Abstract
The cynomolgus monkey is used in drug metabolism studies, because of its evolutionary closeness to human, including cytochrome P450. Cynomolgus monkey CYP2D17, highly homologous to human CYP2D6, has been identified and characterized. Here, we report characterization of another CYP2D, CYP2D44, identified in cynomolgus monkey liver. The CYP2D44 cDNA contained an open reading frame of 497 amino acids sharing high sequence identity (87–93%) with other primate CYP2Ds. CYP2D44 mRNA was predominantly expressed in liver, similar to CYP2D17 mRNA. CYP2D17 and CYP2D44 form a gene cluster in the genome, similar to human CYP2Ds. Metabolic assays of the CYP2D17 and CYP2D44 proteins heterologously expressed in Escherichia coli indicated that CYP2D44 metabolized human CYP2D6 substrates, bufuralol and dextromethorphan (bufuralol 1′-hydroxylation and dextromethorphan O-demethylation) but to a lesser extent than CYP2D17. Kinetic analysis of dextromethorphan metabolism indicated that the apparent Km and Vmax of CYP2D17 and CYP2D44 catalyzed O-demethylation were similar, and, the Vmax values of CYP2D17 and CYP2D44 catalyzed N-demethylation (which human CYP2D6 catalyzes much less effectively) were similar, but the apparent Km of the CYP2D44 reaction was higher. Western blot analysis showed that CYP2D proteins were expressed in cynomolgus and rhesus monkey liver as well as in human and marmoset liver. Similar to CYP2D6, CYP2D44 copy number varied among the eight cynomolgus monkeys and four rhesus monkeys used in this study. These results indicated that CYP2D44, together with CYP2D17, had functional characteristics similar to those of human CYP2D6 but measurably differed in dextromethorphan N-demethylation, suggesting its importance for CYP2D-dependent drug metabolism in macaque.
Introduction
Cytochrome P450 (P450) 2D6 is a drug-metabolizing enzyme in humans, metabolizing approximately 20 to 25% of all prescribed drugs, such as antiarrhythmics, β-blockers, neuroleptics, and antidepressants (Ingelman-Sundberg, 2005). CYP2D6 is a highly variable drug-metabolizing enzyme; the genetic polymorphisms of CYP2D6, including CYP2D6*5 with the entire gene deleted, influence the pharmacokinetics of more than half of all drugs metabolized by CYP2D6 (Gaedigk et al., 1991; Steen et al., 1995). Therefore, CYP2D6 is one of the most clinically important drug-metabolizing enzymes.
Macaques include cynomolgus monkey (Macaca fascicularis), rhesus monkey (Macaca mulatta), and Japanese monkey (Macaca fuscata). The former two are used routinely in preclinical studies. Cynomolgus monkey CYP2D17 (Mankowski et al., 1999) and Japanese monkey CYP2D29 (Hichiya et al., 2002) have been identified and characterized in macaques. Rhesus monkey CYP2D42 cDNA, nearly 98% homologous to cynomolgus monkey CYP2D17 cDNA, has been isolated and its sequence can be found in GenBank. After consulting with the P450 Nomenclature Committee (http://drnelson.uthsc.edu/cytochromeP450), in this article, we designate rhesus monkey CYP2D42 as CYP2D17. Deduced amino acid sequences of these CYP2D cDNAs share high sequence identity (93–96%) with human CYP2D6. Cynomolgus monkey CYP2D17 and Japanese monkey CYP2D29 metabolize typical human CYP2D6 substrates; CYP2D17 metabolizes bufuralol and dextromethorphan, whereas CYP2D29 metabolizes bufuralol and debrisoquine (Mankowski et al., 1999; Hichiya et al., 2002). Moreover, cynomolgus monkey CYP2D17 and Japanese monkey CYP2D29 enzymatic activities are inhibited by the CYP2D6 inhibitor quinidine, indicating that these macaque CYP2Ds have functional characteristics similar to those of human CYP2D6.
