Cytochrome P450 1A1, 2C9, 2C19, and 3A4 Polymorphisms Account

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Article Cite This: Chem. Res. Toxicol. 2018, 31, 1373−1381

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Cytochrome P450 1A1, 2C9, 2C19, and 3A4 Polymorphisms Account for Interindividual Variability of Toxicological Drug Metabolism in Cynomolgus Macaques Yasuhiro Uno,*,† Shotaro Uehara,‡ Norie Murayama,‡ and Hiroshi Yamazaki*,‡ †

Shin Nippon Biomedical Laboratories, Ltd., Kainan, Wakayama 642-0017, Japan Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan

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‡

ABSTRACT: Cytochromes P450 (P450s) and their genetic variants in humans are important drug-metabolizing enzymes partly accounting for interindividual variations in drug metabolism and toxicity. However, these genetic variants in P450s have not been fully investigated in cynomolgus macaques, a nonhuman primate species widely used in toxicological studies. In this study, genetic variants found in cynomolgus CYP1A1, CYP2C9 (formerly CYP2C43), CYP2C19 (CYP2C75), and CYP3A4 (CYP3A8) were assessed on functional importance. Resequencing of CYP1A1 in cynomolgus macaques found 18 nonsynonymous variants, of which M121I and V382I were located in SRSs, domains potentially important for P450 function. By further analyzing these two variants, V382I was significantly associated with lower drug-metabolizing activities in the liver for the heterozygotes than the wild types. Similarly, the heterozygotes or homozygotes of CYP2C9 variants (A82V and H344R) and CYP2C19 variant (A490V) showed significantly lower drug-metabolizing activities in the liver than the wild types. Moreover, the homozygotes of CYP3A4 variant (S437N) showed significantly higher activities than the wild type in the liver. Kinetic analyses using recombinant proteins revealed that CYP2C9 variants (A82V and H344R) showed substantially lower Ks values than the wild type, although CYP1A1 variant (V382I) showed kinetic parameters similar to the wild type. Likewise, CYP2C19 variant (A490V) showed substantially a lower Vmax/Km value than the wild type, whereas CYP3A4 variant (S437N) showed a higher Vmax/Km value than the wild type. These results suggest the toxicologically functional importance of CYP2C9 variants (A82V and H344R), CYP2C19 variant (A490V), and CYP3A4 variant (S437N) for hepatic drug metabolism in cynomolgus macaques.

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INTRODUCTION It is widely appreciated that parent drugs and their metabolites can mediate the adverse effects exhibited by some new therapeutic agents. Much progress has been made in understanding mechanisms of toxicities influenced by intraand interspecies differences in drug metabolism and disposition caused by genetic polymorphisms in drug-metabolizing enzymes.1,2 A typical interspecies difference example is thalidomide teratogenicity in which a toxic arene oxide metabolite was produced by livers from rabbits, monkeys, and humans (all sensitive species to thalidomide teratogenesis) but not produced by livers from rats (not sensitive).3 Cytochrome P450 (P450) is a gene family comprising a large number of drug-metabolizing enzymes including 57 functional genes and 58 pseudogenes in humans (see http:// drnelson.uthsc.edu/cytochromeP450.html). Among the human P450s, CYP2C9 and CYP2C19 are essential drugmetabolizing enzymes, which metabolize prescribed drugs such as flurbiprofen and S-mephenytoin.4 Drug-metabolizing activities mediated by human CYP2C9 and CYP2C19 are diverse, partly accounted for by genetic variants. For example, CYP2C9*2 and 2C9*3 resulted in reduced activities and are more prevalent in Caucasians than in Africans, and CYP2C19*2 and 2C19*3 are responsible for poor metabolizing © 2018 American Chemical Society

phenotypes and are more prevalent in Asians than in Caucasians or Africans.5 Cynomolgus macaques (Macaca fascicularis) are a nonhuman primate species widely used in drug metabolism and toxicological studies. Cynomolgus CYP2C9 (formerly CYP2C43) and CYP2C19 (CYP2C75) are highly homologous to both human CYP2C9 and CYP2C196 and are abundantly expressed in the liver.7 Cynomolgus CYP2C9 and CYP2C19 selectively catalyze efavirenz 8-hydroxylation and R-warfarin 7hydroxylation, respectively,8−10 although these enzymes also metabolize human CYP2C9/19 substrates, S-mephenytoin, flurbiprofen, and tolbutamide.6,11 Cynomolgus CYP1A1, highly homologous to human CYP1A1, metabolizes human CYP1A substrates such as ethoxyresorufin and caffeine.12 In the liver, cynomolgus CYP1A1 mRNA is expressed much more abundantly than cynomolgus CYP1A2 mRNA, the gene of which is most likely under pseudogenization,12,13 indicating that CYP1A1 is a major CYP1A in cynomolgus macaques. Cynomolgus CYP3A4 is an ortholog of human CYP3A4, one of the most important P450s, which is abundantly expressed in Received: September 6, 2018 Published: November 9, 2018 1373

