.25%), and the remaining nine allelic variants showed no statistical difference. In addition, the kinetic parameters of two CYP3A4  variants (CYP3A4*17  and CYP3A4*30 ) could not be detected, due to the defect of the CYP3A4  gene.
Conclusion: These findings were the first evaluation of all these infrequent CYP2C9  and CYP3A4  alleles for sildenafil metabolism; when treating people who carry these CYP2C9 and CYP3A4 variants, there should be more focus on the relation of dose intensity, side effects and therapeutic efficacy when administering sildenafil. The study will provide fundamental data on effect of CYP2C9  and CYP3A4  allelic variation on sildenafil metabolism for further clinical research.
Keywords: allelic variants, CYP2C9  polymorphisms, CYP3A4  polymorphisms, enzymatic activity, sildenafil, individual treatment