Comparisons between humans and cynomolgus monkeys show that monkeys generally catalyze human CYP2D6 marker reactions more efficiently in liver. For example, rates of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation were much higher in cynomolgus monkeys than in human, and the CYP2D proteins detected in liver, by Western blot, seem to be much more abundant in cynomolgus monkey than in humans (Shimada et al., 1997; Weaver et al., 1999). Western blot analysis revealed two CYP2D protein bands in blots of cynomolgus monkey liver microsomes (Jacqz-Aigrain et al., 1991; Weaver et al., 1999), raising the possibility that another CYP2D isoform, other than CYP2D17, is expressed in cynomolgus monkey liver.
To assess this possibility, we attempted to find another CYP2D cDNA in cynomolgus monkey by reanalyzing our expressed sequence tag (EST) data, which were generated by end-sequencing cDNAs of cynomolgus monkey liver (Uno et al., 2008). We successfully identified a novel CYP2D44 cDNA. In this article, we report on the characterization of cynomolgus monkey CYP2D44 performed by sequence analysis, phylogenetic analysis, genomic structure, tissue expression pattern, and metabolic assays. Expression of CYP2D protein was examined in liver microsomes from macaques (cynomolgus monkey and rhesus monkey) as well as human and marmoset. Moreover, gene copy number was assessed in cynomolgus monkeys and rhesus monkeys.
Materials and Methods
Materials.
Bufuralol and dextromethorphan were purchased from Wako (Osaka, Japan). Oligonucleotides and TaqMan probe were synthesized by Invitrogen (Tokyo, Japan) and Applied Biosystems (Foster City, CA), respectively. Pooled hepatic microsomes from humans, cynomolgus monkeys, rhesus monkeys, and marmosets were purchased from BD Gentest (Woburn, MA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise specified.
Tissues and Nucleic Acid Preparation.
Tissue samples were collected from six cynomolgus monkeys (three males and three females from Indochina, 4–5 years of age, 3–5 kg), and total RNA was extracted from the tissues as described previously (Uno et al., 2006). Tissue samples were collected from brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus. Whole blood samples were collected from eight cynomolgus monkeys (four males and four females from Indochina, 4–5 years of age, 3–5 kg) and four rhesus monkeys (two males and two females from China, 7 years of age, weighing 3–5 kg). Genomic DNA was prepared from blood samples with the PUREGENE DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN), according to the manufacturer's instructions. This study was approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd. (Kainan, Japan).
Isolation of CYP2D17 and CYP2D44 cDNAs.
Reverse transcription (RT)-polymerase chain reaction (PCR) was performed using total RNA extracted from cynomolgus monkey liver as described previously (Uno et al., 2006). In brief, the first-strand cDNA was generated in a mixture containing 1 μg of total RNA, oligo(dT), and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) at 37°C for 1 h. The RT product was used as the template for the subsequent PCR performed with KOD Plus DNA polymerase (Toyobo, Osaka, Japan), according to the manufacturer's protocol with a thermal cycler (Applied Biosystems). PCR conditions include an initial denaturation at 95°C for 2 min and 35 cycles of 95°C for 20 s, 58°C for 20 s, and 72°C for 2 min, followed by a final extension at 72°C for 10 min. The primers used were 5′-GCAGTAAGGCAGCTATGGAGCTA-3′ and 5′-CTAGCGGGGCACAGCACA-3′ for CYP2D17 and 5′-GTGTCCTGCCTGGTCCTCT-3′ and 5′-CTAGCGGGGCACAGCACA-3′ for CYP2D44. These primers were designed to amplify the coding regions of CYP2D17 (GenBank accession number U38218), and CYP2D44 found in our EST database (Uno et al., 2008). After addition of an A-overhang, the amplified cDNAs were cloned into pCR2.1 vectors using a TOPO TA Cloning Kit (Invitrogen), and the inserts were sequenced using an ABI PRISM BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 3730 DNA Analyzer (Applied Biosystems).
Exon Amplification.