DOI: 10.1021/acs.chemrestox.8b00257 Chem. Res. Toxicol. 2018, 31, 1373−1381

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Chemical Research in Toxicology Table 1. Primers Used for Site-Directed Mutagenesis gene

variant

CYP1A1

c.1144A > G

CYP2C9

c.245C > T c.1031A > G

CYP2C19

c.1469T > C

CYP3A4

c.1310G > A

a

F R F R F R F R F R

name

sequence (5′ → 3′)b

mfCYP1A1_c1144A_G (5qc1) mfCYP1A1_c1144A_G (3qc1) mfCYP2C43_c245C_T (5qc1) mfCYP2C43_c245C_T (3qc1) mfCYP2C43_c1031A_G (5qc1) mfCYP2C43_c1031A_G (3qc1) mfCYP2C75_c1469T_C (5qc1) mfCYP2C75_c1469T_C (3qc1) mfCYP3A4_c1310G_A (5qc1) mfCYP3A4_c1310G_A (3qc1)

CGACACTCCTCCTTCgTCCCCTTCACCATCC GGATGGTGAAGGGGAcGAAGGAGGAGTGTCG CTGCATGGATATGAAGtGGTGAAGGAAGCCCTG CAGGGCTTCCTTCACCaCTTCATATCCATGCAG GCAGGACAGGAGCCgCATGCCCTACACAG CTGTGTAGGGCATGcGGCTCCTGTCCTGC GCTGTGCTTCATTCCTGcTTGAAGAAGAGCAGATG CATCTGCTCTTCTTCAAgCAGGAATGAAGCACAGC CCTTACATATACACGCCCTTTGGAAaTGGACCCAGAAACTGC GCAGTTTCTGGGTCCAtTTCCAAAGGGCGTGTATATGTAAGG

a

F, forward; R, reverse. bNucleotides to be changed are shown in lower case.

the liver14 and metabolizes a large number of prescribed drugs including midazolam.15 Numerous genetic variants have been found in cynomolgus CYP2C9 and CYP2C19.16,17 Cynomolgus CYP2C9 I112L variant was associated with lower metabolic clearance of efavirenz in vivo.18 Cynomolgus CYP2C19 variants F100N, A103V, and I112L accounted for reduced catalytic activity of the enzyme in flurbiprofen 4′-hydroxylation, omeprazole 5hydroxylation, and R-/S-warfarin 7-hydroxylation in vitro.17 RWarfarin 7-hydroxylation was also affected in vivo by the compound alleles of all these CYP2C19 variants,19 for which a genotyping tool has been developed.20 However, most of the genetic variants identified in cynomolgus CYP2C9 and CYP2C19 remain to be functionally characterized. An increased rate of nifedipine oxidation has been previously observed with recombinant CYP3A4 p.S437N, whereas the rate of midazolam 1′-/4-hydroxylation exhibited by this mutant is comparable to that observed with the wild-type enzyme.21 These apparent differences between the recombinant CYP3A4 p.S437N and the wild type remain to be reevaluated using liver microsomes. In this study, we resequenced the genomes of 107 cynomolgus macaques and four rhesus macaques to identify genetic variants of CYP1A1. For the CYP1A1, CYP2C9, CYP2C19, and CYP3A4 genetic variants, drug-metabolizing activities in liver microsomes were compared between the wild types and mutants (heterozygotes and homozygotes). The genetic variants found to be associated with altered drugmetabolizing activities were further investigated by kinetic analysis using the proteins heterologously expressed in Escherichia coli (E. coli). Among the numerous variants identified, this study focused on the variants, for which the liver samples sufficient for the analysis were available. The present study contributes to the establishment of a valuable model for studying the roles of P450 polymorphisms in interindividual variations in drug metabolism and toxicity using cynomolgus macaques.

