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Pigs are sometimes used in preclinical drug metabolism studies, with growing interest, and thus their drug-metabolizing enzymes, including the cytochromes P450 (P450 or CYP; EC 1.14.14.1), need to be examined. In the present study, novel CYP4A cDNAs were isolated and characterized, namely, pig CYP4A23 and CYP4A90; cat CYP4A37 and CYP4A106; and tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g. For comparison, the following known CYP4A cDNAs were also analyzed: pig CYP4A21 and dog CYP4A37, CYP4A38, and CYP4A39. These CYP4A cDNAs all contained open reading frames of 504-513 amino acids and had high amino acid sequence identity (74%-80%) with human CYP4As. Phylogenetic analysis of amino acid sequences revealed that these CYP4As were clustered in each species. All CYP4A genes contained 12 coding exons and formed a gene cluster in the corresponding genomic regions. A range of tissue types were analyzed, and these CYP4A mRNAs were preferentially expressed in liver and/or kidney, except for pig CYP4A90, which showed preferential expression in lung and duodenum. CYP4A enzymes, heterologously expressed in Escherichia coli , preferentially catalyzed lauric acid 12-hydroxylation and arachidonic acid 20-hydroxylation, just as human CYP4A11 does, with the same regioselectivity (i.e., at the ω -position of fatty acids). These results imply that dog, cat, pig, and tree shrew CYP4As have functional characteristics similar to those of human CYP4A11, with minor differences in lauric acid 12-hydroxylation. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450, CYP) 4As are important P450s in human biological processes because of their fatty acid-metabolizing ability. Pig CYP4A21, CYP4A23, and CYP4A90; cat CYP4A37 and CYP4A106; tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g; and dog CYP4A37, CYP4A38, and CYP4A39 cDNAs were isolated and analyzed. These CYP4A cDNAs shared relatively high sequence identities with human CYP4A11 and CYP4A22. Pig, cat, tree shrew, and dog CYP4As in the liver and kidneys are likely to catalyze the ω -hydroxylation of fatty acids., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

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.

Cynomolgus macaques are used in preclinical studies in part because of their evolutionary closeness to humans. However, drug transporters [including solute carrier (SLC) transporters] essential for the absorption and excretion of drugs have not been fully investigated at the molecular level in cynomolgus macaques. We identified and characterized cynomolgus macaque SLC15A1, SLC15A2, SLC22A1, SLC22A2, SLC22A6, SLC22A8, SLC47A1, and SLC47A2, along with SLCO (formerly SLC21A) transporters SLCO1A2, SLCO1B1, SLCO1B3, and SLCO2B1. These cynomolgus SLC transporters had high amino acid sequence identities (92-97%) with their human orthologs and contained sequence motifs characteristic of SLC transporters. Phylogenetic analysis showed that these cynomolgus SLC transporters were more closely clustered with their human orthologs than with those of dogs, rats, or mice. Gene structure and genomic organization were similar in macaques and humans. Cynomolgus SLC transporter mRNAs showed distinct tissue expression patterns, being most abundantly expressed in jejunum (SLC15A1), liver (SLC22A1, SLCO1B1, and SLCO2B1), and kidney (SLC15A2, SLC22A2, SLC22A6, SLC22A8, SLC47A1, SLC47A2, and SLCO1A2). In contrast, cynomolgus SLCO2B1 mRNA was more ubiquitously expressed. Among these SLC mRNAs, the most abundant in liver was SLCO1B1, in jejunum SLC15A1, and in kidney SLC22A2. These results suggest similar characteristics of SLC transporters in cynomolgus macaques and humans., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2021 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