PCR and sequencing were performed to determine the gene sequence of CYP2D44 from exon 1 to exon 3, which was absent in the rhesus monkey genome assembly. PCR was performed with cynomolgus monkey genomic DNA using 5 pmol of each primer, 0.5 mM dNTPs, 2 mM MgCl2, and 1 unit of LA Taq DNA polymerase (Takara, Tokyo, Japan) in a total volume of 20 μl. The primers were 5′-CCCCTGGCCATGACAGTA-3′ and 5′-TGCTCCAGCGACTTCTTG-3′. Thermal cycler conditions were as follows: 95°C for 1 min followed by 35 cycles of 95°C for 20 s, 55°C for 20 s, and 72°C for 2 min with a final extension at 72°C for 5 min. The PCR products were cloned into vectors using pGEM-T Easy Vector System (Promega, Madison, WI) according to the manufacturer's protocol. The inserts were sequenced by the primer walking method as described earlier using the primers, 5′-TGGGCAGCATATGTTATGGA-3′, 5′-ACGAGGTCAGGAGATCGAGA-3′, 5′-GACCCACAGTGCAAAAGGTT-3′, and 5′-ACCCACTCTCTGGCCTTTTT-3′.
Sequence Analysis.
Sequence data were analyzed with DNASIS Pro (Hitachi Software, Tokyo, Japan) and a Genetyx system (Software Development, Tokyo, Japan), including multiple alignment of amino acid sequences and drawing of a phylogenetic tree. A homology search was conducted using the BLAST program (National Center for Biotechnology Information, Bethesda, MD). CYP2D amino acid sequences used in this analysis included human CYP2D6 (NP_000097), cynomolgus monkey CYP2D17 (AAA79722), rhesus monkey CYP2D17 (NP_001035308), Japanese monkey CYP2D29 (AAL73443), marmoset CYP2D19 (O18992) and CYP2D30 (AAL92448), dog CYP2D15 (NP_001003333), and rat CYP2D1 (NP_695225), CYP2D2 (NP_036862), CYP2D3 (NP_775116), CYP2D4 (NP_612524), and CYP2D5 (NP_775426), found in GenBank. The cynomolgus monkey CYP2D44 amino acid sequence was deduced from the cDNA identified in this study.
Measurement of mRNA Expression.
CYP2D17 and CYP2D44 mRNA expression levels were measured by real-time RT-PCR, as reported previously (Uno et al., 2006), using gene-specific primers and TaqMan MGB probes. In brief, RT reactions were performed as described above using random primers (Invitrogen). One twenty-fifth of the reaction volume was used for the subsequent PCR. The amplification was performed in a total volume of 25 μl using TaqMan Universal PCR Master Mix (Applied Biosystems) with the ABI PRISM 7500 sequence detection system (Applied Biosystems) following the manufacturer's protocol. The primers used were 5′-CACCGACCAAGCCGGAC-3′ and 5′-AGGCGATCACGTTGCTCACT-3′ for CYP2D17 and 5′-GCCGACCAAGCCGGAT-3′ and 5′-GGAGGCAATTACGTTGCTCG-3′ for CYP2D44. The probes used were 5′-FAM-ACCCTTTCGCCCAAAC-MGB-3′ for CYP2D17 and 5′-FAM-CCCCTTTCGCCCCAGT-MGB-3′ for CYP2D44. The final concentrations of the primer set and the probe were 300 and 200 nM, respectively. The relative expression level was determined by normalization of the raw data to the 18S ribosomal RNA level based on three independent amplifications.
Heterologous Protein Expression in Escherichia coli.