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Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd. (Kainan, Japan). Amplification and DNA Sequencing. Direct sequencing was carried out using cynomolgus and rhesus macaque genomes for CYP1A1, CYP2C9, CYP2C19, and CYP3A4 as described previously.13,16,17,21 Briefly, each 20 μL reaction of polymerase chain reaction (PCR) mixture contained 1 ng of genomic DNA, 5 pmole of forward and reverse primers, and 1 unit of ExTaq HS polymerase (Takara, Otsu, Japan). Amplifications were performed as follows: 95 °C for 2 min; 35 cycles of 20 s at 95 °C, 30 s at 60 °C, and 0.5−1.5 min at 72 °C; and 7 min at 72 °C. Sequencing was performed using an ABI PRISM BigDye Terminator version 3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), followed by electrophoresis by an ABI PRISM 3730 DNA Analyzer (Applied Biosystems). PCR and sequencing primers were as described previously for CYP1A1, CYP2C9, CYP2C19, and CYP3A4.13,16,17,21 Sequence data were analyzed using DNASIS Pro (Hitachi Software, Tokyo, Japan) and Sequencher (Gene Codes Corporation, Ann Arbor, MI). Site-Directed Mutagenesis and Protein Expression. For functional analysis of CYP1A1, CYP2C9, CYP2C19, and CYP3A4 variants, proteins were heterologously coexpressed with NADPHcytochrome P450 reductase in E. coli using expression plasmids, and membrane preparation was performed, as previously described.6,12,21 Briefly, each mutation was introduced into the expression plasmid using the QuikChange Lightning kit (Agilent, Santa Clara, CA) according to the manufacturer’s instructions. The entire sequence of the inset was confirmed by sequencing as described earlier. Primers used are listed in Table 1. Enzyme Assays. Ethoxyresorufin O-deethylation, efavirenz 8hydroxylation, R-warfarin 7-hydroxylation, and midazolam 1′- and 4hydroxylation were measured as drug-metabolizing activities of CYP1A1, CYP2C9, CYP2C19, and CYP3A4 proteins, respectively, as described previously.9,21,24,25 Briefly, a typical mixture (0.25 mL) contained recombinant P450 protein (2.5−20 pmol) or liver microsomes (0.20 or 0.50 mg/mL), an NADPH-generating system (0.25 mM NADP+, 2.5 mM glucose 6-phosphate, and 0.25 unit/mL glucose-6-phosphate dehydrogenase), and substrate (0.050−10 μM ethoxyresorufin, 20−200 μM efavirenz, 1.0−200 μM R-warfarin, or 5.0−200 μM midazolam) in 50−100 mM potassium phosphate buffer (pH 7.4). The mixtures were incubated at 37 °C for 10−30 min within the linearity ranges, followed by adding 17% perchloric acid or acetonitrile to terminate reactions. The supernatants or extracts obtained by centrifugation at 10 000 × g for 5 min were analyzed by high-performance liquid chromatography with an ultraviolet or fluorescence detector. Kinetic analysis was done using nonlinear regression analysis (Prism 5.0, GraphPad Software, La Jolla, CA). Michaelis constant Km and S50 and substrate inhibition Ks values were determined using hyperbolic or substrate concentration-dependent velocity curves. Docking Simulation. Cynomolgus CYP2C9 and CYP3A4 primary sequences were aligned with crystal structures of human CYP2C9 (Protein Data Bank code IR90) and human CYP3A4 (code

EXPERIMENTAL PROCEDURES

Preparation of Genomes and Liver Microsomes. Whole blood samples were collected from 107 cynomolgus macaques (36 males and 71 females, 3−9 years of age, weighing 3−5 kg) including 40, 27, and 40 bred in Cambodia, China, and Indonesia, respectively, and four rhesus macaques (from China, 2 males and 2 females, 2−5 years of age, weighing 3−5 kg). Genomic DNA was prepared from these blood samples using the Puregene DNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Liver microsomes of the genotyped animals were prepared as described previously.7,22,23 The study was reviewed and approved by the 1374

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Chemical Research in Toxicology Table 2. Nonsynonymous Variants Identified in Cynomolgus and Rhesus CYP1A1 allele frequency cynomolgus variant

exon

nucleotide changeb

amino acid change

Cambodia

China

Indonesia

rhesus

c.217G > T c.230G > C c.260G > A c.266T > A c.286C > G c.299G > T c.363G > C c.473A > T c.541C > T c.617G > A c.682G > A c.722G > A c.749G > A c.1015G > A c.1067G > A c.1144G > A c.1324G > T c.1451C > G

1 1 1 1 1 1 1 1 1 1 1 1 1 3 4 4 6 6

GGGAC(G > T)TGCTG GATTC(G > C)CATTG GCTGA(G > A)CGGCC CGGCC(T > A)GGACA AGGCC(C > G)TGGTG GCAGG(G > T)CGATG AGCAT(G > C)TCCTT CTGCT(A > T)CCTGG CAGGG(C > T)CTGGG CCAGC(G > A)CTATG AGGTG(G > A)TTGGC TCTTC(G > A)CTACC TAATG(G > A)CTTCA CCAGG(G > A)TACAG ACGGC(G > A)CCCCC CCTTC(G > A)TCCCC ACAAG(G > T)TGCTA GCCAC(C > G)GGGTG

V73L R77P S87N L89Q L96V G100V M121I Y158F P181S R206H V228I R241H G250D V339I R356H V382I V442L P484R

1/80 0/80 0/80 0/80 0/80 0/80 0/80 2/80 1/80 0/80 0/80 0/80 0/80 0/80 0/80 8/80 N.D.a N.D.

0/50 0/50 0/50 0/50 0/50 0/50 0/50 0/50 0/50 1/50 0/50 0/50 0/50 0/54 0/54 7/54 0/12 0/12

0/76 0/76 0/76 1/76 1/76 1/76 1/76 0/76 0/76 0/76 1/76 1/76 5/76 8/80 1/80 0/80 2/16 1/16

0/8 1/8 1/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 3/8 0/8 0/8 0/8 0/8

site

SRS1

SRS5

a N.D., not determined. bNucleotide changes were detected by comparing with cynomolgus CYP1A1 cDNA sequence (GenBank accession no. D17575).

Figure 1. Ethoxyresurufin O-deethylation activities mediated by liver microsomes from cynomolgus macaques genotyped for CYP1A1 p.V382I. Individual cynomolgus macaque liver microsomes (0.20 mg protein/mL) were incubated with ethoxyresorufin (A, 0.10 μM; B, 1.0 μM; C, 10 μM) for 10 min for 12 wild-type, three heterozygous, and one homozygous cynomolgus macaques for CYP1A1 p.V382I. Individual (plots) and mean (bars) values of cynomolgus macaques were statistically analyzed: ∗P < 0.05, two-way ANOVA with Dunnett’s post-tests, compared with the wildtype group.

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5TE8), respectively, using MOE software (ver. 2018.0101, Computing Group, Montreal, Canada) for modeling of the three-dimensional structures. Models of the cynomolgus CYP2C9 p.A82V and p.H344R, CYP2C19 p.A490V, and CYP3A4 p.S437N were established by incorporating the mutations into their respective wild types; CYP2C9, CYP2C19, and CYP3A4 models as described previously.9 Prior to docking simulation, the energy of these cynomolgus CYP2C9, CYP2C19, and CYP3A4 models was minimized using the Amber 10 force field. Docking simulation was carried out for efavirenz, Rwarfarin, and midazolam binding to cynomolgus CYP2C9, CYP2C19, and CYP3A4 models, respectively, in the ASE Dock software (Ryoka Systems, Tokyo, Japan). Solutions were generated for each docking experiment and ranked according to the total interaction energy (U value, kcal/mol) as described previously.26 Statistical Analysis. To compare catalytic activities of the wild types and variants in liver microsomes, two-way ANOVA with Dunnett’s post-tests was performed using Prism software (GraphPad Software, La Jolla, CA).

RESULTS Resequencing of CYP1A1 and Analysis of p.V382I. To identify genetic variants, CYP1A1 was resequenced in 107 cynomolgus macaques and four rhesus macaques. The analysis identified a total of 18 nonsynonymous variants (Table 1), among which p.M121I and p.V382I were in substrate recognition sites (SRS), the important regions for protein function.27 One of the 18 variants identified was found in both cynomolgus and rhesus macaques. Fifteen of the 16 variants found in cynomolgus macaques were unique to the animals bred in Cambodia, China, or Indonesia (Table 2). The variant c.1468G > A (p.V490M) was found in all the animals analyzed, likely because this is a rare allele contained in cDNA (D17575) used as a reference sequence, and thus was not counted as a nonsynonymous variant. M121I was excluded from further analysis due to unavailability of the samples for the metabolic analysis. Influence of the CYP1A1 variant p.V382I on ethoxyresorufin O-deethylation was assessed using liver microsomes of the 1375

DOI: 10.1021/acs.chemrestox.8b00257 Chem. Res. Toxicol. 2018, 31, 1373−1381

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Chemical Research in Toxicology genotyped cynomolgus macaques. By comparison of liver microsomes from 12 wild-type animals, three heterozygotes, and one homozygote, the heterozygotes showed significantly (p < 0.05) reduced catalytic activities compared with the wild types (Figure 1). However, analysis of recombinant proteins showed that catalytic activity was similar between the variant and the wild-type protein in ethoxyresorufin O-deethylation (Figure 2). Similarly, kinetic analysis did not show a difference

Figure 3. Efavirenz 8-hydroxylation activities mediated by liver microsomes from cynomolgus macaques genotyped for CYP2C9 p.A82V (A) and p.H344R (B). Individual liver microsomes (0.50 mg protein/mL) of cynomolgus macaques were incubated with efavirenz (open, 20 μM; and closed, 200 μM) at 37 °C for 30 min. Individual (plots) and mean values with SD values (bars) of cynomolgus macaques were statistically analyzed: ∗P < 0.05, two-way ANOVA with Dunnett’s post-tests, compared with the wild-type group.

wild types (Figure 3B). The heterozygotes were not found in the cynomolgus macaques analyzed. Analysis of recombinant proteins showed reduction in efavirenz 8-hydroxylation for p.A82V and p.H344R compared with the wild type at a substrate concentration of 10 μM (Figure 4), but inhibition of enzyme activity for p.A82V and p.H344R was commonly observed at high substrate concentrations (Figure 4). By kinetic analysis of efavirenz 8-hydroxylation using recombinant proteins, p.A82V and p.H344R showed substantially lower Ks than the wild-type protein (Figure 4, Table 3), supporting the results with liver microsomes. The results suggest the importance of p.A82V and p.H344R on the catalytic function of CYP2C9 protein. p.A82V and p.H344R Endow CYP2C19 Reduced Catalytic Activity. Influence of the CYP2C19 variant p.A490V on R-warfarin 7-hydroxylation was assessed using liver microsomes of the cynomolgus macaques genotyped for CYP2C19 p.[F100N; A103V; I112L]. By comparing liver microsomes of 10 wild-type, three heterozygous, and five homozygous animals for CYP2C19 variant p.A490V, the homozygotes showed significantly (p < 0.05) reduced catalytic

Figure 2. Ethoxyresurufin O-deethylation activities of CYP1A1 wildtype and p.V382I variant. Ethoxyresurufin O-deethylation activities were measured using heterologously expressed proteins (the wild type and p.V382I) at substrate concentrations of 0.20 (closed bars) and 2.0 (open bars) μM as described in the Experimental Procedures. Values are averages of triplicate determinations.

in kinetic parameters between the variant and the wild-type protein (Table 3). The results suggest that p.V382I does not influence CYP1A1 activity. p.A82V and p.H344R Account for Reduced Activity of CYP2C9. Effects of the CYP2C9 variants p.A82V and p.H344R on efavirenz 8-hydroxylation were assessed using liver microsomes of the genotyped cynomolgus macaques. By comparison of liver microsomes from 18 wild-type animals, three heterozygotes, and one homozygote, the heterozygotes for p.A82V showed significantly (p < 0.05) reduced catalytic activities compared with the wild types (Figure 3A). Similarly, for liver microsomes from 18 wild-type animals and four homozygotes, the homozygotes for p.H344R showed significantly (p < 0.05) reduced catalytic activities compared with the

Table 3. Kinetic Parameters of Drug Oxidation Activities of Cynomolgus CYP1A1, CYP2C9, CYP2C19, and CYP3A4 and Their Variantsa variant CYP1A1 wild-type CYP1A1 V382I CYP2C9 wild-type CYP2C9 A82V CYP2C9 H344R CYP2C19 wild-type CYP2C19 A490V CYP3A4 wild-type CYP3A4 S437N

Km or S50 μM 0.51 0.18 97 40 14 50 58 5.3 9.6 1.1 12

± ± ± ± ± ± ± ± ± ± ±

0.09 0.05 31 23 7 13 15 1.0 (1′-) 1.8 (4-) 0.6 (1′-) 2 (4-)

KS μM

88 ± 30 28 ± 16 31 ± 15

220 ± 47 (1′-) 290 ± 85 (1′-)

Vmax nmol/min/nmol of P450 7.1 6.5 6.5 7.7 2.6 3.7 1.1 26 12 29 21

± ± ± ± ± ± ± ± ± ± ±

0.3 0.4 1.6 3.3 0.8 0.4 0.1 2 (1′-) 1 (4-) 2 (1′-) 1 (4-)

Hill coefficient, n

1.2 ± 0.2 1.2 ± 0.2

Vmax/Km mL/min/nmol of P450 14 36 0.067 0.19 0.19 74 19 4.9 1.3 26 1.8

a ́ and 4- (4-) hydroxylation activities Ethoxyresorufin O-deethylation, efavirenz 8-hydroxylation, R-warfarin 7-hydroxylation, and midazolam 1'-́ (1'-) of CYP1A1, CYP2C9, CYP2C19, and CYP3A4, respectively, were determined as described in the Experimental Procedures. Kinetic parameters determined by nonlinear regression analyses by employing Michaelis−Menten equation, v = Vmax × [S]/(Km + [S]), are shown as means ± standard errors. For CYP2C9 and CYP3A4, kinetic parameters were determined using the equations, v = Vmax × [S]/(Km S+ [S] + [S]2/Ks) for substrate inhibition and v = Vmax × [S]n/(Sn50 + [S]n) for substrate activation, respectively.

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Chemical Research in Toxicology

Figure 4. Efavirenz 8-hydroxylation activities of CYP2C9 wild-type, p.A82V, and p.H344R variants. Efavirenz 8-hydroxylation activities were measured using heterologously expressed proteins (the wild type, open; p.A82V, triangles; and p.H344R, squares) at substrate concentrations of 3.4−380 μM as described in the Experimental Procedures. Values are averages of triplicate determinations.

Figure 5. R-Warfarin 7-hydroxylation activities mediated by liver microsomes from cynomolgus macaques genotyped for CYP2C19 p.A490V. R-Warfarin 7-hydroxylation activities were measured in liver microsomes of cynomolgus macaques genotyped for CYP2C19 p.A490V in the background of CYP2C19 p.[F100N; A103V; I112L] including the wild types (A and D), heterozygotes (B and E), and homozygotes (C and F). Individual cynomolgus macaque liver microsomes (0.20 mg protein/mL) were incubated with Rwarfarin at 1.0 μM (A, B, and C) or 10 μM (D, E, and F) for 15 min. Individual (plots) and mean (bars) values of cynomolgus macaques were statistically analyzed: ∗P < 0.05, two-way ANOVA with Dunnett’s post-tests, compared with the wild-type group in each background of CYP2C19 p.[F100N; A103V; I112L].

activities compared with the wild types in the wild-type background of p.[F100N; A103V; I112L] (Figure 5). Analysis of recombinant proteins showed substantial reduction in Rwarfarin 7-hydroxylation for the variant protein p.A490V compared with the wild-type protein (Figure 6). By kinetic analysis of R-warfarin 7-hydroxylation using recombinant proteins, the variant protein (p.A490V) showed substantially lower Vmax and Vmax/Km than the wild-type protein (Table 3), which supported the result of the liver microsomes. The results suggest the importance of p.A490V for the catalytic function of CYP2C19 protein. CYP3A4 p.S437N Is Involved in Reduction of Midazolam 1′-Hydroxylation. Effects of the CYP3A4 variant p.S437N on enzyme activities were assessed using midazolam as substrate in liver microsomes of the genotyped cynomolgus macaques. By comparison of liver microsomes from five wild-type animals, three heterozygotes, and two homozygotes, the heterozygotes and homozygotes showed increased catalytic activities compared with the wild types in midazolam 1′- and 4-hydroxylation, of which the difference between homozygotes and the wild type reached statistical significance (p < 0.05) in midazolam 1′-hydroxylation (Figure 7). Analysis of recombinant proteins showed an increase in midazolam 1′- and 4-hydroxylation for the variant protein p.S437N compared with the wild-type protein (Figure 8). Kinetic analysis of midazolam 1′- and 4-hydroxylation by recombinant CYP3A4 p.S437N exhibited the difference between the mutant and the wild-type enzyme in the values of Vmax/Km ratio by a factor of 5.3 and 1.4, respectively (Table 3). This observation supports the results obtained with liver microsomes. The results suggest the importance of p.S437N for catalytic function of CYP3A4 protein. Docking Simulation of Cynomolgus CYP2C9, CYP2C19, and CYP3A4 Variants. Molecular interactions of efavirenz, R-warfarin, and midazolam, respectively, were investigated with CYP2C9, CYP2C19, and CYP3A4 variants

Figure 6. R-Warfarin 7-hydroxylation activities of the CYP2C19 wild type and p.A490V variant.R-warfarin 7-hydroxylation was measured using heterologously expressed proteins (the wild type and p.A490V) at substrate concentrations of 1, 10, and 50 μM as described in the Experimental Procedures. Values are averages of triplicate determinations.

(Figure 9). The ligand-interaction energy (U value) of interaction of efavirenz with the active site of CYP2C9 was found to be −23.0 kcal/mol (Figure 9A). Although the ligandinteraction energy of the interaction of efavirenz with CYP2C9 p.A62V was calculated to be −24.1, that of CYP2C9 p.H344R was low (−14.2), which suggested an unstable interaction of efavirenz and the iron center of the CYP2C9 p.H344R variant. The ligand-interaction energy of interaction of the first 1377

DOI: 10.1021/acs.chemrestox.8b00257 Chem. Res. Toxicol. 2018, 31, 1373−1381

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Chemical Research in Toxicology

Figure 7. Midazolam 1′- and 4-hydroxylation activities mediated by liver microsomes from cynomolgus macaques genotyped for the CYP3A4 p.S437N. Individual cynomolgus macaque liver microsomes (0.20 mg protein/mL) were incubated with 100 μM midazolam for 15 min. Individual (plots) and mean (bars) values of cynomolgus macaques were statistically analyzed: ∗P < 0.05, two-way ANOVA with Dunnett’s post-tests, compared with the wild-type group.

Figure 8. Midazolam 1′- and 4-hydroxylation activities of CYP3A4 wild-type and p.S437N variant. Midazolam 1′- (circles) and 4(squares) hydroxylation activities were measured using heterologously expressed CYP3A4 proteins (the wild type, open symbols; and p.S437N, closed symbols) at six points of substrate concentration ranging 5.0−150 μM as described in the Experimental Procedures. Kinetic parameters determined by nonlinear regression analysis are shown in Table 3. Each point indicates mean of duplicate determinations.

Figure 9. Molecular docking of cynomolgus CYP2C9, CYP2C19, and CYP3A4 variants. Molecular docking was performed as described in Experimental Procedures to assess interactions of efavirenz (A), Rwarfarin (B), and midazolam (C) with the iron center of cynomolgus CYP2C9, CYP2C19, and CYP3A4 (and their variants), respectively.

for these animals allowed comparisons of drug-metabolizing activity between the wild-type and mutant animals. Analysis of CYP1A1 variants found a total of 18 nonsynonymous variants, including p.M121I and p.V382I in SRSs, important regions for protein function (Table 2). Most of the variants were unevenly distributed in cynomolgus macaques bred in Cambodia, China, and Indonesia, as has been reported for other cynomolgus P450s.16,17,21,28−32 p.V382I was only the variant shared by cynomolgus macaques bred in Cambodia and China, possibly because the founder animals of the latter were originated from Indochina (including Cambodia).33 Of the two CYP1A1 nonsynonymous variants analyzed (p.M121I and p.V382I), p.V382I was associated with reduced drug-metabolizing activity in liver microsomes (Figure 1). However, the variant protein (p.V382I) showed drugmetabolizing activity and kinetics similar to the wild-type protein (Figure 2, Table 3). In human CYP1A1, substitution of residue 382 resulted in reduced catalytic activity of the enzyme, depending on the substrate, and exchanging this residue between CYP1A1 and CYP1A2 interconverts substrate preference of the enzymes.34,35 This is consistent with differences between these enzymes in volume near residue 382, a key residue for the active site.36 Similarly, an amino acid change at residue 382 increases catalytic efficiency of human

molecule of R-warfarin with the active site of CYP2C19 and CYP2C19 p.A490V was found to be −31.1 and −28.2, respectively (Figure 9B). As reported previously in terms of homotropic cooperativity of the wild-type CYP2C19,9 although the ligand-interaction energy of interaction of the second molecule of R-warfarin with CYP2C19 was calculated to be −47.0, that of CYP2C19 p.A490V was low (+19.9), suggesting impossible interaction of the second molecule of Rwarfarin and the iron center of CYP2C19 p.A490V variant. On the other hand, the ligand-interaction energy of midazolam with the active site of CYP3A4 was found to be −23.0 (Figure 9C). However, the ligand-interaction energy of midazolam with CYP3A4 p.S437N was calculated to be −59.2, indicating a stable interaction of midazolam and the iron center of CYP3A4 p.S437N.

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DISCUSSION Numerous genetic variants of CYP1A1, CYP2C9, CYP2C19, and CYP3A4 have been identified in cynomolgus macaques;13,16,17 however, most genetic variants remain to be functionally characterized, but preparation of liver microsomes 1378

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Chemical Research in Toxicology CYP1A2 in phenacetin metabolism.37 Therefore, residue 382 might also play an important role in catalytic activity and substrate preference of cynomolgus CYP1A1. It is of great interest to investigate p.V382I using different substrates. Cynomolgus CYP2C9 variants p.A82V and p.H344R were associated with lower efavirenz 8-hydroxylation in liver microsomes (Figure 3), which is supported by kinetic analysis using recombinant proteins (Table 3). Near residue 82, the nonsynonymous variant of human CYP2C9 at residue 78 (2C9*31) showed reduced catalytic activity compared with the wild type when analyzing recombinant protein using bosentan, carvedilol, diclofenac, fluoxetine, glimepiride, losartan, mestranol, phenytoin, propofol, tolbutamide, and S-warfarin as substrates.38−48 Similarly, in the same studies, near residue 344, the human CYP2C9 variant at residue 343 (2C9*54) showed lower catalytic activity with bosentan, carvedilol, diclofenac, fluoxetine, losartan, mestranol, and tolbutamide, and higher catalytic activity with phenytoin compared with the wild-type protein. By docking simulation, a low ligandinteraction energy for efavirenz with CYP2C9 p.H344R suggested an unstable interaction between efavirenz and the iron center of the variant protein (Figure 9A), supporting results of the enzyme assays and kinetic analysis. CYP2C19 variant p.A490V was associated with lower R-warfarin 7hydroxylation in liver microsomes (Figure 5), supported by kinetic analysis using recombinant proteins (Table 3). However, an effect of p.A490V was not evident in the background of CYP2C19 p.[F100N; A103V; I112L] (Figure 5), indicating that p.[F100N; A103V; I112L] exerts a larger effect on catalytic activity than p.A490V. In humans, CYP2C9*32 (p.V490F) was predicted to exert possible functional change in modeling. 49 Near residue 490, CYP2C9*12 (p.R489S) showed moderately reduced catalytic activity with tolbutamide compared with the wild-type when analyzing recombinant proteins.50 By docking simulation, a low ligand-interaction energy for interaction of the second molecule of R-warfarin with CYP2C19 p.A490V suggested impossible interaction between the second molecule of Rwarfarin and the iron center of the variant protein (Figure 9B), supporting the enzyme assays and kinetic analysis results. CYP3A4 variant p.S437N was associated with higher midazolam 1′-hydroxylation in liver microsomes (Figure 7), supported by kinetic analysis using recombinant proteins (Table 3). Ser437 is located in the heme-binding region of the enzyme, potentially important for enzyme function. By docking simulation, the higher ligand-interaction energy of midazolam with CYP3A4 p.S437N suggested a stable interaction of midazolam with the iron center of the enzyme, supporting the enzyme assays and kinetic analysis results. A previous study did not show a substantial difference of S437N compared with the wild type in midazolam 1′-hydroxylation,21 possibly due to determination at only one substrate concentration (100 μM) as shown in Figure 8 of the present study. CYP1A1 variant (p.V382I), CYP2C9 variants (p.A82V and p.H344R), CYP2C19 variant (p.A490V), and CYP3A4 variant (p.S437N) are distributed unevenly in different groups of cynomolgus macaques and rhesus macaques. CYP1A1 variant (p.V382I) was found in cynomolgus macaques bred in Cambodia and China but not in Indonesia (Table 2). CYP2C9 variants (p.A82V and p.H344R) are prevalent in cynomolgus macaques bred in Indochina and rhesus macaques but not in Indonesian cynomolgus macaques.16 CYP2C19 variant (p.A490V) is prevalent in all three groups of

cynomolgus macaques, and rhesus macaques, but its frequency is much higher in Indonesian cynomolgus macaques and rhesus macaques, most of which possess this variant.17 CYP3A4 variant (p.S437N) was found in cynomolgus macaques bred in Indochina and Indonesia but not in rhesus macaques.21 Similarly, CYP2C9 variant p.I112L, previously determined to be functionally relevant, was found in rhesus macaques, and cynomolgus macaques bred in Indochina, but not Indonesia.16 Another functional variant CYP2C19 p. [F100N; A103V; I112L] was found in far more frequently in cynomolgus macaques bred in Indochina, compared with cynomolgus macaques bred in Indonesia and rhesus macaques.17 Therefore, these variants might account for the variable catalytic activities in cynomolgus macaques bred in Indochina and Indonesia, and rhesus macaques. An interindividual variability of CYP2C-dependent metabolism has been noted; urinary excretion of 4-hydroxymephenytoin varied in 64 cynomolgus macaques, which could be divided into extensive and poor metabolizers.51 Such a variable metabolism might be accounted for by genetic variants of cynomolgus CYP2C9 or CYP2C19 analyzed in this study because these enzymes catalyze S-mephenytoin 4′-hydroxylation.6,11,52 Moreover, the CYP2C9 p.I112L and CYP2C19 p. [F100N; A103V; I112L] relevant to the enzyme function are responsible for metabolism variations.17−19 Because numerous variants of CYP2C9 and CYP2C19 remain to be characterized, further study of those variants might reveal more variants relevant to the enzyme function. For such important variants, a genotyping tool could be developed to aid drug metabolism studies using cynomolgus macaques, as was done for the CYP2C9 and CYP2C19 variants.20,53 It is established that multiple forms of cynomolgus P450 enzymes have generally similar substrate recognition functionality to human P450 enzymes.51 Genetic/acquired individual differences in cynomolgus P450 enzymes involved in drug oxidation could be associated with toxicological effects. Therefore, genotyping the animals for drug-metabolizing P450 enzyme genes before and after preclinical studies might be beneficial for pharmaceutical industry to obtain less biased data by reduced interindividual variations and to understand the reasons for genotype-oriented biased data. In conclusion, 18 nonsynonymous variants of CYP1A1 were found in cynomolgus and rhesus macaques. CYP1A1 (p.V382I), CYP2C9 (p.A82V, p.H344R), and CYP2C19 (p.A490V) variants were significantly associated with reduced drug-metabolizing activities of the enzymes in the genotyped liver microsomes, of which the CYP2C9 and CYP2C19 variants also influenced kinetic parameters. In contrast, the CYP3A4 variant S437N was significantly associated with increased drug-metabolizing activity. These results suggest the functional importance of CYP2C9 variants (p.A82V and p.H344R), CYP2C19 variant (p.A490V), CYP3A4 variant (p.437N), and potentially CYP1A1 (p.V382I), which might be important variants to be considered in toxicological drug metabolism studies using cynomolgus macaques. This work represents another step toward establishing cynomolgus macaque as a valuable model for studying the roles of P450 polymorphisms in interindividual variations in drug metabolism and toxicity. 1379

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +81-73-483-8881. Fax: + 81-73-483-7377 *E-mail: [email protected]. Phone: +81-42-7211406. Fax: +81-42-721-1406. ORCID

Hiroshi Yamazaki: 0000-0002-1068-4261 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors greatly thank Masahiro Utoh for the support to this work and Lance Bell for the advice on English writing. REFERENCES

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