For drug development, species differences in drug-metabolism reactions present obstacles for predicting pharmacokinetics in humans. We characterized the species differences in hydrolases among humans and mice, rats, dogs, and cynomolgus monkeys. In this study, to expand the series of such studies, we attempted to characterize marmoset hydrolases. We measured hydrolase activities for 24 compounds using marmoset liver and intestinal microsomes, as well as recombinant marmoset carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC). The contributions of CES1, CES2, and AADAC to hydrolysis in marmoset liver microsomes were estimated by correcting the activities by using the ratios of hydrolase protein levels in the liver microsomes and those in recombinant systems. For six out of eight human CES1 substrates, the activities in marmoset liver microsomes were lower than those in human liver microsomes. For two human CES2 substrates and three out of seven human AADAC substrates, the activities in marmoset liver microsomes were higher than those in human liver microsomes. Notably, among the three rifamycins, only rifabutin was hydrolyzed by marmoset tissue microsomes and recombinant AADAC. The activities for all substrates in marmoset intestinal microsomes tended to be lower than those in liver microsomes, which suggests that the first-pass effects of the CES and AADAC substrates are due to hepatic hydrolysis. In most cases, the sums of the values of the contributions of CES1, CES2, and AADAC were below 100%, which indicated the involvement of other hydrolases in marmosets. In conclusion, we clarified the substrate preferences of hydrolases in marmosets. SIGNIFICANCE STATEMENT: This study confirmed that there are large differences in hydrolase activities between humans and marmosets by characterizing marmoset hydrolase activities for compounds that are substrates of human CES1, CES2, or arylacetamide deacetylase. The data obtained in this study may be useful for considering whether marmosets are appropriate for examining the pharmacokinetics and efficacies of new chemical entities in preclinical studies., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Paraoxonase (PON) plays roles in the metabolism of organophosphate xenobiotics and drugs. Despite the importance of marmosets for research into drug metabolism and pharmacokinetics, marmoset paraoxonase has not yet been fully characterized. Consequently, we identified the PON1 gene in the marmoset genome by sequence homology analysis. Marmoset PON1 cDNA containing an open reading frame (1065 bp) was successfully cloned from marmoset liver by reverse transcription-polymerase chain reaction. The deduced amino acid sequence (355 amino acids) has approximately 93% identity with the human ortholog and contains important amino acid residues for substrate binding and calcium ion coordination. According to a phylogenetic tree of PON1 amino acid sequences constructed using data from seven animal species, marmoset PON1 is closer to human PON1 than it is to the PON1 orthologs of experimental animals such as pigs, rabbits, rats, and mice. Marmoset PON1 mRNA was predominantly expressed in liver among the five tissues examined. Marmoset PON1 protein secreted into plasma was detected by immunoblotting. The paraoxon-hydrolyzing activity in plasma was higher in marmosets than in humans. Based on these data, we concluded that marmoset and human PON1 have similar characteristics with regard to genomic structure, amino acid sequences, and tissue distribution., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2021 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Polymorphic human and cynomolgus macaque flavin-containing monooxygenases (FMO) 3 are important oxygenation enzymes for nitrogen-containing drugs. Inter-animal variability of FMO3-dependent drug oxygenations in vivo is suspected in cynomolgus macaques because such variability is evident in humans. Therefore, this follow-up study was performed to investigate the pharmacokinetics of orally administered deuterium-labeled trimethylamine in three cynomolgus macaques genotyped for FMO3. Trimethylamine-d 9 was rapidly absorbed and attained plasma concentrations greater than the background levels of non-labeled trimethylamine. Trimethylamine-d 9 was then converted to trimethylamine-d 9 N-oxide. The half-lives, maximum plasma concentrations, and areas under the curve for trimethylamine-d 9 and its N-oxygenated metabolite and the total clearance for orally administered trimethylamine-d 9 were not different among the heterozygote for Q506K FMO3, the heterozygote for V325I FMO3, and the heterozygote for both S99N and F510S FMO3. Trimethylamine N-oxygenation activities mediated by liver microsomes prepared from the same three animals were not substantially different. However, recombinant proteins of the corresponding cynomolgus FMO3 variants showed apparent reduced trimethylamine N-oxygenation activities compared with the wild-type proteins. This study suggests only limited polymorphic effects on the in vivo catalytic function of cynomolgus FMO3. These findings yield important insights in terms of both quantitative and qualitative variations of polymorphic FMO3 in cynomolgus liver., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Common marmoset (Callithrix jacchus) is an attractive animal model primate species for potential use in drug metabolism and pharmacokinetic studies. In this study, marmoset cytochrome P450 (P450) 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 cDNAs were isolated from marmoset tissues (brains, lungs, livers, kidneys, and jejunums). Deduced amino acid sequences (89-98% homologous) of the marmoset P450 gene suggested similarity of molecular characteristics of marmoset P450s to human counterparts, compared with those of pig, rabbit, and rodents. Phylogenetic analysis using amino acid sequences indicated 11 marmoset P450 forms clustered with those of human and other primate counterparts, suggesting marmoset P450s have an evolutionary close relationship to human and other primate counterparts. Tissue expression patterns of these P450 mRNAs except for P450 7B1 mRNA were generally similar to those of human P450s in the five tissue types analyzed. These results suggest similarity of molecular characteristics for P450 2S1, 4V2, 7A1, 7B1, 8B1, 24A1, 26A1, 26C1, 27A1, 39A1, and 51A1 between marmosets and humans, in addition to the orthologs of human P450 1, 2, 3, and 4 families previously identified and characterized in marmosets., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker of hematopoietic stem cells (HSCs). Although we previously elucidated the functional importance of ESAM in HSCs in stress-induced hematopoiesis in adults, it is unclear how ESAM affects hematopoietic development during fetal life. To address this issue, we analyzed fetuses from conventional or conditional ESAM-knockout mice. Approximately half of ESAM-null fetuses died after mid-gestation due to anemia. RNA sequencing analyses revealed downregulation of adult-type globins and Alas2, a heme biosynthesis enzyme, in ESAM-null fetal livers. These abnormalities were attributed to malfunction of ESAM-null HSCs, which was demonstrated in culture and transplantation experiments. Although crosslinking ESAM directly influenced gene transcription in HSCs, observations in conditional ESAM-knockout fetuses revealed the critical involvement of ESAM expressed in endothelial cells in fetal lethality. Thus, we showed that ESAM had important roles in developing definitive hematopoiesis. Furthermore, we unveiled the importance of endothelial ESAM in this process., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

1. Compared to information for herbivores and omnivores, knowledge on xenobiotic metabolism in carnivores is limited. The cytochrome P450 2C (CYP2C) subfamily is recognized as one of the most important CYP groups in human and dog. We identified and characterized CYP2C isoforms and variants in cat, which is an obligate carnivore. 2. Quantitative RT-PCR and immunoblot analyses were carried out to evaluate the expression of CYP2C in the liver and small intestine. A functional CYP2C isoform was heterologously expressed in yeast microsomes to determine the enzymatic activity. 3. Cat had two CYP2C genes, 21 and 41, in the genome; however, CYP2C21P was a pseudogene that had many stop codons. Three splicing variants of CYP2C41 were identified (v1-v3), but only one of them (v1) showed a complete deduced amino acid sequence as CYP2C protein. Transcripts of feline CYP2C41v1 were detected but the amounts were negligible or very small in the liver and small intestine. Immunoreactivity to an antihuman CYP2C antibody was confirmed in the recombinant feline CYP2C41v1 but not in the feline liver. 4. Recombinant feline CYP2C41v1 metabolized several substrates, including dibenzylfluorescein that is specific to human CYP2C. 5. The results suggest a limited role of functional CYP2C isoforms in xenobiotic metabolism in cat.