To characterize CYP2D proteins, expression plasmids were generated with the CYP2D17 and CYP2D44 cDNAs isolated in this study, and the proteins were expressed as reported previously (Iwata et al., 1998; Uno et al., 2006). For CYP2D17, the internal NdeI site was mutated without altering the amino acid residue using the QuikChange XL II Kit (Stratagene, La Jolla, CA), according to the manufacturer's instructions, because the NdeI site was subsequently used to subclone the PCR products. The primers used were 5′-CCTGGGTGTGACCCAcATGACATCCCGTGACATCGAAC-3′ and 5′-GTTCGATGTCACGGGATGTCATgTGGGTCACACCCAGG-3′, where a lower case letter indicates the nucleotide to be mutated. To enhance protein expression, the modification of the N terminus was performed by PCR amplification of the coding region using KOD Plus DNA polymerase with the CYP2D cDNA as the template. The primers used were 5′-GGAATTCCATATGGCTCTGTTATTAGCAGTTTTTCTGGCTGTGACAGTGG-3′ and 5′-GCTCTAGACTAGCGGGGCACAGCACA-3′ for CYP2D17 and 5′-GGAATTCCATATGGCTCTGTTATTAGCAGTTTTTCTGGCCATGACAGTAGC-3′ and 5′-GCTCTAGACTAGCGGGGCACAGCACA-3′ for CYP2D44. The NdeI and XbaI sites of the forward and reverse primers, respectively, facilitate subcloning into the pCW vector, which contained human NADPH-P450 reductase cDNA. Likewise, the expression plasmid of human CYP2D6 was prepared as described previously (Iwata et al., 1998). Protein expression using the generated expression plasmids and membrane preparation was performed as described previously (Iwata et al., 1998; Uno et al., 2006). The CYP2D protein content and NAPDH-P450 reductase content in membrane preparations were determined as described previously by Omura and Sato (1964) and by Phillips and Langdon (1962), respectively.
Measurement of Drug-Metabolizing Activity.
Drug-metabolizing capabilities of cynomolgus monkey CYP2D17 and CYP2D44, and human CYP2D6, were evaluated using human CYP2D6 substrates, bufuralol and dextromethorphan, as described previously (Yamazaki et al., 2002). In brief, each mixture (0.25 ml) contained recombinant CYP2D protein (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 (400 μM bufuralol or 400 μM dextromethorphan) in 50 mM potassium phosphate buffer (pH 7.4). After incubation at 37°C for 15 min, reactions were terminated by addition of 0.25 ml of ice-cold acetonitrile. After centrifugation at 900g for 5 min, the supernatant was analyzed by high-performance liquid chromatography with an ultraviolet detector. Kinetic parameters were determined by nonlinear regression analysis to estimate apparent Km and Vmax. Mixtures were incubated in duplicate for 15 min using seven concentrations of the substrate, dextromethorphan, between 1 and 800 μM.
Western Blot Analysis.
Cynomolgus monkey CYP2D17 and CYP2D44 proteins (1.0 pmol), and liver microsomes (15 mg) were run on 10% SDS polyacrylamide gels and transferred to Hybond-P filters (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Human, cynomolgus monkey, rhesus monkey, and marmoset liver microsomes were analyzed. The filters were immunoblotted with rabbit anti-human CYP2D6 (1:5000) (BD Gentest) and sheep anti-rabbit IgG conjugated with horseradish peroxidase (Surmodics, Eden Prairie, MN). A specific band was visualized using an ECL Western blotting detection reagent (GE Healthcare) per the manufacturer's instructions.
Analysis of CYP2D44 Copy Number.
Copy number variation (CNV) of CYP2D44 was analyzed in eight cynomolgus monkeys and four rhesus monkeys using real-time PCR as described previously (Rotger et al., 2007) except that ALB was used as the reference gene. The reaction mixtures were prepared, and the PCR was carried out as described earlier for real-time RT-PCR. For CYP2D44, the primers used were 5′-CGCTGGAGCAGTGGGTG-3′ and 5′-GGAGGCAATTACGTTGCTCG-3′, and the probe used was 5′-FAM-CCCCTTTCGCCCCAGT-MGB-3′. For ALB, the primers used were 5′-TGTTGCATGAGAAAACGCCA-3′ and 5′-GTCGCCTGTTCACCAAGGAT-3′, and the probe used was 5′-VIC-AAGCGAGAAAGTCACCAAATGCTGCACGG-TAMRA-3′. Primer and probe concentrations were 600 and 200 nM, respectively, for both genes. To generate standard curves for quantification, six serial dilutions (40–0.039 ng) were made using calibrator DNA. All amplifications were performed in duplicate. The normalized copy number of CYP2D44 in the haploid genome was described as N = CYP2D44 copy number/ALB copy number.
Results
Identification of CYP2D44 cDNA.
