Novel Cytochrome P450 2C119 Enzymes in Cynomolgus and Rhesus Macaques Metabolize Progesterone, Diclofenac, and Omeprazole.

Cynomolgus and rhesus macaques are used in drug metabolism studies due to their evolutionary and phylogenetic closeness to humans. Cytochromes P450 (P450s or CYPs), including the CYP2C family enzyme, are important endogenous and exogenous substrate-metabolizing enzymes and play major roles in drug metabolism. In cynomolgus and rhesus macaques, six CYP2Cs have been identified and characterized, namely, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2C76, and CYP2C93. In this study, CYP2C119, a new CYP2C, was identified and characterized in cynomolgus and rhesus macaques. Cynomolgus and rhesus CYP2C119 contained open reading frames of 489 amino acids with high sequence identities to human CYP2C8 and to cynomolgus and rhesus CYP2C8. Phylogenetic analysis showed that cynomolgus and rhesus CYP2C119 were closely related to cynomolgus and rhesus CYP2C8. In cynomolgus and rhesus genomes, CYP2C genes, including CYP2C119, form a cluster. Among the tissues analyzed, cynomolgus CYP2C119 mRNA was predominantly expressed in liver. Hepatic expressions of CYP2C119 mRNA in four cynomolgus and two rhesus macaques varied, with no expression in one rhesus macaque. Among the CYP2C mRNAs, CYP2C119 mRNA was expressed less abundantly than CYP2C8, CYP2C9, CYP2C19, and CYP2C76 mRNAs but more abundantly than CYP2C18 mRNA. Recombinant cynomolgus and rhesus CYP2C119 catalyzed progesterone 16α-, 17α-, and 21-hydroxylation and diclofenac and omeprazole oxidations, indicating that CYP2C119 is a functional enzyme. Therefore, the novel CYP2C119 gene, expressed in macaque liver, encodes a functional enzyme that metabolizes human CYP2C substrates and is likely responsible for drug clearances. SIGNIFICANCE STATEMENT: Cytochrome P450 2C119 was found in cynomolgus and rhesus macaques, in addition to the known P450 2C8, 2C9, 2C18, 2C19, 2C76, and 2C93. Cynomolgus and rhesus CYP2C119 contain open reading frames of 489 amino acids with high sequence identity to human CYP2C8. Cynomolgus CYP2C119 mRNA is predominantly expressed in the liver. Recombinant CYP2C119 catalyzed progesterone hydroxylation and diclofenac and omeprazole oxidations. Therefore, the novel CYP2C119 gene expressed in the macaque liver encodes a functional enzyme that metabolizes human CYP2C substrates.

Liver microsomal cytochrome P450 3A-dependent drug oxidation activities in individual dogs.

Drug oxidations are mediated mainly by cytochromes P450 (P450s or CYPs). CYP3As are an important P450 subfamily and include liver-specific CYP3A12 and intestine-specific CYP3A98 in dogs. Individual differences in drug oxidation activities were investigated, including correlations with immunoreactive CYP3A protein intensities and CYP3A mRNA expression levels in livers.Pooled and individual dog liver microsomes showed activities towards nifedipine, midazolam, alprazolam, and estradiol, but the levels of catalytic activities varied approximately twofold among the individual dogs. One dog harboured a CYP1A2 variant causing protein deletion but showed higher activities than the other dogs towards nifedipine oxidation, midazolam 1'-hydroxylation, alprazolam 4-hydroxylation, estradiol 16α-hydroxylation activities, and caffeine C8-hydroxylation; the latter is used as a reference reaction for CYP1A.In individual dog liver microsomes, the intensities of the immunochemical bands with anti-human CYP3A4 and anti-rat CYP3A2 antibodies along with CYP3A12 and CYP3A26 mRNA expression levels showed good correlations (p < 0.05) with nifedipine oxidation, midazolam 1'- and 4-hydroxylation, alprazolam 1'- and 4-hydroxylation, and estradiol 16α-hydroxylation activities.These results suggest that the oxidation activities of dog liver microsomes towards nifedipine and other typical CYP3A-catalyzed drugs exhibit approximately twofold individual differences and were predominantly mediated by liver-specific CYP3A12 in the dogs.