The common marmoset (Callithrix jacchus) is a useful experimental animal to evaluate the pharmacokinetics of drug candidates. Cytochrome P450 (P450) 2B enzyme in marmoset livers has been identified; however, only limited information on the enzymatic properties and distribution has been available. Marmoset P450 2B6 amino acids showed high sequence identities (>86%) with those of primates including humans and cynomolgus monkeys. Phylogenetic analysis using amino acid sequences indicated that marmoset P450 2B6 was closer to human and cynomolgus monkey P450 2B6 than to P450 2B orthologs of other species, including pigs, dogs, rabbits and rodents. Quantitative polymerase chain reaction analysis using specific primers showed P450 2B6 mRNA predominantly expressed in livers among the five marmoset tissues, similar to those of humans and cynomolgus monkeys. Marmoset P450 2B6 heterologously expressed in Escherichia coli membranes oxidized 7-ethoxycoumarin, pentoxyresorufin, propofol and testosterone, at roughly similar rates to those of humans and/or cynomolgus monkeys. A high capacity of marmoset P450 2B6 with propofol 4-hydroxylation (at low ionic strength conditions) with a low K m value was relatively comparable to that for marmoset livers. These results collectively indicated a high propofol 4-hydroxylation activity of P450 2B6 expressed in marmoset livers.

Carboxylesterase (CES) is important for the detoxification of a wide range of drugs and xenobiotics. In this study, the hepatic level of CES2 mRNA was examined in cynomolgus macaques used widely in preclinical studies for drug metabolism. Three CES2 mRNAs were present in cynomolgus macaque liver. The mRNA level was highest for cynomolgus CES2A (formerly CES2v3), much lower for cynomolgus CES2B (formerly CES2v1) and extremely low for cynomolgus CES2C (formerly CES2v2). Most various transcript variants produced from cynomolgus CES2B gene did not contain a complete coding region. Thus, CES2A is the major CES2 enzyme in cynomolgus liver. A new transcript variant of CES2A, CES2Av2, was identified. CES2Av2 contained exon 3 region different from wild-type (CES2Av1). In cynomolgus macaques expressing only CES2Av2 transcript, CES2A contained the sequence of CES2B in exon 3 and vicinity, probably due to gene conversion. On genotyping, this CES2Av2 allele was prevalent in Indochinese cynomolgus macaques, but not in Indonesian cynomolgus or rhesus macaques. CES2Av2 recombinant protein showed similar activity to CES2Av1 protein for several substrates. It is concluded that CES2A is the major cynomolgus hepatic CES2, and new transcript variant, CES2Av2, has similar functions to CES2Av1.

Background: Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical studies. Information for major drug-metabolizing cytochrome P450 (P450) enzymes is now available that supports the use of this primate species as an animal model for drug development. Here, we collect and provide an overview of information on the activities of common marmoset hepatic and intestinal microsomes with respect to 28 typical human P450 probe oxidations., Results: Marmoset P450 2D6/8-dependent R-metoprolol O-demethylation activities in hepatic microsomes were significantly correlated with those of midazolam 1'- and 4-hydroxylations, testosterone 6β-hydroxylation, and progesterone 6β-hydroxylation, which are probe reactions for marmoset P450 3A4/5/90. In marmosets, the oxidation activities of hepatic microsomes and intestinal microsomes were roughly comparable for midazolam and terfenadine. Overall, multiple forms of marmoset P450 enzymes in livers and intestines had generally similar substrate recognition functionalities to those of human and/or cynomolgus monkey P450 enzymes., Conclusion: The marmoset could be a model animal for humans with respect to the first-pass extraction of terfenadine and related substrates. These findings provide a foundation for understanding individual pharmacokinetic and toxicological results in nonhuman primates as preclinical models and will help to further support understanding of the molecular mechanisms of human P450 function., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

1. Simulated clearances of R-warfarin and efavirenz were recently reported for individual cynomolgus monkeys genotyped for cytochrome P450 2C19 and 2C9, respectively. To expand and verify this modeling procedure, simulations of R/S-omeprazole and R/S-warfarin clearances after oral administrations in individual marmosets were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling consisting of gut, liver and central compartments. 2. Pharmacokinetics of R/S-omeprazole were chirally determined using the previously reported plasma microsamples in this study. The areas under the plasma concentration/time curves (AUC) of R-omeprazole and S-warfarin, but not S-omeprazole and R-warfarin, after oral administrations in the P450 2C19 homozygous mutant group were significantly higher than those in the wild-type group. These modeled hepatic intrinsic clearances were also significantly associated with the marmoset P450 2C19 genotypes. Other parameter values, e.g. absorption rate constants or systemic circulation volumes, were not likely determining factors. 3. The reported individual AUC values measured in 4-6 marmosets after oral R-omeprazole and S-warfarin administrations were significantly correlated with the AUC values predicted using the PBPK models after virtual administrations. 4. This study indicates that clearances of R-omeprazole, S-warfarin and related medicines associated with polymorphic P450 2C19 in individual marmosets can be simulated using simplified individual PBPK models.

1. A potentially useful animal model for preclinical studies is the common marmoset (Callithrix jacchus). In this study, using reverse-transcription polymerase chain reaction from marmoset livers, we identified a novel cytochrome P450 (P450) 2F1 cDNA with an open reading frame of 1473 bp. 2. High sequence identities of 92-94% with primate P450 2 F amino acid sequences were indicated by deduced amino acid sequences of P450 2F1 cDNA. Phylogenetic analysis indicates that marmoset P450 2F1 is more congruent with primate P450 2 F forms than those of other species such as rodents. 3. Among five tissue types examined, abundant expression of marmoset P450 2F1 mRNA and P450 2F1 protein in lungs was shown. Cynomolgus monkey P450 2F1 mRNA was abundantly expressed in lungs as well as testes and ovaries in 10 tissue types. 4. Similar to those of humans and cynomolgus monkeys, marmoset P450 2F1 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation and biphenyl hydroxylation, however unlike human P450 2F1, marmoset P450 2F1 exhibited hydroxylation activity toward coumarin and chlorzoxazone. 5. These findings indicated that P450 2F1 enzyme expressed in marmoset lungs and also catalyzed metabolism of xenobiotics, suggesting the importance of P450 2 F-dependent drug metabolism in marmoset lungs.