Reanalysis of our in-house cynomolgus monkey liver EST database identified a CYP2D cDNA sequence that did not match the CYP2D17 cDNA sequence. Because the clones we found did not contain the complete cDNA sequence, RT-PCR was performed using cynomolgus monkey liver total RNA to isolate the complete cDNA of this novel CYP2D, named CYP2D44 by the P450 Nomenclature Committee (http://drnelson.uthsc.edu/cytochromeP450). The CYP2D44 cDNA contained an open reading frame of 497 amino acids with primary sequence structures characteristic of P450 proteins, such as six substrate recognition sites (SRSs) (Gotoh, 1992) and a heme-binding region (Fig. 1). Deduced amino acids of CYP2D44 in primates shared high sequence identity (91–93%) with human and chimpanzee CYP2D6, and cynomolgus monkey and rhesus monkey CYP2D17 and to a lesser extent (87–88%) with Japanese monkey CYP2D29 and marmoset CYP2D19 and CYP2D30, among the CYP2Ds in GenBank (Table 1). In contrast, CYP2D44 shared lower sequence identity (70–73%) with dog and rat CYP2Ds. A phylogenetic analysis using the CYP2D amino acid sequences from human, macaque, dog, and rat indicated that cynomolgus monkey CYP2D44 and other macaque CYP2Ds were most closely clustered with human CYP2D6 (Fig. 2). These results suggest the evolutionary closeness of CYP2D44 to other primate CYP2Ds, compared with CYP2Ds of dog and rat, which are also used in preclinical studies. The cynomolgus monkey CYP2D44 cDNA sequence has been deposited into GenBank under the accession number DQ297684. Deduced amino acid sequences of our CYP2D17 cDNA differed from another entry (U38218) in six amino acid residues, probably due to genetic polymorphisms. The sequence of our CYP2D17 cDNA was verified by sequencing the inserts of multiple clones to avoid possible sequencing errors.
Genome Organization and Gene Structure of CYP2D44.
Human and rhesus monkey (a species closely related to cynomolgus monkey) genomes were analyzed using BLAT to determine the genomic location of CYP2D44. The analysis indicated that CYP2D17 and CYP2D44 are located in the CYP2D cluster, as in humans (Fig. 3). In the macaque CYP2D cluster, only two genes, CYP2D17 and CYP2D44, were found, whereas three genes, CYP2D6, CYP2D7P, and CYP2D8P, are present in the human genome. CYP2D44 sequences from exon 1 to exon 3 were absent in the genome. This is probably because of the misassembly of genome sequences in the CYP2D cluster. To complete the gene sequence, the sequences from exon 1 to exon 3 were amplified by PCR and sequenced. The complete gene sequence was aligned with the cynomolgus monkey CYP2D44 cDNA sequence to determine the exon-intron structure of CYP2D44. This gene contained nine exons, and the sizes of the exons and introns ranged from 142 to 188 bp and 88 to 1620 bp, respectively (Table 2). The exact sizes of exon 1 and exon 9 are currently not known, because the entire lengths of the 5′ and 3′ untranslated regions were not determined.
CYP2D44 mRNA Expression in Tissues.
To measure mRNA expression of CYP2D17 and CYP2D44, real-time RT-PCR was performed with gene-specific primers and probes using total RNAs prepared from cynomolgus monkey tissues: brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus. Of these tissues, both CYP2D17 and CYP2D44 mRNAs were expressed predominantly in the liver (Fig. 4), and CYP2D44 mRNA expression was approximately 4.4-fold lower than that of CYP2D17.
Drug-Metabolizing Capability of CYP2D44 Protein.
