CYP2C19 polymorphisms account for inter-individual variability of drug metabolism in cynomolgus macaques
Graphical abstract
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).
Section snippets
Preparation of DNA and microsome samples
Whole blood samples were collected from 78 cynomolgus macaques (38 from Indochina and 40 from Indonesia, 4–5 years of age, weighing 3–5 kg) and 36 rhesus macaques (from China, 7 years of age, weighing 3–5 kg). From these samples, genomic DNA was prepared using the Puregene DNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Microsome samples were prepared from the livers of 7 Indochinese cynomolgus macaques as described previously [11]. The study was reviewed
Identification of genetic variants
To identify genetic variants, CYP2C19 was re-sequenced in 78 cynomolgus macaques and 36 rhesus macaques. The analysis identified a total of 34 non-synonymous variants, among which 6 were in substrate recognition sites (SRS), the important regions for protein function; Phe100Asn, Ala103Val, and Ile112Leu in SRS1, Ile237Val and Leu240Met in SRS3, and Leu361Phe in SRS5 (Table 2). Three variants (Phe100Asn, Ala103Val, and Ile112Leu) completely co-segregated and mostly co-segregated in the
Discussion
Cynomolgus macaques are important animal species for drug metabolism studies. However, genetic variants, which might result in the inter-individual variability of catalytic activity, have not been investigated in cynomolgus CYP2C19, which encodes the enzyme metabolizing human CYP2C substrates such as diclofenac, flurbiprofen, omeprazole, and warfarin. In the present study, a total of 34 non-synonymous genetic variants were found by screening the genome samples of 78 cynomolgus and 36 rhesus
Acknowledgements
This work was supported in part by the Ministry of Education, Science, Sports and Culture of Japan and the Takeda Science Foundation, Japan. Authors greatly thank Mr. Masahiro Utoh for his support to this work, Drs. Shotaro Uehara and Makiko Shimizu for their technical assistance, and Mr. Lance Bell for his advice on English writing.
References (21)
- et al.
Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects
Pharmacol Ther
(2007) - et al.
CYP2C76 non-synonymous variants in cynomolgus and rhesus macaques
Drug Metab Pharmacokinet
(2012) - et al.
A discovery of genetic variants in CYP1D1: an implication for functional integrity of CYP1D1 in cynomolgus macaques and rhesus macaques
Drug Metab Pharmacokinet
(2011) - et al.
Regional distribution of drug-metabolizing enzyme activities in the liver and small intestine of cynomolgus monkey
Drug Metab Pharmacokinet
(2011) - et al.
Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation
Biochem Pharmacol
(2012) - et al.
Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli
Protein Expr Purif
(2002) - et al.
Diclofenac hydroxylation in monkeys: efficiency, regioselectivity, and response to inhibitors
Biochem Pharmacol
(2007) - et al.
Structural characterization of human cytochrome P450 2C19: active site differences between P450s 2C8, 2C9, and 2C19
J Biol Chem
(2012) - et al.
Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity
J Biol Chem
(1996) Clinical relevance of genetic polymorphisms in the human CYP2C subfamily
Br J Clin Pharmacol
(2001)
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2018, Biochemical PharmacologyCitation Excerpt :Metabolic clearance of human P450 cocktail probes (caffeine, S-warfarin, omeprazole, metoprolol, and midazolam in combination) [28] was compared to those of the same cassette dosing in cynomolgus monkeys [29]. Cynomolgus monkey P450 2C19, highly homologous to human P450 2C19, catalyzed R-warfarin 7-hydroxylation [6,30]. In vivo pharmacokinetics of racemic warfarin and its metabolites after oral and intravenous administrations were studied in male cynomolgus monkeys (n = 11, from Indochina) [17] which had been genotyped for the compound P450 2C19 [F100N; A103V; I112L] variant [10] (Table 2).