Newly identified cytochrome P450 3A genes of tree shrews and pigs are expressed and encode functional enzymes.

Novel cytochrome P450 3A5 (CYP3A5) cDNA in tree shrews (which are non-rodent primate-like species) and pig CYP3A227 cDNA were identified, along with known pig CYP3A22, CYP3A29, and CYP3A46 cDNAs. All five cDNAs contained open reading frames encoding a polypeptide of 503 amino acids that shared high sequence identity (72-78 %) with human CYP3A4 and were more closely related to human CYP3As than rat CYP3As by phylogenetic analysis. CYP3A5 was the only CYP3A in the tree shrew genome, but pig CYP3A genes formed a CYP3A gene cluster in the genomic region corresponding to that of human CYP3A genes. Tree shrew CYP3A5 mRNA was predominantly expressed in liver and small intestine, among the tissues analyzed, whereas pig CYP3A227 mRNA was most abundantly expressed in jejunum, followed by liver. Metabolic assays established that tree shrew CYP3A5 and pig CYP3A proteins heterologously expressed in Escherichia coli metabolized typical human CYP3A4 substrates nifedipine and midazolam. These results suggest that novel tree shrew CYP3A5 and pig CYP3A227 were functional enzymes able to metabolize human CYP3A4 substrates in liver and small intestine, similar to human CYP3A4, although pig CYP3A227 mRNA was minimally expressed in all tissues analyzed.

Newly Identified Tree Shrew Cytochrome P450 2A13 is Expressed in Liver and Lung and Encodes a Functional Drug-Metabolizing Enzyme Similar to Dog Cytochrome P450 2A13 and Pig Cytochrome P450 2A19.

The tree shrew, a non-rodent primate-like species, is used in various fields of biomedical research, including hepatitis virus infection, myopia, depression, and toxicology. Recent genome analysis found that the numbers of cytochrome P450 (P450 or CYP) genes are similar in tree shrews and humans and their sequence identities are high. Although the P450s are a family of important drug-metabolizing enzymes, they have not yet been fully investigated in tree shrews. In the current study, tree shrew CYP2A13 cDNA was isolated from liver, and its characteristics were compared with those of pig, dog, and human CYP2As. Tree shrew CYP2A13 amino acid sequences were highly identical (87-92%) to the human CYP2As and contained sequence motifs characteristic of P450s. Phylogenetic analysis revealed that tree shrew CYP2A13 was more closely related to human CYP2As than to rat CYP2As, similar to dog and pig CYP2As. Among the tissue types analyzed, tree shrew CYP2A13 mRNA was preferentially expressed in liver and lung, similar to dog CYP2A13 mRNA, whereas dog CYP2A25 and pig CYP2A19 mRNAs were predominantly expressed in liver. Tree shrew liver microsomes and tree shrew CYP2A13 proteins heterologously expressed in Escherichia coli catalyzed coumarin 7-hydroxylation and phenacetin O-deethylation, just as human, dog, and pig CYP2A proteins and liver microsomes do. These results demonstrate that tree shrew CYP2A13 is expressed in liver and lung and encodes a functional drug-metabolizing enzyme. SIGNIFICANCE STATEMENT: Novel tree shrew cytochrome P450 2A13 (CYP2A13) was identified and characterized in comparison with human, dog, and pig CYP2As. Tree shrew CYP2A13 isolated from liver had high sequence identities and close phylogenetic relationships to its human homologs and was abundantly expressed in liver and lung at the mRNA level. Tree shrew CYP2A13 metabolized coumarin and phenacetin, human selective CYP2A6 and CYP2A13 substrates, respectively, similar to dog and pig CYP2As, and is a functional drug-metabolizing enzyme likely responsible for drug clearances.