To determine the metabolic properties of CYP2D44, metabolic assays were performed using typical human CYP2D6 substrates, bufuralol and dextromethorphan, with cynomolgus monkey CYP2D17 and CYP2D44 proteins heterologously expressed in E. coli. Both CYP2D17 and CYP2D44 catalyzed bufuralol 1′-hydroxylation and dextromethorphan O-demethylation, reactions also catalyzed efficiently by human CYP2D6, and CYP2D44 activity was 55 to 61% of CYP2D17 activity (Table 3). Kinetic analysis of dextromethorphan O-demethylation indicated that the Km and Vmax for CYP2D17 were, respectively, 2.7 μM and 14.6 nmol/(min · nmol) of P450, and the Km and Vmax for CYP2D44 were, respectively, 3.8 μM and 10.1 nmol/(min · nmol) of P450 (Table 4). The intrinsic clearance (Vmax/Km) of dextromethorphan O-demethylation for CYP2D17 and CYP2D44 was comparable to that of human CYP2D6. Km and Vmax for dextromethorphan N-demethylation, which human CYP2D6 catalyzes much less effectively than O-demethylation, were, respectively, 7.2 μM and 5.4 nmol/(min · nmol) of P450 for CYP2D17 and 19 μM and 5.5 nmol/(min · nmol) of P450 for CYP2D44 (Table 4). The intrinsic clearance (Vmax/Km) of dextromethorphan N-demethylation for CYP2D17 and CYP2D44 was, respectively, 0.75 and 0.29 ml/(min · nmol) of P450, higher than that of human CYP2D6, 0.03 ml/(min · nmol) of P450, supporting higher efficiency of cynomolgus monkey CYP2Ds than human CYP2D6 in this reaction. These results indicated that the metabolic properties of both cynomolgus monkey CYP2Ds are similar to those of human CYP2D6 but differ in dextromethorphan N-demethylation.
Western Blot Analysis.
To see whether CYP2D17 and CYP2D44 proteins are expressed in liver, Western blot analysis was performed using anti-human CYP2D6 antibody. First, the reactivity of the anti-human CYP2D6 antibody was examined using cynomolgus monkey CYP2D17 and CYP2D44 recombinant proteins. The strong bands were successfully detected in CYP2D17 and CYP2D44, indicating that these CYP2D proteins can be detected with the anti-human CYP2D6 antibody (Fig. 5A). Next, to detect expression of CYP2D proteins in liver, Western blot analysis was performed with the anti-human CYP2D6 antibody using liver microsomes from human, cynomolgus monkey, rhesus monkey, and marmoset. The reactive bands were detected in all samples (Fig. 5B), suggesting that CYP2D proteins were expressed in cynomolgus and rhesus monkey liver (probably including CYP2D17 and CYP2D44), similar to human and marmoset liver.
Copy Number Variation.
Interindividual differences in human CYP2D6 copy numbers in single alleles are well documented. To assess the possibility of such variations in macaques, as a first step, CYP2D44 copy number was determined in eight cynomolgus monkeys and four rhesus monkeys using real-time PCR (Fig. 6). The method was confirmed to detect CYP2D44 specifically not CYP2D17. The copy numbers, defined as mean CYP2D44 quantity/mean ALB quantity, were close to 0.5, 1.0, 1.5, or 2.0, indicating that CYP2D44 copies most likely vary among the animals tested (one, two, three, or four copies per diploid genome).
Discussion
Human CYP2D6 is highly polymorphic and metabolizes antiarrhythmics, β-blockers, neuroleptics, and antidepressants. Previous reports indicated higher CYP2D activity in cynomolgus monkey liver than in human liver (Shimada et al., 1997; Weaver et al., 1999). This was possibly due to additional CYP2D isoforms present in cynomolgus monkey because two CYP2D bands were detected in cynomolgus monkey liver by Western blot analysis (Jacqz-Aigrain et al., 1991; Weaver et al., 1999), whereas humans possess only a single CYP2D, CYP2D6. This finding prompted us to search for novel CYP2D cDNAs in our in-house cynomolgus monkey liver EST database (Uno et al., 2008), which led to the successful identification of CYP2D44 cDNA. CYP2D44 amino acid sequence identity with human and other primate CYP2Ds was much higher than with those of dog and rat, which are also used in preclinical studies, reflecting the evolutionary closeness of CYP2D44 to human CYP2D6 and other primate CYP2Ds.