1. Roles of human cytochrome P450 (P450) 3A4 in oxidation of an antihistaminic drug terfenadine have been previously investigated in association with terfenadine-ketoconazole interaction. Several antihistamine drugs have been recently identified as substrates for multiple P450 enzymes. In this study, overall roles of P450 3A4, 2J2, and 4F12 enzymes in terfenadine t-butyl hydroxylation were investigated in small intestines and livers from humans, marmosets, and/or cynomolgus monkeys. 2. Human liver microsomes and liver and small intestine microsomes from marmosets and cynomolgus monkeys effectively mediated terfenadine t-butyl hydroxylation. Ketoconazole and N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (a P450 4A/F inhibitor) almost completely and moderately inhibited these activities, respectively, in human liver microsomes; however, these chemicals did not show substantially suppression in marmoset liver. Anti-human P450 3A and 4F antibodies showed the roughly supportive inhibitory effects. 3. Recombinant P450 3A4/90 and 4F12 showed high terfenadine t-butyl hydroxylation activities with substrate inhibition constants of 84-144 μM (under 26-76 μM of K m values), in similar manners to liver and intestine microsomes. 4. These results suggest that human and marmoset P450 3A4/90 and 4F12 in livers or small intestines played important roles in terfenadine t-butyl hydroxylation. Marmosets could be a model for humans during first pass extraction of terfenadine and related substrates.

Cytochromes P450 (P450) largely remain to be characterized in great apes. Comparative immunochemical detection of drug metabolizing forms of P450s 1A, 2A, 2B, 2C, 2D, 2E, 2J, 3A, 4A, and 4F in liver microsomes from chimpanzees, gorillas, orangutans, gibbons, cynomolgus and rhesus macaques, and common marmosets were carried out., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

A β -blocker, metoprolol, is one of the in vivo probes for human cytochrome P450 (P450) 2D6. Investigation of nonhuman primate P450 enzymes helps to improve the accuracy of the extrapolation of pharmacokinetic data from animals into humans. Common marmosets ( Callithrix jacchus ) are a potential primate model for preclinical research, but the detailed roles of marmoset P450 enzymes in metoprolol oxidation remain unknown. In this study, regio- and stereo-selectivity of metoprolol oxidations by a variety of P450 enzymes in marmoset and human livers were investigated in vitro. Although liver microsomes from cynomolgus monkeys and rats preferentially mediated S -metoprolol O -demethylation and R -metoprolol α -hydroxylation, respectively, those from humans, marmosets, minipigs, and dogs preferentially mediated R -metoprolol O -demethylation, in contrast to the slow rates of R - and S -metoprolol oxidation in mouse liver microsomes. R - and S -metoprolol O -demethylation activities in marmoset livers were strongly inhibited by quinidine and ketoconazole, and were significantly correlated with bufuralol 1'-hydroxylation and midazolam 1'-hydroxylation activities and also with P450 2D and 3A4 contents, which is different from the case in human livers that did not have any correlations with P450 3A-mediated midazolam 1'-hydroxylation. Recombinant human P450 2D6 enzyme and marmoset P450 2D6/3A4 enzymes effectively catalyzed R -metoprolol O -demethylation, comparable to the activities of human and marmoset liver microsomes, respectively. These results indicated that the major roles of P450 2D enzymes for the regio- and stereo-selectivity of metoprolol oxidation were similar between human and marmoset livers, but the minor roles of P450 3A enzymes were unique to marmosets., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

1. A cDNA encoding novel cytochrome P450 (P450) 4A enzyme was cloned from marmoset livers by reverse transcription (RT)-polymerase chain reaction (PCR) based on the marmoset genome sequences. The amino acid sequence deduced from P450 4A11 cDNA contained consensus sequences of six substrate recognition sites and one heme-binding domain. 2. Marmoset P450 4A11, highly identical (85-88%) to cynomolgus monkey and human P450 4A enzymes, was grouped into the same cluster as cynomolgus monkey and human P450 4A enzymes by phylogenetic analysis. 3. The tissue distribution analyses by real-time RT PCR and immunoblotting demonstrated that marmoset P450 4A11 mRNA and proteins were expressed in kidneys and livers. Marmoset P450 4A11 enzyme heterologously expressed in Escherichia coli preferentially catalyzed the ω-hydroxylation of arachidonic acid and lauric acid, similar to cynomolgus monkey and human P450 4A11 enzymes. However, lauric acid ω-hydroxylation activity of marmoset P450 4A11 was low compared with those of marmoset liver microsomes. 4. These results indicated that novel marmoset P450 4A11 was also a fatty acid ω-hydroxylase expressed in kidneys and livers, with the same regioselectivity (at ω-position of fatty acid) as cynomolgus monkey and human P450 4A enzymes.