Novel cytochrome P450 1 (CYP1) genes in tree shrews are expressed and encode functional drug-metabolizing enzymes.

Tree shrews (Tupaia belangeri) are a non-rodent primate-like species sometimes used for biomedical research involving hepatitis virus infections and toxicology. Genome analysis has indicated similarities between tree shrews and humans in the numbers of cytochromes P450 (P450 or CYP), which constitute a family of important drug-metabolizing enzymes; however, P450s have not been fully investigated in tree shrews. In this study, we identified CYP1A1, CYP1A2, CYP1B1, and CYP1D1 cDNAs from tree shrew liver and compared their characteristics with dog, pig, and human CYP1As. The deduced amino acid sequences of tree shrew CYP1s were highly identical (82-87 %) to human CYP1s. In tree shrews, CYP1A1 and CYP1A2 mRNAs were preferentially expressed in liver, whereas CYP1D1 mRNA was preferentially expressed in kidney and lung. In contrast, CYP1B1 mRNA was expressed in various tissues, with the most abundant expression in spleen. Among the tree shrew CYP1 mRNAs, CYP1A2 mRNA was most abundant in liver, and CYP1B1 mRNA was most abundant in kidney, small intestine, and lung. All tree shrew CYP1 proteins heterologously expressed in Escherichia coli catalyzed caffeine and estradiol in a similar manner to tree shrew liver microsomes and human, dog, and pig CYP1 proteins. These results suggest that tree shrew CYP1A1, CYP1A2, CYP1B1, and CYP1D1 genes, different form human pseudogene CYP1D1P, are expressed in liver, small intestine, lung, and/or kidney and encode functional drug-metabolizing enzymes important in toxicology.

A comprehensive analysis of six forms of cytochrome P450 2C (CYP2C) in pigs.

Pigs are an important species used in drug metabolism studies; however, the cytochromes P450 (P450s or CYPs) have not been fully investigated in pigs.In this study, pig CYP2C32, CYP2C33, CYP2C34, CYP2C36, CYP2C42, and CYP2C49 cDNAs were isolated and found to contain open reading frames of 490 or 494 amino acids that shared 64-82% sequence identity with human CYP2C8/9/18/19.Pig CYP2C genes formed a gene cluster in a genomic region that corresponded to that of the human CYP2C cluster; an additional gene cluster was formed by pig CYP2C33a and CYP2C33b distant from the first cluster but located in the same chromosome.Among the tissues analysed, these pig CYP2C mRNAs were preferentially expressed in liver, small intestine, and/or kidney; pig CYP2C49, CYP2C32, CYP2C34, and CYP2C33 mRNAs were the most abundant CYP2C mRNAs in liver, jejunum, ileum, and kidney, respectively.Metabolic assays showed that pig CYP2C proteins (heterologously expressed in Escherichia coli) metabolised typical human CYP2C substrates diclofenac, warfarin, and/or omeprazole.The results suggest that these pig CYP2Cs are functional enzymes able to metabolise human CYP2C substrates in liver and small intestine, just as human CYP2Cs do.

Molecular cloning and tissue distribution of marmoset thiopurine S-methyltransferase.

The common marmoset (Callithrix jacchus) is a New World monkey that is increasingly used in pharmacological and toxicological studies. Thiopurine S-methyltransferase (TPMT) plays roles in the metabolism of widely used anticancer and anti-inflammatory drugs. Here, we report the isolation and molecular characterization of marmoset TPMT cDNA, which was found to contain an open-reading frame of 245 amino acids that was approximately 92% identical to its human ortholog. Marmoset TPMT was phylogenetically closer to other primate orthologs than to its pig, dog, rabbit, or rodent orthologs. Among the five marmoset tissue types analyzed, marmoset TPMT mRNA was most abundant in kidney and liver, just as human TPMT is. These results suggest that marmoset and human TPMT are similar at the molecular level.

Regional distributions of UDP-glucuronosyltransferase activities toward estradiol and serotonin in the liver and small intestine of cynomolgus macaques.

The cynomolgus macaque is a nonhuman primate species that is often used in drug metabolism studies during drug development. However, the localization of UDP-glucuronosyltransferases (UGTs), essential drug-metabolizing enzymes, has not been fully investigated in the liver and small intestine of cynomolgus macaques. In this study, UGT activities were analyzed in liver (five lobes) and small intestine (the duodenum and six sections from the proximal jejunum to the distal ileum) using typical probe substrates of human UGTs: 7-hydroxycoumarin, estradiol, serotonin, propofol, and zidovudine. In liver, UGT activities with respect to all substrates were detected, and the activity levels were similar in all liver lobes of the cynomolgus macaques tested. In contrast, in the small intestine, UGT activities toward all substrates were detected, but their levels generally decreased from jejunum to ileum in cynomolgus macaques. The localization of estradiol 3-O-glucuronosyltransferases and serotonin O-glucuronosyltransferases (which are mainly UGT1A enzymes) appear to be different in liver and small intestine. These results collectively suggest that, in cynomolgus macaques, UGT1As are differentially localized in the small intestine but are relatively homogeneously distributed in the liver.

Interleukin-1ß and tumor necrosis factor-α affect cytochrome P450 expression in cynomolgus macaque hepatocytes.

The cynomolgus macaque, partly due to its evolutionary closeness to humans, is an important nonhuman primate species used in drug metabolism studies. In humans, expressions of cytochromes P450 (P450s), including the important drug-metabolizing enzyme P450 3A4, are affected by various cytokines. However, this phenomenon has not been fully investigated in cynomolgus macaques. In this study, the effects of cytokines on P450 expression were investigated using the quantitative polymerase chain reaction to evaluate mRNA expression. Hepatocytes from cynomolgus macaques were treated with lipopolysaccharide and various cytokines, including interleukin (IL)-1ß, IL-2, IL-6, interferon-γ, and tumor necrosis factor-α, and the expression levels of 11 P450s were compared with those of solvent-treated controls. Tumor necrosis factor-α significantly decreased cynomolgus P450 2C8 and 2C76 mRNA expression in multiple lots of cynomolgus hepatocytes investigated. IL-1ß significantly decreased cynomolgus P450 1A1, 2C8, 2C19, and 2C76 mRNA expression, but increased P450 3A5 mRNA expression in multiple lots of hepatocytes. Moreover, P450 1A1-and 2C19-mediated drug oxidations were significantly and dose-dependently suppressed by IL-1ß, under the present limited conditions. These results suggest that cytokines can influence hepatic P450 mRNA expression levels in cynomolgus macaques, just as cytokines are reported to affect P450 expression in humans.

Functional characterization for polymorphic organic anion transporting polypeptides (OATP/SLCO1B1, 1B3, 2B1) of monkeys recombinantly expressed with various OATP probes.

The hepatic uptake of clinical drugs mediated by human hepatic organic anion transporting polypeptides (OATP/SLCO) has been reported extensively. In this study, hepatic uptake by recombinantly expressed monkey OATP1B1, OATP1B3 and OATP2B1 was investigated using three human OATP1B1 and OATP1B3 substrates (pitavastatin, pravastatin and rosuvastatin) and one OATP1B3 substrate (telmisartan), as the governmental drug interaction guidelines recommend, and seven reported clinical drugs. The uptake of known human probes into recombinant OATP-expressing cells was significantly greater than that of mock cells. Consequently, pitavastatin, pravastatin and rosuvastatin were suggested to be substrates of recombinant monkey OATP1B1 and OATP1B3, and telmisartan was suggested to be a substrate of recombinant monkey OATP1B3, in a manner similar to human OATPs. In contrast, atorvastatin, bosentan, etoposide, fexofenadine, fluvastatin, glibenclamide and simeprevir were broadly transported by recombinant monkey OATP1B1, OATP1B3 and OATP2B1. Furthermore, some of the 16 non-synonymous monkey OATP1B1 variants found in 64 cynomolgus and 32 rhesus monkeys mediated up to a 1.6-fold [3 H]pitavastatin uptake (with low Michaelis constant values) in comparison with the wild type under the present conditions. Despite sequences of monkey recombinant OATPs not being totally reflective of those of human OATPs, our results collectively suggested that OATP1B1, OATP1B3 or OATP2B1 in monkeys could mediate roughly a similar hepatic uptake of various OATP probes. Recombinant monkey OATPs would be good experimental tools for in vitro hepatic uptake in cell systems.

