CYP2C19 polymorphisms account for inter-individual variability of drug metabolism in cynomolgus macaques

Abstract

CYP2C19 (formerly known as CYP2C75), highly homologous to human CYP2C19, has been identified in cynomolgus and rhesus macaques, non-human primate species widely used in drug metabolism studies. CYP2C19 is predominantly expressed in liver and encodes a functional drug-metabolizing enzyme. Genetic variants in human CYP2C genes account for the inter-individual variability in drug metabolism; however, genetic variants have not been investigated in macaque CYP2C19. In the present study, re-sequencing of CYP2C19 in 78 cynomolgus and 36 rhesus macaques identified 34 non-synonymous variants. Among these, 6 were located in substrate recognition sites, the domains important for protein function. Eighteen and 6 variants were unique to cynomolgus and rhesus macaques, respectively. Four variants were characterized by site-directed mutagenesis and metabolic assays, and 3 variants (p.Phe100Asn, p.Ala103Val, and p.Ile112Leu) showed substantially reduced activity as compared with wild type in flurbiprofen 4′-hydroxylation, omeprazole 5-hydroxylation, and R-/S-warfarin 7-hydroxylation. These variants, co-segregating in the animals analyzed, influenced metabolic activities because the homozygotes and/or heterozygotes showed significantly reduced catalytic activities in liver toward flurbiprofen 4′-hydroxylation and omeprazole 5-hydroxylation as compared with wild type. Kinetic analysis for R-warfarin 7-hydroxylation and docking simulation indicated that CYP2C19 Ala103Val would change the function and conformation of this enzyme. Ala103Val variation diminished homotropic cooperativity of CYP2C19 with R-warfarin yielding low metabolic capacity. These results indicated that the interindividual variability of CYP2C-dependent drug metabolism is at least partly accounted for by CYP2C19 variants in cynomolgus macaques.

Introduction

Human CYP2Cs, including CYP2C8, CYP2C9, and CYP2C19, are essential drug-metabolizing enzymes, involving the metabolism of approximately 20% of the prescribed drugs such as flurbiprofen and S-mephenytoin [1]. In the human CYP2C genes, a number of genetic variants have been identified (see http://www.cypalleles.ki.se/), including CYP2C9*2 and CYP2C9*3. These alleles, resulting in the reduced activity of the enzyme, are more prevalent in Caucasians than in Africans while Asians do not appear to possess CYP2C9*2 [2]. In human CYP2C19, CYP2C19*2 and CYP2C19*3, accounting for the majority of poor metabolizer phenotypes, are more prevalent in Asians than in Caucasians or Africans, among which CYP2C19*3 is largely found in Asians [2]. These defective alleles of human CYP2C9 and CYP2C19 genes need to be considered in drug metabolism studies during drug development.

Cynomolgus macaques (Macaca fascicularis), and rhesus macaques (Macaca mulatta), are non-human primate species widely used in drug metabolism studies. Cynomolgus CYP2C19, formerly known as CYP2C75, is highly homologous to human CYP2C9 and CYP2C19, and the gene is abundantly expressed in liver [3]. Cynomolgus CYP2C19 metabolizes human CYP2C substrates; S-mephenytoin, flurbiprofen, and tolbutamide [3], [4]. The previous study reported that pharmacokinetics of S-mephenytoin was highly variable among 64 cynomolgus macaques [5], and such inter-animal variations might be accounted for by genetic variants. Genetic polymorphisms have been found in macaque P450 genes [6], [7], [8], [9], [10]: however, the genetic variants have not been investigated in macaque CYP2C19.

In the present study, the genomes of 78 cynomolgus macaques (38 from Indochina and 40 from Indonesia) and 36 rhesus macaques were analyzed to identify CYP2C19 genetic variants. Several identified alleles were analyzed by metabolic assays using flurbiprofen, omeprazole, and S-/R-warfarin as substrate with monkey liver microsomes and the proteins heterologously expressed in Escherichia coli (E. coli).