Common marmosets ( Callithrix jacchus ) are potentially primate models for preclinical drug metabolism studies because there are similarities in the molecular characteristics of cytochrome P450 enzymes between this species and humans. However, characterization of non-cytochrome P450 enzymes has not been clarified in marmosets. Here, we report characterization of flavin-containing monooxygenases FMO1-FMO5 identified in marmoset tissues. Marmoset FMO forms shared high amino acid sequence identities (93%-95%) and phylogenetic closeness with human homologous FMO forms. FMO1 and FMO3 mRNA were abundantly expressed in the liver and kidneys among five marmoset tissues examined, where FMO3 protein was detected by immunoblotting. FMO inhibition assays using preheated tissue microsomes indicated that benzydamine N -oxygenation and sulindac sulfide S -oxygenation in the marmoset liver was mainly catalyzed by FMO3, the major hepatic FMO. Marmoset FMO3 protein heterologously expressed in Escherichia coli effectively catalyzed benzydamine N -oxygenation and sulindac sulfide S -oxygenation comparable to marmoset liver microsomes. These results indicate that the FMO3 enzyme expressed in marmoset livers mainly metabolizes benzydamine and sulindac sulfide (typical human FMO substrates), suggesting its importance for FMO-dependent drug metabolism in marmosets., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Common marmosets ( Callithrix jacchus ), small New World primates, are increasingly attracting attention as potentially useful animal models for drug development. However, characterization of cytochrome P450 (P450) 3A enzymes involved in the metabolism of a wide variety of drugs has not investigated in marmosets. In this study, sequence homology, tissue distribution, and enzymatic properties of marmoset P450 3A4 ortholog, 3A5 ortholog, and 3A90 were investigated. Marmoset P450 3A forms exhibited high amino acid sequence identities (88-90%) to the human and cynomolgus monkey P450 3A orthologs and evolutionary closeness to human and cynomolgus monkey P450 3A orthologs compared with other P450 3A enzymes. Among the five marmoset tissues examined, P450 3A4 ortholog mRNA was abundant in livers and small intestines where P450 3A4 ortholog proteins were immunologically detected. Three marmoset P450 3A proteins heterologously expressed in Escherichia coli membranes catalyzed midazolam 1'- and 4-hydroxylation, alprazolam 4-hydroxylation, nifedipine oxidation, and testosterone 6 β -hydroxylation, similar to cynomolgus monkey and human P450 3A enzymes. Among the marmoset P450 3A enzymes, P450 3A4 ortholog effectively catalyzed midazolam 1'-hydroxylation, comparable to microsomes from marmoset livers and small intestines. Correlation analyses with 23 individual marmoset liver microsomes suggested contributions of P450 3A enzymes to 1'-hydroxylation of both midazolam (human P450 3A probe) and bufuralol (human P450 2D6 probe), similar to cynomolgus monkey P450 3A enzymes. These results indicated that marmoset P450 3A forms had functional characteristics roughly similar to cynomolgus monkeys and humans in terms of tissue expression patterns and catalytic activities, suggesting marmosets as suitable animal models for P450 3A-dependent drug metabolism., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Sex and age differences in hepatic expression of drug-metabolizing enzyme genes could cause variations in drug metabolism, but has not been fully elucidated, especially in Asian population. In this study, the global expression of human hepatic genes was analyzed by microarrays in 40 Japanese subjects (27 males and 13 females). Thirty-five sex-biased genes were identified (P < 0.005). Whereas, 60 age-biased genes in two age groups, <60 years and ≥70 years (P < 0.001), were identified in males. By Gene Ontology analysis, the sex-biased genes were related to protein catabolism and modification, while the age-biased genes were related to transcription regulation and cell death. Quantitative polymerase chain reaction confirmed the female-biased expression of drug-metabolizing enzyme genes BChE, CYP4X1, and SULT1E1 (≥1.5-fold, P < 0.05). Further analysis of drug-metabolizing enzyme genes indicated that expression of CYP2A6 and CYP3A4 in females in the ≥70 age group was less than in the <60 age group (≥1.5-fold, P < 0.05), and this trend was also observed for PXR expression in males (≥1.5-fold, P < 0.05). The results presented provide important insights into hepatic physiology and function, especially drug metabolism, with respect to sex and age., (Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Marmoset cytochrome P450 2C19, highly homologous to human P450 2C9 and 2C19, has been identified in common marmosets (Callithrix jacchus), a nonhuman primate species used in drug metabolism studies. Although genetic variants in human and macaque P450 2C genes account for the interindividual variability in drug metabolism, genetic variants have not been investigated in the marmoset P450 2C19 In this study, sequencing of P450 2C19 in 24 marmosets identified three variants p.[(Phe7Leu; Ser254Leu; Ile469Thr)], which showed substantially reduced metabolic capacity of S-warfarin compared with the wild-type group in vivo and in vitro. Although mean plasma concentrations of R-warfarin in marmosets determined after chiral separation were similar between the homozygous mutant and wild-type groups up to 24 hours after the intravenous and oral administrations of racemic warfarin, S-warfarin depletion from plasma was significantly faster in the three wild-type marmosets compared with the three homozygous mutant marmosets. These variants, cosegregating in the marmosets analyzed, influenced metabolic activities in 18 marmoset liver microsomes because the homozygotes and heterozygotes showed significantly reduced catalytic activities in liver microsomes toward S-warfarin 7-hydroxylation compared with the wild-type group. Kinetic analysis for S-warfarin 7-hydroxylation indicated that the recombinant P450 2C19 Ser254Leu variant would change the metabolic capacity. These results indicated that the interindividual variability of P450 2C-dependent drug metabolism such as S-warfarin clearance is at least partly accounted for by P450 2C19 variants in marmosets, suggesting that polymorphic P450 2C-dependent catalytic functions are relatively similar between marmosets and humans., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Common marmosets (Callithrix jacchus) are attracting attention as animal models in preclinical studies for drug development. However, cytochrome P450s (P450s), major drug-metabolizing enzymes, have not been fully identified and characterized in marmosets. In this study, based on the four novel P450 4F genes found on the marmoset genome, we successfully isolated P450 4F2, 4F3B, 4F11, and 4F12 cDNAs in marmoset livers. Deduced amino acid sequences of the four marmoset P450 4F forms exhibited high sequence identities (87%-93%) to the human and cynomolgus monkey P450 4F homologs. Marmoset P450 4F3B and 4F11 mRNAs were predominantly expressed in livers, whereas marmoset P450 4F2 and 4F12 mRNAs were highly expressed in small intestines and livers. Four marmoset P450 4F proteins heterologously expressed in Escherichia coli catalyzed the ω-hydroxylation of leukotriene B4 In addition, marmoset P450 4F12 effectively catalyzed the hydroxylation of antiallergy drug ebastine, a human P450 2J/4F probe substrate. Ebastine hydroxylation activities by small intestine and liver microsomes from marmosets and cynomolgus monkeys showed greatly higher values than those of humans. Ebastine hydroxylation activities by marmoset and cynomolgus monkey small intestine microsomes were inhibited (approximately 60%) by anti-P450 4F antibodies, unlike human small intestine microsomes, suggesting that contribution of P450 4F enzymes for ebastine hydroxylation in the small intestine might be different between marmosets/cynomolgus monkeys and humans. These results indicated that marmoset P450 4F2, 4F3B, 4F11, and 4F12 were expressed in livers and/or small intestines and were functional in the metabolism of endogenous and exogenous compounds, similar to those of cynomolgus monkeys and humans., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

The microminipig is one of the smallest minipigs that has emerged as a possible experimental animal model, because it shares many anatomical and/or physiological similarities with humans, including the coronary artery distribution in the heart, the digestive physiology, the kidney size and its structure, and so on. However, information on gene expression profiles, including those on drug-metabolizing phase I and II enzymes, in the microminipig is limited. Therefore, the aim of the present study was to identify transcripts in microminipig livers and to determine gene expression profiles. De novo assembly and expression analyses of microminipig transcripts were conducted with liver samples from three male and three female microminipigs using parallel long-read and short-read sequencing technologies. After unique sequences had been automatically aligned by assembling software, the mean contig length of 50843 transcripts was 707 bp. The expression profiles of cytochrome P450 (P450) 1A2, 2C, 2E1 and 3A genes in livers in microminipigs were similar to those in humans. Liver carboxylesterase (CES) precursor, liver CES-like, UDP-glucuronosyltransferase (UGT) 2C1-like, amine sulfotransferase (SULT)-like, N-acetyltransferases (NAT8) and glutathione S-transferase (GST) A2 genes, which are relatively unknown genes in pigs and/or humans, were expressed strongly. Furthermore, no significant gender differences were observed in the gene expression profiles of phase I enzymes, whereas UGT2B17, SULT1E1, SULT2A1, amine SULT-like, NAT8 and GSTT4 genes were different between males and females among phase II enzyme genes under the present sample conditions. These results provide a foundation for mechanistic studies and the use of microminipigs as model animals for drug development in the future. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Macaques, including cynomolgus and rhesus macaques, are important animal species used in drug metabolism studies. CYP2D44 is expressed in cynomolgus macaque liver and encodes a functional drug metabolizing enzyme, metabolizing typical human CYP2D substrates such as bufuralol and dextromethorphan. CYP2D44 is highly homologous to human CYP2D6 that is known to be polymorphic with a large inter-individual variation in metabolic activities, however, genetic polymorphisms have not been investigated in macaque CYP2D44. In the present study, screening of 78 cynomolgus and 40 rhesus macaques found a total of 67 variants, including 64 non-synonymous variants, 1 nonsense mutation, and 2 frameshift mutations, and 1 gene conversion, of which 14, 19, and 15 variants were unique to Indochinese cynomolgus macaques, Indonesian cynomolgus macaques, and Chinese rhesus macaques, respectively. Eleven of the 64 non-synonymous variants were located in substrate recognition sites, the regions important for protein function. By site-directed mutagenesis and metabolic assays, S175N, V185L, A235G, R242G, R245K, and N337D showed substantially decreased activity in bufuralol 1'-hydroxylation as compared with wild-type proteins. Moreover, two null alleles (c.128T>del and c.664G>T) were found in Indonesian cynomolgus macaques, but not in Indochinese cynomolgus macaques or Chinese rhesus macaques. These results suggest that genetic polymorphisms might account for the variability of CYP2D44-dependent metabolism in macaques.

At the 6th International Workshop on Genotoxicity Testing (IWGT), the liver micronucleus test working group discussed practical aspects of the in vivo rodent liver micronucleus test (LMNT). The group members focused on the three methodologies currently used, i.e., a partial hepatectomy (PH) method, a juvenile/young rat (JR) method, and a repeated-dose (RD) method in adult rodents. Since the liver is the main organ that metabolizes chemicals, the LMNT is expected to detect clastogens, especially those that need metabolic activation in the liver, and aneugens. Based on current data the three methods seem to have a high sensitivity and specificity, but more data, especially on non-genotoxic but toxic substances, would be needed to fully evaluate the test performance. The three methods can be combined with the micronucleus test (MNT) using bone marrow (BM) and/or peripheral blood (PB). The ability of the PH method to detect both clastogens and aneugens has already been established, but the methodology is technically challenging. The JR method is relatively straightforward, but animal metabolism might not be fully comparable to adult animals, and data on aneugens are limited. These two methods also have the advantage of a short testing period. The RD method is also straightforward and can be integrated into repeated-dose (e.g. 2 or 4 weeks) toxicity studies, but again data on aneugens are limited. The working group concluded that the LMNT could be used as a second in vivo test when a relevant positive result in in vitro mammalian cell genotoxicity tests is noted (especially under the condition of metabolic activation), and a negative result is observed in the in vivo BM/PB-MNT. The group members discussed LMNT protocols and reached consensus about many aspects of test procedures. However, data gaps as mentioned above remain, and further data are needed to fully establish the LMNT protocol., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A repeated-dose liver micronucleus assay using young adult rats was conducted with methyl methanesulfonate (MMS) as a part of a collaborative study supported by the Collaborative Study Group for the Micronucleus Test/the Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group. MMS is a classical DNA-reactive carcinogen, but it is not a liver carcinogen. In the first experiment (14-day study), MMS was administered per os to 6-week-old male Crl:CD (SD) rats every day for 14 days at a dose of 12.5, 25, or 50mg/kg/day. In the second experiment (28-day study), 6-week-old male SD rats were treated with MMS at 7.5, 15, or 30mg/kg/day for 28 days, because the highest dose used in the 14-day study (50mg/kg/day) caused mortality. Hepatocyte and bone marrow cell specimens were prepared on the day after the final dose. The frequency of micronucleated hepatocytes (MNHEPs) in the liver and that of micronucleated immature erythrocytes (MNIMEs) in the bone marrow were evaluated. Exposure to 50mg/kg/day MMS for 14 days resulted in an increased frequency of MNHEPs, but MMS had no effect on the frequency of MNHEPs in the rats exposed to the chemical for 28 days at doses up to 30mg/kg/day. MMS induced MNIMEs production at doses of 25 and 50mg/kg/day in the 14-day study and at doses of 15 and 30mg/kg/day in the 28-day study. Overall, the effect of MMS on the frequency of MNHEPs was considered to be equivocal., (Copyright © 2014 Elsevier B.V. All rights reserved.)

1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates.

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.

1. Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient. 2. A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83-86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat. 3. Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting. 4. By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1'-hydroxylation. 5. These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6.

Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

Drug-drug interactions (DDIs) caused by the inhibition of hepatic uptake transporters such as organic anion transporting polypeptide (OATP) can affect therapeutic efficacy and cause adverse reactions. We investigated the potential utility of pitavastatin as an in vivo probe substrate for preclinically studying OATP-mediated DDIs using cynomolgus monkeys. Cyclosporine A (CsA) and rifampicin (RIF), typical OATP inhibitors, inhibited active uptake of pitavastatin into monkey hepatocytes with half-maximal inhibitory concentration values comparable with those in human hepatocytes. CsA and RIF increased the area under the plasma concentration-time curve (AUC) of intravenously administered pitavastatin in cynomolgus monkeys by 3.2- and 3.6-fold, respectively. In addition, there was no apparent prolongation of the elimination half-life of pitavastatin due to the decrease in both hepatic clearance and volume of distribution. These findings suggest that DDIs were caused by the inhibition of hepatic uptake of pitavastatin. CsA and RIF increased the AUC of orally administered pitavastatin by 10.6- and 14.8-fold, respectively, which was additionally caused by the effect of the CsA and RIF in the gastrointestinal tract. Hepatic contribution to the overall DDI for oral pitavastatin with CsA was calculated from the changes in hepatic availability and clearance, and it was shown that the magnitude of hepatic DDI was comparable between the present study and the clinical study. In conclusion, pharmacokinetic studies using pitavastatin as a probe in combination with drug candidates in cynomolgus monkeys are useful to support the assessment of potential clinical DDIs involving hepatic uptake transporters.

The glutathione S-transferase (GST) family comprises cytosolic, mitochondrial, and microsomal GSTs, all essential enzymes that metabolize a wide range of endogenous and exogenous substrates. Among the microsomal GSTs (MGSTs) in humans, MGST1, MGST2, and MGST3 are involved in detoxification; however, MGSTs have not been fully investigated in cynomolgus macaque, an important primate species widely used in drug metabolism and toxicity studies. In the present study, cynomolgus MGST2 and MGST3 cDNAs were isolated from liver tissue and characterized along with previously isolated cynomolgus MGST1. For comparison with the cynomolgus cDNAs, MGST2 and MGST3 cDNAs were also isolated from rhesus macaque (closely related to cynomolgus macaque) liver. Cynomolgus MGST2 and MGST3, respectively, were highly identical (99 and 98%) to human MGST2 and MGST3 and nearly identical to the amino acid sequences of the rhesus orthologs, and they were closely clustered with human MGST2 and MGST3 by phylogenetic analysis. The analysis of genome data indicated that MGST1, MGST2, and MGST3, respectively, had similar gene structures and genomic organization in macaque and human. Therefore, cynomolgus MGSTs have molecular similarities to the corresponding human MGSTs. Cynomolgus MGST2 and MGST3 were expressed in liver, jejunum, and kidney, but at lower levels than MGST1. GST activities were measured with 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane as substrates, using proteins heterologously expressed in Escherichia coli. Cynomolgus MGST1, MGST2, and MGST3 conjugated 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane, indicating that cynomolgus MGST1, MGST2, and MGST3 are functional enzymes. These results suggest that these functional cynomolgus MGST enzymes and the corresponding human MGSTs are molecularly similar.

Estrogen plays important roles in estrogen-responsive tissues, such as mammary glands, ovaries, and the uterus. In the liver, the major drug metabolizing organ, estrogen is known to regulate expression of some drug-metabolizing enzymes. Due to the lack of information on the role of estrogen in hepatic gene expression in primate species, we previously investigated the late response of hepatic gene expression to estradiol in cynomolgus macaques. To understand the early response of hepatic gene expression to estradiol, in this study, microarray analysis was conducted using cynomolgus macaque liver samples collected at 1 h and 5 h after estradiol injection. Comparison of expression profiles in estradiol and solvent (control)-treated ovariectomized cynomolgus macaques revealed 27 differentially expressed genes (>2.0-fold), including 18 at 1 h and 9 at 5 h after estradiol injection. As indicated by Gene Ontology analysis, these genes were related to oxidoreductase activity and transferase activity, partly representing important aspects of drug-metabolizing enzymes. Further analysis by quantitative polymerase chain reaction revealed that estradiol down-regulated CYP2A24, CYP2C76, and CYP2E1 (>2.0-fold) at 1 h and up-regulated GSTM5 (>2.0-fold) at 5 h after estradiol injection. These results suggest that the short-term estradiol treatment influenced expression of hepatic genes, including drug-metabolizing enzyme genes, in cynomolgus macaque liver.

Cytochrome P450 3A (CYP3A) is the major subfamily of CYP, one of the most important metabolizing enzymes for drugs in humans and other mammals. We found two novel CYP3A genes, CYP3A131 and CYP3A132 in domestic cats (Felis catus). Both feline CYP3A proteins consist of 504 deduced amino acids and show high identity with canine CYP3A homologues and those of some artiodactyls. CYP3A131 transcripts were expressed predominantly in liver and small intestine, and to a negligible extent in other tissues, including brain, heart, kidney and lung. CYP3A132 expression was only detected in liver with much lesser amount. These results suggest the possible major role of CYP3A131 in xenobiotic metabolism including first-pass effects in domestic cats.