Functionally relevant genetic variants of glutathione S-transferase GSTM5 in cynomolgus and rhesus macaques.

Glutathione S-transferase (GST) is a family of enzymes important for conjugation with glutathione of endogenous and exogenous compounds. Human GSTM1 null allele is associated with toxicity and cancers. Cynomolgus and rhesus macaques have molecular and enzymatic similarities of GSTs to humans; however, genetic variants have not been investigated. In macaques, instead of pseudogenized GSTM1, GSTM5 is a predominant GSTM isoform. In this study, re-sequencing of GSTM5 in 64 cynomolgus and 31 rhesus macaques found 6 non-synonymous variants, and 1 variant (IVS5 + 1) causing exon skip. Of these 7 variants, 3 and 1 were found only in Indochinese and Indonesian cynomolgus macaques, respectively. Cynomolgus GSTM5-mediated styrene 7,8-oxide and trans-stilbene oxide conjugation activities correlated with GSTM protein levels immunochemically quantified in cynomolgus liver samples. Using recombinant GSTM5 proteins, 4 of the 6 non-synonymous variants including E29Q, L96R, M166V and S201N showed substantially lower metabolic activities. Moreover, a homozygote for E29Q and heterozygotes for S201N or IVS5 + 1 showed significantly lower conjugation activities in liver cytosolic fractions as compared with wild-type animals. Therefore, the present results suggest that inter-animal variability of GST-dependent drug metabolism is at least partly accounted for by GSTM5 variants in cynomolgus and rhesus macaques as pre-clinical animal models.

Genetic Variants of Glutathione S-Transferase GSTT1 and GSTT2 in Cynomolgus Macaques: Identification of GSTT Substrates and Functionally Relevant Alleles.

Glutathione S-transferase (GST) is a family of important drug-metabolizing enzymes, conjugating endogenous and exogenous compounds. Genetic polymorphisms result in the inter-individual variability of GST activity in humans. Especially, human GSTT1 and GSTT2 null alleles are associated with toxicity and various cancers derived from chemicals. Cynomolgus macaque, a nonhuman primate species widely used in drug metabolism studies, has molecular and enzymatic similarities of GSTs to the human orthologs; however, genetic polymorphisms have not been investigated in this species. In this study, resequencing of GSTT1 and GSTT2 in 64 cynomolgus and 32 rhesus macaques found 15 nonsynonymous variants and 1 nonsense variant for GSTT1 and 15 nonsynonymous variants for GSTT2. Some of these GSTT variants were distributed differently in Indochinese and Indonesian cynomolgus macaques and rhesus macaques. For analysis of functional relevance of the GSTT variants, 1-iodohexane and dibromomethane were determined to be suitable substrates for cynomolgus GSTT1 and GSTT2. However, the conjugation activities were roughly correlated with GSTT protein levels immunochemically quantified in cynomolgus liver samples with no statistical significances, implying the contributions of the GST genetic variants. Among the GSTT1 variants identified, the animals carrying R76C and D125G mutations showed lower conjugation activities toward dibromomethane than those of the wild-type in liver cytosolic fractions. Moreover, the recombinant R76C/D125G and D125G GSTT variant proteins showed significantly lower 1-iodohexane or dibromomethane conjugation activities than those of the wild-type protein. Therefore, inter-animal variability of GSTT-dependent drug metabolism is at least partly accounted for by GSTT1 and possibly GSTT2 variants in cynomolgus and rhesus macaques.

Molecular and functional characterization of UDP-glucuronosyltransferase 1A in cynomolgus macaques.

UDP-glucuronosyltransferases (UGTs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics. Molecular characteristics of UGTs have been extensively investigated in humans, but in cynomolgus macaques, a non-human primate species widely used in drug metabolism studies, remain to be investigated. In this study, 12 UGT1A cDNAs (UGT1A1, 1A2, 1A4A, 1A4B, 1A5A, 1A5B, 1A5C, 1A6, 1A7, 1A8, 1A9, and 1A10) were isolated and characterized in cynomolgus macaques. UGT1A5C cDNA did not contain a complete coding region due to nonsense mutations, and was excluded from further analysis. Amino acid sequences of all 11 cynomolgus UGT1As had high sequence identities (92-95%) with human UGT1As and were phylogenetically close to human UGT1As. These cynomolgus UGT1A genes shared exons 2-5, and contained a variable exon 1 unique to each gene, similar to human UGT1A genes. Moreover, cynomolgus and human UGT1A gene clusters were located in corresponding regions in the genome. Among the 10 tissue types analyzed, cynomolgus UGT1A mRNAs were most abundantly expressed in the liver, jejunum, and/or kidney, the drug-metabolizing organs, similar to human UGT1As. Among these 11 cynomolgus UGT1A mRNAs, cynomolgus UGT1A2, UGT1A9, and UGT1A10 mRNAs were most abundantly expressed in the liver, kidney, and jejunum, respectively. Cynomolgus liver microsomes and UGT1A proteins catalyzed glucuronidation of the substrates human UGT1As catalyze, including 4-methylumbelliferone, 4-nitrophenol, estradiol, trifluoperazine, serotonin, and propofol, although trifluoperazine glucuronidation was not catalyzed by any cynomolgus UGT1A proteins. These results suggest that cynomolgus UGT1As are functional enzymes with molecular similarities to human UGT1As.

Progesterone hydroxylation by cytochromes P450 2C and 3A enzymes in marmoset liver microsomes.

1. Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical drug metabolism studies. However, the roles of marmoset cytochrome P450 (P450) isoforms in the oxidation of endobiotic progesterone have not been fully investigated. In this study, the roles of marmoset P450 isoforms in progesterone hydroxylation were extensively determined. 2. The activities of liver microsomes from individual marmosets with respect to progesterone 21/17α- and 16α/6ß-hydroxylation were significantly correlated with those for flurbiprofen 4-hydroxylation and midazolam 1'-hydroxylation, respectively, as similar correlations have been found in humans. Anti-P450 2 C and 3 A antibodies suppressed progesterone 21/17α- and 16α/6ß-hydroxylation, respectively, in marmoset liver microsomes. 3. Recombinant marmoset P450 2C58 and 2C19 catalyzed progesterone to form 21-hydroxyprogesterone and 16α-hydroxyprogesterone, respectively, as major products with high maximum velocity/Km values of 0.53 and 0.089 mL/min/nmol, respectively. Recombinant marmoset P450 3A4/90 oxidized progesterone to form 6ß-hydroxyprogesterone as a major product with homotropic cooperativity (>1 of Hill coefficients). 4. These results indicate that the overall activities and roles of liver microsomal P450 enzymes in marmoset livers are similar to those in humans, especially for progesterone 21/17α- and 16α/6ß-hydroxylation by marmoset P450 2 C and 3 A enzymes, respectively, suggesting important roles for these P450 enzymes in the metabolism of endobiotics in marmosets.

Functional expression and comparative characterization of four feline P450 cytochromes using fluorescent substrates.

1. Cytochrome P450s (CYP) are a major group of metabolizing enzymes for xenobiotics in humans and other mammals. The properties of CYP isoforms in the domestic cat, an obligate carnivore, are largely unknown at present. In this study, we studied relative expression in tissues and enzymatic properties of nine significant feline CYP isoforms. 2. CYP2E2 transcript was most abundant in the feline liver, followed by CYP2A13 and 2E1. Transcripts of CYP3A131, 1A2 and 1A1 were also present in the liver, while CYP2D6 and 3A132 were only slightly expressed. CYP3A131 was a major transcript in the small intestine. 3. Four major CYP isoforms in the feline liver and small intestine (CYP1A2, CYP2A13, CYP2E2 and CYP3A131) were heterologously expressed in Escherichia coli to generate functional monooxygenase systems. We carried out screenings of 17 test compounds known to be inhibitors of CYP isoforms in other mammals as well as two anticancer drugs to assess the activity modulation of feline CYP isoforms using fluorogenic substrates. These CYP isoforms showed similar selectivity to counterparts in other mammals against inhibitors as a whole but with many exceptions. 4. The present study suggests the usefulness of the feline CYP recombinant system to obtain chemical affinity information and possible drug interactions in CYP metabolism of domestic cats.

Pre-incubation with cyclosporine A potentiates its inhibitory effects on pitavastatin uptake mediated by recombinantly expressed cynomolgus monkey hepatic organic anion transporting polypeptide.

Cyclosporine A, an inhibitor of hepatic organic anion transporting polypeptides (OATPs), reportedly increased plasma concentrations of probe substrates, although its maximum unbound blood concentrations were lower than the experimental half-maximal inhibitory (IC50 ) concentrations. Pre-incubation with cyclosporine A in vitro before simultaneous incubation with probes has been reported to potentiate its inhibitory effects on recombinant human OATP-mediated probe uptake. In the present study, the effects of cyclosporine A and rifampicin on recombinant cynomolgus monkey OATP-mediated pitavastatin uptake were investigated in pre- and simultaneous incubation systems. Pre-incubation with cyclosporine A, but not with rifampicin, decreased the apparent IC50 values on recombinant cynomolgus monkey OATP1B1- and OATP1B3-mediated pitavastatin uptake. Application of the co-incubated IC50 values toward R values (1 + [unbound inhibitor]inlet to the liver, theoretically maximum /inhibition constant) in static models, 1.1 in monkeys and 1.3 in humans, for recombinant cynomolgus monkey and human OATP1B1-mediated pitavastatin uptake might result in the poor prediction of drug interaction magnitudes. In contrast, the lowered IC50 values after pre-incubation with cyclosporine A provided better prediction with R values of 3.9 for monkeys and 2.7 for humans when the estimated maximum cyclosporine A concentrations at the inlet to the liver were used. These results suggest that the enhanced inhibitory potential of perpetrator medicines by pre-incubation on cynomolgus monkey OATP-mediated pitavastatin uptake in vitro could be of value for the precise estimation of drug interaction magnitudes in silico, in accordance with the findings from pre-administration of inhibitors on pitavastatin pharmacokinetics validated in monkeys. Copyright © 2016 John Wiley & Sons, Ltd.

Molecular Cloning, Tissue Distribution, and Functional Characterization of Marmoset Cytochrome P450 1A1, 1A2, and 1B1.

The common marmoset (Callithrix jacchus), a New World monkey, has potential to be an animal model for drug metabolism studies. In this study, we identified and characterized cytochrome P450 (P450) 1A1 and 1B1 in addition to the known P450 1A2 in marmosets. Marmoset P450 1A1 and 1B1 cDNA contained open reading frames encoding 512 and 543 amino acids, respectively, with high sequence identities (90%-93%) to other primate P450 1A1s and 1B1s. A phylogenetic tree based on amino acid sequences showed close evolutionary relationships among marmoset, macaque, and human P450 1A and 1B enzymes. By mRNA quantification and immunoblot analyses in five marmoset tissues, P450 1A1 was mainly expressed in lungs and small intestines, and P450 1A2 was expressed predominantly in livers. In contrast, P450 1B1 was expressed in all tissues tested. Marmoset P450 1A1, 1A2, and 1B1 heterologously expressed in Escherichia coli catalyzed 7-ethoxyresorufin O-deethylation, 7-ethoxycoumarin O-deethylation, and phenacetin O-deethylation, similar to those of humans and cynomolgus monkeys. Notably, marmoset P450 1A1 and 1A2 more efficiently catalyzed 7-ethoxyresorufin O-deethylation than those of the human homologs, but were comparable to those of the cynomolgus monkey homologs. Additionally, marmoset P450 1B1 preferentially catalyzed estradiol 4-hydroxylation; however, rat P450 1B1 more favorably catalyzed estradiol 2-hydroxylation, indicating that the estradiol hydroxylation specificity of marmoset P450 1B1 was similar to those of human and cynomolgus monkey P450 1B1. These results indicated that marmoset P450 1A and 1B enzymes had functional characteristics similar to those of humans and cynomolgus monkeys, suggesting that P450 1A and 1B-dependent metabolism was similar among marmosets, cynomolgus monkeys, and humans.

Substrate Selectivities and Catalytic Activities of Marmoset Liver Cytochrome P450 2A6 Differed from Those of Human P450 2A6.

The common marmoset (Callithrix jacchus), a New World primate species, is potentially a useful animal model for preclinical studies in drug development. However, cytochrome P450 (P450) enzymes have not been fully identified and characterized in marmosets. In this study, we identified P450 2A6 cDNA with the sequence highly identical (91-94%) to human P450 2A6, 2A7, and 2A13 cDNA and cynomolgus monkey P450 2A23, 2A24, and 2A26 cDNA. Among the tissue types examined, marmoset P450 2A6 mRNA was most abundantly expressed in livers where P450 2A6 protein was also detected by immunoblotting. Phylogenetic analysis showed that marmoset P450 2A6 was more closely clustered with human and cynomolgus monkey P450 2As than P450 2As of dog, rat, and mouse (the species also used in drug metabolism). Marmoset P450 2A6 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation, similar to human P450 2A6 and 2A13 and cynomolgus monkey P450 2A23, 2A24, and 2A26, but much less effectively coumarin 7-hydroxylation, showing some difference as well. Interestingly, marmoset P450 2A6 and cynomolgus monkey P450 2A23 catalyzed phenacetin O-deethylation, which is catalyzed by human P450 1A2 and 2A13, but not by P450 2A6. Marmoset P450 2A6 also exhibited catalytic activity toward testosterone by the multiple sites, but not rat P450 2A-specific testosterone 7α-hydroxylation activity. These results indicated that marmoset P450 2A6 had functional characteristics different from those of human and cynomolgus monkey P450 2As in terms of partially different substrate specificities and catalytic activities, indicating its importance of further studies for P450 2A-dependent drug metabolism in marmosets.

Identification and functional characterization of novel feline cytochrome P450 2A.

1. Cytochrome P450s are the major metabolizing enzymes for xenobiotics in humans and other mammals. Although the domestic cat Felis catus, an obligate carnivore, is the most common companion animal, the properties of cytochrome P450 subfamilies are largely unknown. 2. We newly identified the feline CYP2A13, which consists of 494 deduced amino acids, showing the highest identity to CYP2As of dogs, followed by those of pigs, cattle and humans. 3. The feline CYP2A13 transcript and protein were expressed almost exclusively in the liver without particular sex-dependent differences. 4. The feline CYP2A13 protein heterogeneously expressed in Escherichia coli showed metabolic activity similar to those of human and canine CYP2As for coumarin, 7-ethoxycoumarin and nicotine. 5. The results indicate the importance of CYP2A13 in systemic metabolism of xenobiotics in cats.

Evaluation of 89 compounds for identification of substrates for cynomolgus monkey CYP2C76, a new bupropion/nifedipine oxidase.

Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity.