Comparative study on the interaction between 3 CYP2C9 allelic isoforms and benzbromarone by using LC-MS/MS method

Benzbromarone is a uricosuric drug metabolized predominantly by cytochrome P450 2C9 from in vitro findings. Human CYP2C9 exhibits extensive genetic polymorphism and numbers of clinic studies have demonstrated that CYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of benzbromarone. But in vitro study on the interaction between CYP2C9 allelic isoforms and benzbromarone was rare. Here, an LC-MS/MS method was established and validated to determine the concentration of benzbromarone in different CYP2C9 enzyme incubation systems for the drug-enzyme interaction study. By selecting appropriate internal standard and optimizing separation system, including mobile phase, sample solvent and gradient elution condition, this LC-MS/MS method was developed with fine linearity (r2 ≥ 0.996), good reproducibility (RSD ≤ 6.6%), high stability (92.37-114.67%), efficient recovery (91.23-109.82%) and acceptable matrix effect (110.54-115.31%). Based on this method, the interaction between 3 CYP2C9 allelic isoforms and benzbromarone was researched by kinetics parameters (Km, Vmax, Clint). As a result, CYP2C9*1 displayed the highest metabolic activity towards benzbromarone, CYP2C9*2 showed a little lower catalytic activity than CYP2C9*1 (relative clearance/*1 = 85.86%), CYP2C9*3 showed the lowest catalytic activity (relative clearance/*1 = 21.57%). The result illustrated that various CYP2C9 allelic isoforms showed different enzymatic activities towards benzbromarone, which could offer effective consultation for personalized administration in clinic.