Short Communication Genetic Variants of CYP3A4 and CYP3A5 in Cynomolgus and Rhesus Macaques

Cynomolgus and rhesus macaques are frequently used in preclinical trials due to their close evolutionary relationships to humans. We conducted an initial screening for genetic variants in cynomolgus and rhesus macaque genes orthologous to human CYP3A4 and CYP3A5. Genetic screening of 78 Indochinese and Indonesian cynomolgus macaques and 34 Chinese rhesus macaques revealed a combined total of 42 CYP3A4 genetic variants, including 12 nonsynonymous variants, and 34 CYP3A5 genetic variants, including nine nonsynonymous variants. Four of these nonsynonymous variants were located at substrate recognition sites or the hemebinding region, domains essential for protein function, including c.886G>A (V296M) and c.1310G>A (S437N) in CYP3A4 and c.1437C>G (N479K) and c.1310G>C (T437S) in CYP3A5. The mutant proteins of these genetic variants were expressed in Escherichia coli and purified. Metabolic activity of these proteins measured using midazolam and nifedipine as substrates showed that none of these protein variants substantially influences the drugmetabolizing capacity of CYP3A4 or CYP3A5 protein. In Indonesian cynomolgus macaques, we also found IVS3 1delG in CYP3A4 and c.625A>T in CYP3A5, with which an intact protein cannot be produced due to a frameshift generated. Screening additional genomes revealed that two of 239 animals and three of 258 animals were heterozygous for IVS3 1delG of CYP3A4 and c.625A>T of CYP3A5, respectively. Some genetic variants were unevenly distributed between Indochinese and Indonesian cynomolgus macaques and between cynomolgus and rhesus macaques. Information on genetic diversity of macaque CYP3A4 and CYP3A5 presented here could be useful for successful drug metabolism studies conducted in macaques. Cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) macaques have been used to predict the metabolic fate of drugs in humans due to their evolutionary closeness to humans. As with humans, macaques have a diverse genetic background as evidenced by numerous genetic polymorphisms that have been reported (Ferguson et al., 2007; Hernandez et al., 2007; Street et al., 2007). In cynomolgus macaques, interanimal differences have been noted in drug metabolism by in vivo analysis using dextromethorphan and S-mephenytoin as probe substrates (Jacqz et al., 1988), which could be, in some part, attributable to genetic variability of drug-metabolizing enzymes, because genetic variants of cytochromes P450 (P450s) such as CYP2C76 have been identified (Uno et al., 2009). Human CYP3As are considered the major drug-metabolizing cytochrome P450 (P450) subfamily, comprised of CYP3A4, CYP3A5, CYP3A7, and CYP3A43 (Gellner et al., 2001). In humans, CYP3As account for more than half of the total P450 content in human liver (Thummel and Wilkinson, 1998) and metabolize more than half of all prescription drugs, such as nifedipine, midazolam, and testosterone (Thummel and Wilkinson, 1998; Evans and Relling, 1999). Numerous interindividual differences in drug-metabolizing capability mediated by CYP3A4 and CYP3A5 have been reported in humans, some of which are partially caused by genetic polymorphisms (see http:// www.imm.ki.se/CYPalleles/). For example, hepatic CYP3A5 protein is present at detectable levels in 10 to 30% of whites and 60% of African Americans (Hustert et al., 2001; Kuehl et al., 2001; Lin et al., 2002). This interindividual variability in CYP3A5 protein expression is highly correlated with CYP3A5*3, a defective allele, because the aberrant splicing imposed by the mutant allele gives rise to a nonfunctional protein (Kuehl et al., 2001). Identification and characterization of such genetic variants are essential for understanding drugmetabolizing properties of CYP3A4 and CYP3A5 enzymes. Several groups, including ours, have identified cDNAs highly homologous to human CYP3A4 or CYP3A5 cDNA in cynomolgus and rhesus macaques (Komori et al., 1992; Carr et al., 2006; Uno et al., 2007). Between these macaque species and humans, sequence identity of CYP3A4 and CYP3A5 is approximately 95 and 94% in cDNA and 93 and 91% in amino acid sequence, respectively. After consulting with the P450 Nomenclature Committee (http://drnelson.utmem.edu/ cytochromeP450), in this article, we designate cynomolgus CYP3A8 and rhesus CYP3A64, both orthologous to human CYP3A4, as CYP3A4, and rhesus CYP3A66, orthologous to human CYP3A5, as CYP3A5. Rhesus CYP3A4 possesses metabolic capabilities and induction properties similar to human CYP3A4 (Carr et al., 2006). Cynomolgus CYP3A4 and CYP3A5 exhibited testosterone 6 -hydroxylation (Uno et al., 2007), similar to their human P450 counterparts, indicating similar drug-metabolizing properties of macaque and human CYP3A. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.109.029710. □S The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. ABBREVIATIONS: P450, cytochrome P450; PCR, polymerase chain reaction; UTR, untranslated region; RT, reverse transcription; SRS; substrate recognition site. 0090-9556/10/3802-209–214$20.00 DRUG METABOLISM AND DISPOSITION Vol. 38, No. 2 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 29710/3553968 DMD 38:209–214, 2010 Printed in U.S.A. 209 http://dmd.aspetjournals.org/content/suppl/2009/11/12/dmd.109.029710.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from In this study, we attempted to comprehensively identify and characterize genetic variants for CYP3A4 and CYP3A5 in cynomolgus and rhesus macaques. Genome samples from Indochinese and Indonesian cynomolgus macaques and from Chinese rhesus macaques were used to elucidate the regional and lineage differences in allele frequency of the identified variants. Metabolic activities were measured by using the human CYP3A substrates, midazolam and nifedipine, to characterize potentially important variants. Materials and Methods Animals and Genomic DNA Extraction. Genomic DNA was prepared from whole blood samples by using the PUREGENE DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer’s instructions. The blood samples used in this study were collected from 296 cynomolgus macaques (38 from Indochina and 258 from Indonesia) and 34 rhesus macaques (from China). This study was reviewed and approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd. DNA Sequencing. Genetic variants were identified by polymerase chain reaction (PCR) amplification and sequencing of all CYP3A4 and CYP3A5 exons, including the 5 -untranslated region (UTR), the coding region, and the 3 UTR, using genome samples from 78 cynomolgus macaques (38 from Indochina, 40 from Indonesia) and 34 rhesus macaques. For IVS3 1delG of CYP3A4 and c.625A T of CYP3A5, 199 and 218 Indonesian cynomolgus macaques were also genotyped, respectively. A 20l PCR reaction contained 1 ng of genomic DNA, 5 pmol of forward and reverse primers, and 1 unit of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). The amplification was performed in a thermal cycler (Applied Biosystems) with an initial denaturation at 95°C for 10 min and 30 cycles of 20 s at 95°C, 30 s at 55°C, and 1 min at 72°C, followed by a final extension step of 10 min at 72°C. Sequencing was performed by using an ABI PRISM BigDye Terminator version 3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems), followed by electrophoresis on an ABI PRISM 3730 DNA Analyzer (Applied Biosystems). The primers used for PCR and sequencing are listed in Supplemental Material Table 1 (supplemental tables are available at http://dmd. aspet.journals.org). Sequence data were analyzed by using DNASIS Pro (Hitachi Software, Tokyo, Japan). Genetic variants of CYP3A4 were identified by comparison with CYP3A4 cDNA sequences of cynomolgus macaque (GenBank accession number S53047) and rhesus macaque (GenBank accession number NM_017460), whereas CYP3A5 variants were identified by comparison with CYP3A5 cDNA sequences of cynomolgus macaque (GenBank accession number DQ074795) and rhesus macaque (GenBank accession number NM_001040219). Preparation of Expression Plasmids and Protein Expression. For functional characterization of the genetic variants, expression plasmids were prepared as described previously (Iwata et al., 1998; Uno et al., 2006). The mutation was introduced into the expression plasmid containing cynomolgus CYP3A4 cDNA (GenBank accession number S53047) by using the QuikChang XL II kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. The primer pairs used were as follows: 5 -ATGGAAAAGTGTGGGCTTTTATGATGGTC-3 and 5 -GACCATCATAAAAGCCCACACTTTTCCAT-3 for IVS3 1delG; 5 GTCTGATCTGGAGCTCATGGCCCAATCAATTATCTT-3 and 5 -AAGATAATTGATTGGGCCATGAGCTCCAGATCAGAC-3 for c.886G A; and 5 -CCTTACATATACACGCCCTTTGGAAATGGACCCAGAAACTGC-3 and 5 -GCAGTTTCTGGGTCCATTTCCAAAGGGCGTGTATATGTAAGG-3 for c.1310G A. Likewise, the mutation was introduced into the expression plasmid containing cynomolgus CYP3A5 cDNA (GenBank accession number DQ074795) by using the following primers: 5 -GGAAAGCGTTAAGTAGTTCCTAAAATTTG-3 and 5 -CAAATTTTAGGAACTACTTAACGCTTTCC-3 for c.625A T; 5 CATATACACACCCTTTGGAACTGGACCCAGAAACTGCATTGG-3 and 5 CCAATGCAGTTTCTGGGTCCAGTTCCAAAGGGTGTGTATATG-3 for c.1310G C; and 5 -TGTAGATCCCCTTGAAATTAGGCAAGCAAGGCCTTCTTCAATCAG-3 and 5 -CTGATTGAAGAAGGCCTTGCTTGCCTAATTTCAAGGGGATCTACA-3 for c.1437C G. The entire sequence of the cDNA insert was confirmed by sequencing. The PCR products were cloned into a pCW vector containing human NADPH-P450 reductase cDNA. The plasmids were used for protein expression in Escherichia coli performed according to the method of Iwata et al. (1998). Membrane fractions were prepared from bacterial cells as described previously (Sandhu et al., 1994). Measurement of P450 protein content in the membrane preparations was determined spectrally, according to the method reported by Omura and Sato (1964). The yield of NADPH-P450 reductase was estimated as described previously (Phillips and Langdon, 1962). Measurement of Enzyme Activity. Midazolam hydroxylation and nifedipine oxidation were determined as described previously (Yamazaki et al., 1999). In brief, a typical incubation mixture (0.25 ml) contained recombinant CYP3A4 or CYP3A5 proteins (5 pmol), 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 (100 M midazolam or 100 M nifedipine) in 0.10 M potassium phosphate buffer (pH 7.4). Midazolam reactions were incubated at 37°C for 10 min and terminated by addition of 0.25 ml of ice-cold acetonitrile. After centrifugation at 2000g for 10 min, the supernatant was analyzed by high-performance liquid chromatography with an ultraviolet detector. Nifedipine reactions were incubated at 37°C for 5 min and terminated by addition of 1.5 ml of CH2Cl2, 0.2 M NaCl, and 0.1 M Na2CO3. Organic phases were evaporated under a stream of nitrogen gas, and product formation was determined by high-performance liquid chromatography with an ultraviolet detector. RNA Preparation and Reverse Transcription-PCR. To examine transcripts of macaque CYP3A4 and CYP3A5, total RNA was extracted from livers of seven cynomolgus macaques and reverse transcription (RT)-PCR was carried out as described previously (Uno et al., 2006). In brief, the first-strand cDNA was generated in a mixture containing 1 g of total RNA, oligo(dT), and SuperScript II RT reverse transcriptase (Invitrogen, Carlsbad, CA) at 37°C for 1 h. One twenty-fifth of this reaction was used for the subsequent PCR that was carried out by using AccuPrime Taq DNA polymerase (Invitrogen) according to the manufacturer’s protocol. PCR conditions include an initial denaturation at 94°C for 2 min and 35 cycles of 94°C for 30 s, 65°C for 30 s, and 68°C for 2 min, followed by a final extension at 68°C for 5 min. The primers used were as follows: 5 -CACACACAGCCCAGCAAAC-3 and 5 -CCGTCTTCATTTCAGGGTTC-3 for CYP3A4; and 5 -CGATGGACCTCATCCCAAAT-3 and 5 -CTCATTCTCCACTTAGGGTTCC-3 for CYP3A5. The amplified cDNAs were cloned into vectors, and the inserts were sequenced as described earlier. Results and Discussion To identify genetic variants, all CYP3A4 and CYP3A5 exons including the 5 UTR, the coding region, and the 3 UTR, were amplified and sequenced by using genome samples from 78 cynomolgus macaques (38 from Indochina and 40 from Indonesia) and 34 rhesus macaques. A total of 42 variants was identified in CYP3A4 exons, including five in the 5 UTR, 27 in the coding region, and 10 in the 3 UTR (Table 1). c.886G A in substrate recognition site (SRS) 4 and c.1310G A in the heme-binding region were among 12 nonsynonymous variants found. A total of 34 variants was found in CYP3A5 exons, including five in the 5 UTR and 29 in the coding region (Table 2). No variants were found in the 3 UTR. Nine of the variants found were nonsynonymous including c.1310G C in the heme-binding region and c.1437C G in SRS6. c.387T A and c.1310G C in CYP3A5 were found to be major alleles in the animals analyzed, probably reflecting genetic differences between the animals used to identify CYP3A5 cDNA and those used in this study. The frequency of all genotypes was in Hardy-Weinberg equilibrium within each population ( 2 test). A comparison of allele frequency between cynomolgus and rhesus macaques revealed that 10 (23.8%) of total 42 variants for CYP3A4 and 6 (17.6%) of total 34 variants for CYP3A5 were shared by both macaque lineages. Likewise, a recent study indicated that approximately half of genetic polymorphisms were shared between the two lineages (Street et al., 2007), suggesting that genetic variants identified in either macaque lineage can be partly used for analysis of the other. On the other hand, 24 and 8 of CYP3A4 alleles and 22 and 6 of CYP3A5 alleles were unique to cynomolgus and rhesus macaques, respectively. Among the variants found only in cynomolgus macaques, 10 CYP3A4 variants, including three nonsynonymous vari210 UNO ET AL. at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from