Investigation of the CYP2D genomic organization is important because the position and direction of each gene in the gene cluster provides essential information to assess CYP2D orthologous relationships between species. The CYP2D cluster is formed in humans and macaques, respectively, by CYP2D6, CYP2D7P, and CYP2D8P and by CYP2D17 and CYP2D44, in corresponding genomic regions. The number of genes in the CYP2D cluster is different in humans and macaques. This is probably due to gene duplication of P450s, including CYP2Ds, in each species during evolution (Nelson et al., 2004). Alternatively, an additional CYP2D might be present in the genome but excluded in the assembled genome sequence because of the potential genomic misassembly in this region. The lack of the CYP2D44 5′ and 3′ ends of the assembled macaque genome supports this notion. Although a 1:1 orthologous relationship between macaque and human CYP2Ds was not evident in this study, further assemblies and refinement of the genome sequences might identify an additional CYP2D in the macaque genome, which could help assess the orthologous relationship.
Similar to CYP2D17, CYP2D44 mRNA was predominantly expressed in liver, indicating its functional role in liver. Although CYP2D44 mRNA expression in liver was 4.4-fold lower than that of CYP2D17, CYP2D44 mRNA might be expressed at a similar or higher level than CYP2D17 mRNA, depending on the animal, because mRNA expression can be varied among individual animals. Such variability in CYP2D expression, especially at a protein level, is important, because it could account for the possible interanimal variability in CYP2D-dependent drug metabolism. By Western blot analysis using anti-human CYP2D6 antibody, which reacted with both cynomolgus monkey CYP2D17 and CYP2D44, CYP2D protein bands were detected in cynomolgus and rhesus monkey liver. In our hands, the CYP2D44-specific antibodies could not be produced; the antibodies generated using the synthetic peptides corresponding to the CYP2D44 amino acid residues less conserved with CYP2D17 reacted with both CYP2D17 and CYP2D44. The isoform-specific antibodies for use in Western blots, if generated, would allow separate detection of CYP2D17 and CYP2D44, making it possible to assess the interanimal variability of CYP2D17 and CYP2D44 protein expression in cynomolgus monkey liver.
Cynomolgus monkey CYP2D44 effectively catalyzed bufuralol 1′-hydroxylation and dextromethorphan O-demethylation, human CYP2D6 marker reactions, which were also shown to be catalyzed by cynomolgus monkey CYP2D17 in this and previous studies (Mankowski et al., 1999). In this study, CYP2D44 catalysis of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation was approximately 55 and 60% of CYP2D17 catalysis rates, respectively. The lower activity of CYP2D44 dextromethorphan O-demethylation catalysis might be due to CYP2D44 having a higher Km than CYP2D17. CYP2D17 and CYP2D44 efficiently catalyzed dextromethorphan N-demethylation, which human CYP2D6 also catalyzes, but much less efficiently (Yu and Haining, 2001; Yu et al., 2001). The intrinsic clearance of both cynomolgus monkey CYP2Ds was comparable to that of human CYP2D6 in dextromethorphan O-demethylation but much higher than that of CYP2D6 in dextromethorphan N-demethylation. These results suggest that CYP2D17 and CYP2D44 have metabolic properties similar to those of human CYP2D6 but differ with respect to dextromethorphan N-demethylation. Further investigation will identify additional similarities and differences in metabolic properties between human CYP2D6 and cynomolgus monkey CYP2D17 and CYP2D44.
Site-directed mutagenesis of human CYP2D6 has shown the importance of Phe120, Glu216, Asp301, Phe481, and Phe483 in determining the interaction between CYP2D6 and its ligands (de Graaf et al., 2007). These amino acid residues are conserved between human CYP2D6, cynomolgus monkey CYP2D17 and CYP2D44, rhesus monkey CYP2D17, Japanese monkey CYP2D29, and marmoset CYP2D19 and CYP2D30, except that cynomolgus monkey CYP2D44 contains Val481 in SRS6. F481N and F481G cause the CYP2D6 catalytic efficiency of debrisoquine and dextromethorphan metabolism to decrease (Hayhurst et al., 2001; de Graaf et al., 2007). Phe481 and Phe483 are located in the loop between the two β-strands of the fourth sheet region. Several studies proposed that Phe483 is oriented into the active cavity, whereas Phe481 is in a position far from any possible interaction with ligands; however, Phe481 was oriented into the active cavity by constrained molecular dynamics simulations, although Phe481 did not appear to be involved in the reaction site binding (Rowland et al., 2006). It is of great interest to investigate F481V to determine whether it accounts for the lower CYP2D44 catalytic efficiency of bufuralol and dextromethorphan metabolism in comparison with CYP2D17.
Human CYP2D6 is highly variable, partly because of genetic polymorphisms, including CYP2D6*5 with the entire gene deleted (Gaedigk et al., 1991; Steen et al., 1995). Some individuals possess more than one CYP2D6 per allele, especially those in North East Africa and Western Europe, which accounts, at least partly, for ultrarapid metabolizers of CYP2D6 drugs (Ingelman-Sundberg, 2005). Likewise, in this study, CYP2D44 showed CNVs among cynomolgus monkeys and rhesus monkeys (one to four copies per diploid genome). It should be noted that single nucleotide polymorphisms (SNPs) have not been analyzed in CYP2D44, and, thus, SNPs at the annealing sites of the primers used in this analysis, if any, could affect the resultant copy numbers. SNPs, especially nonsynonymous SNPs, can also affect metabolic activity of P450 proteins. Because the possibility of interanimal differences in CYP2D-dependent drug metabolism has been reported in macaques (Jacqz et al., 1988), the CNVs and SNPs in CYP2D44 might account for such differences. Investigation of genetic polymorphisms (including CNVs and SNPs) and knowledge of the genetic background for the animals would be highly helpful for understanding interanimal differences in CYP2D-dependent drug metabolism in macaques.
The CYP2D44 cDNA sequence was successfully identified in our cynomolgus monkey liver EST database (Uno et al., 2008). The genomic information, including genome sequence and EST data, has become increasingly available to the public and has been successfully applied to the identification of novel genes in macaques (Magness et al., 2005), including CYP2C76 (Uno et al., 2006). CYP2C76, not orthologous to any human P450 (Uno et al., 2006), is partly responsible for differences in drug metabolism between cynomolgus monkeys and humans (Uno et al., 2007). This article further supports the importance of genomic information, which was used to identify CYP2D44, relevant to drug metabolism in cynomolgus monkey. As more genomic information becomes available, identification of additional P450s essential for drug metabolism in macaques can be expected in the future.
In conclusion, we identified a novel CYP2D, CYP2D44, in cynomolgus monkey, which shares high sequence identity with human CYP2D6 and cynomolgus monkey CYP2D17. CYP2D17 and CYP2D44 form the macaque CYP2D cluster in a region corresponding to the human CYP2D cluster. CYP2D44 mRNA was predominantly expressed in liver, and a CYP2D band (expected to contain CYP2D44) was detected in cynomolgus monkey liver by Western blot. CYP2D44, together with CYP2D17, efficiently catalyzed bufuralol 1′-hydroxylation and dextromethorphan O-demethylation, which human CYP2D6 also catalyzes efficiently. CYP2D17 and CYP2D44 also efficiently catalyzed dextromethorphan N-demethylation, which human CYP2D6 catalyzes much less efficiently. These results indicated a resemblance in CYP2D metabolic properties between cynomolgus monkeys and humans, with some differences in dextromethorphan N-demethylation. Moreover, the polymorphic nature of CYP2D44 was partly implied by detection of CNVs in the cynomolgus and rhesus monkeys analyzed. The information presented in this study will help the prediction of in vivo drug clearance in monkeys by scaling clearance, when further investigation determines hepatic protein levels of two CYP2D isoforms in individual animals. Consequently, the present results qualitatively indicate a functional importance of both macaque CYP2D44 and CYP2D17 in CYP2D-dependent drug metabolism.
Acknowledgments.
We greatly appreciate Masahiro Utoh, Dr. Koichirio Fukuzaki, and Dr. Ryoichi Nagata for their interest and encouragement in this study; Ryo Koizumi for technical assistance; and Patrick Gray for reviewing the manuscript.
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.033274.
-
ABBREVIATIONS:
- P450
- cytochrome P450
- EST
- expressed sequence tag
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- CNV
- copy number variation
- SRS
- substrate recognition site
- SNPs
- single nucleotide polymorphisms.
- Received March 15, 2010.
- Accepted May 25, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics