Functional Measurement of CYP2C9 and CYP3A4 Allelic Polymorphism on Sildenafil Metabolism

Abstract

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

Acknowledgments

The authors thank the members of the Beijing Institute of Geriatrics of the Ministry of Health for their advice and assistance. This work was supported by National Natural Science Foundation of China (NSFC 81973397) and supported by grants from the Jinhua Science and Technology Bureau (No. 2019jzk014). In addition, Peng-fei Tang wants to thank Xue-meng Wu, in particular, for the care and support received during the process of writing and in life.

Disclosure

The authors report no conflicts of interest in this work.