In vitro studies and in silico predictions of fluconazole and CYP2C9 genetic polymorphism impact on siponimod metabolism and pharmacokinetics

The purpose of the study is to investigate the enzyme(s) responsible for siponimod metabolism and to predict the inhibitory effects of fluconazole as well as the impact of cytochrome P450 (CYP) 2C9 genetic polymorphism on siponimod pharmacokinetics (PK) and metabolism. In vitro metabolism studies were conducted using human liver microsomes (HLM), and enzyme phenotyping was assessed using a correlation analysis method. SimCYP, a physiologically based PK model, was developed and used to predict the effects of fluconazole and CYP2C9 genetic polymorphism on siponimod metabolism. Primary PK parameters were generated using the SimCYP and WinNonlin software. Correlation analysis suggested that CYP2C9 is the main enzyme responsible for siponimod metabolism in humans. Compared with the CYP2C9*1/*1 genotype, HLM incubations from CYP2C9*3/*3 and CYP2C9*2/*2 donors showed similar to 10- and 3-fold decrease in siponimod metabolism, respectively. Simulations of enzyme contribution predicted that in the CYP2C9*1/*1 genotype, CYP2C9 is predominantly responsible for siponimod metabolism (similar to 81%), whereas in the CYP2C9*3/*3 genotype, its contribution is reduced to 11%. The predicted exposure increase of siponimod with fluconazole 200 mg was 2.0-2.4-fold for CYP2C9*1/*1 genotype. In context of single dosing, the predicted mean area under the curve (AUC) is 2.7-, 3.0- and 4.5-fold higher in the CYP2C9*2/*2, CYP2C9*2/*3 and CYP2C9*3/*3 genotypes, respectively, compared with the CYP2C9*1/*1 genotype. .Enzyme phenotyping with correlation analysis confirmed the predominant role of CYP2C9 in the biotransformation of siponimod and demonstrated the functional consequence of CYP2C9 genetic polymorphism on siponimod metabolism. Simulation of fluconazole inhibition closely predicted a 2-fold AUC change (ratio within similar to 20% deviation) to the observed value. In silico simulation predicted a significant reduction in siponimod clearance in the CYP2C9*2/*2 and CYP2C9*3/*3 genotypes based on the in vitro metabolism data; the predicted exposure was close (within 30%) to the observed results for the CYP2C9*2/*3 and CYP2C9*3/*3 genotypes.