Functional Measurement of CYP2C9 and CYP3A4 Allelic Polymorphism on Sildenafil Metabolism.

Aim: We aimed to systematically examine the effects of enzymatic activity of 38 human alleles and 21 human alleles, including wild-type and , which contain the 24 novel alleles ( and 6 novel alleles () newly found in the Chinese population, on sildenafil metabolism through in vitro experiment.

Methods: The recombinant cytochrome P450 alleles protein of and 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 variants were significantly altered when compared with the wild-type , with most of these variants exhibiting either reduced Vmax and/or increased Km values. Four alleles () 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 metabolic pathway, when compared with the wild-type , the relative clearance values of four variants ( and ) showed significantly higher relative clearance (130.7-134.9%), while five variants ( and ) 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 variants ( and ) could not be detected, due to the defect of the gene.

Conclusion: These findings were the first evaluation of all these infrequent and 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 and allelic variation on sildenafil metabolism for further clinical research.