Estrogen regulation of gene expression is essential for physiological function of estrogen-responsive tissues, such as mammary glands, ovaries, and the uterus. In the liver, estrogen is responsible for sex-dependent gene expression of drug-metabolizing enzymes in rodents. However, the influence of estrogen on hepatic gene expression has not been fully investigated in primates, including human. Macaque, including cynomolgus macaque, is an important species for comparative studies aimed at understanding human physiology due to its evolutionary closeness to human. To identify estrogen-responsive genes in primate liver, therefore, hepatic gene expression was compared, by microarray analysis, in ovariectomized cynomolgus macaques treated with estradiol or solvent (control). The analysis identified 98 estradiol-responsive genes; 47 and 51 were up- and down-regulated by estradiol, respectively (≥2.0-fold, P < 0.05). Expression of drug-metabolizing enzyme genes was also influenced by estradiol treatment; estradiol enhanced expression of GSTM5 (3.8-fold, P < 0.05) and CYP3A8(4) (2.7-fold, P < 0.01), but lowered expression of CYP4F12 (2.2-fold, P < 0.01), as verified by quantitative polymerase chain reaction. In particular, CYP3A8(4), orthologous to human CYP3A4, is an essential drug-metabolizing enzyme in cynomolgus macaque liver. These results suggest that expression of hepatic genes, including drug-metabolizing enzyme genes, is at least partly regulated by estradiol in cynomolgus macaque.

The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation, and testosterone 6β-, 16α-, 16β-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1'-hydroxylation and testosterone 6β-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.

Preclinical data of fetal, infant, and juvenile animals are important for the prediction of drug toxicity in fetuses and children. However, expression of drug-metabolizing enzymes, including cytochromes P450 (CYPs), have not been fully investigated in fetal, infant, or juvenile liver of the cynomolgus macaque, an animal species important for preclinical studies. In this study, hepatic expression of 20 cynomolgus macaque CYPs (mfCYPs) in the CYP1-4 subfamilies that are relevant to drug metabolism was measured in fetuses, infants, and juveniles using DNA microarrays. Expression of most mfCYPs, including those moderately or abundantly expressed in postnatal livers such as mfCYP2A23, mfCYP2A24, mfCYP2B6, mfCYP2C9, mfCYP2C19, mfCYP2C76, mfCYP2D17, mfCYP2E1 mfCYP3A4, and mfCYP3A5, was much less abundant in fetal livers, but increased substantially after birth. In contrast, expression of mfCYP2C8 in fetal livers was not substantially different from postnatal livers. Since human CYP3A7 is expressed more abundantly in fetal livers than in adult livers, mfCYP3A7, an ortholog of human CYP3A7, was analyzed by quantitative polymerase chain reaction. Expression of mfCYP3A7 in fetal livers was much lower than that in postnatal livers, and greatly increased after birth, unlike the expression of human CYP3A7. These results indicate that expression of most mfCYPs examined was low in fetal livers, but increased greatly in postnatal livers, with a few exceptions such as mfCYP2C8.

Induction of the cytochrome P450 (P450) enzyme is a major concern in the drug discovery processes. To predict the clinical significance of enzyme induction, it is helpful to investigate pharmacokinetic alterations of a coadministered drug in a suitable animal model. In this study, we focus on the induction of CYP3A, which is involved in the metabolism of approximately 50% of marketed drugs and is inducible in both the liver and intestine. As a marker substrate for CYP3A activity, alprazolam (APZ) was selected and characterized using recombinant CYP3A enzymes expressed in Escherichia coli. Both human CYP3A4 and its cynomolgus P450 ortholog predominantly catalyzed APZ 4-hydroxylation with sigmoidal kinetics. When administered intravenously and orally to cynomolgus monkeys, APZ had moderate clearance; its first-pass extraction ratio after oral dosing was estimated to be 0.09 in the liver and 0.45 in the intestine. Pretreatment with multiple doses of rifampicin (20 mg/kg p.o. for 5 days), a known CYP3A inducer, significantly decreased plasma concentrations of APZ after intravenous and oral administrations (0.5 mg/kg), and first-pass extraction ratios were increased to 0.39 in the liver and 0.63 in the intestine. The results were comparable to those obtained in clinical drug-drug interaction (DDI) reports related to CYP3A induction, although the rate of recovery of CYP3A activity seemed to be slower than rates estimated in clinical studies. In conclusion, pharmacokinetic studies using APZ as a probe in monkeys may provide useful information regarding the prediction of clinical DDIs due to CYP3A induction.

A novel cytochrome P450 (CYP), CYP2A26, was identified and characterized in cynomolgus monkey, one of the animal species used in preclinical studies. Deduced amino acid sequences of CYP2A26 cDNA showed high sequence identities (91–95%) with cynomolgus monkey CYP2A23 and CYP2A24, and human CYP2A6 and CYP2A13. Phylogenetic analysis showed that macaque CYP2As (CYP2A26, CYP2A23, and CYP2A24) were most closely clustered with human CYP2As, unlike CYP2As of dog, rat, and mouse (other species also used in drug metabolism). Quantitative polymerase chain reaction analysis showed that CYP2A26 mRNA, along with CYP2A23 and CYP2A24 mRNAs, was expressed predominantly in the liver, where CYP2A proteins were also detected by immunoblotting. Drug-metabolizing assays using the CYP2A26 protein heterologously expressed in Escherichia coli indicated that CYP2A26 catalyzed coumarin 7-hydroxylation with its apparent K(m) lower than that of CYP2A24, but similar to those of CYP2A6 and CYP2A23. These results suggest an evolutionary closeness and functional similarity of cynomolgus monkey CYP2A26 (together with CYP2A23 and CYP2A24) to human CYP2A6, and its functional role as a drug-metabolizing enzyme in the liver.

The macaque is widely used for investigation of drug metabolism due to its evolutionary closeness to the human. However, the genetic backgrounds of drug-metabolizing enzymes have not been fully investigated; therefore, identification and characterization of drug-metabolizing enzyme genes are important for understanding drug metabolism in this species. In this study, we isolated and characterized a novel cytochrome P450 2C18 (CYP2C18) cDNA in cynomolgus macaques. This cDNA was highly homologous (96%) to human CYP2C18 cDNA. Cynomolgus CYP2C18 was preferentially expressed in the liver and kidney. Moreover, a metabolic assay using cynomolgus CYP2C18 protein heterologously expressed in Escherichia coli revealed its activity toward S-mephenytoin 4'-hydroxylation. These results suggest that cynomolgus CYP2C18 could function as a drug-metabolizing enzyme in the liver.

The cynomolgus monkey is used to study drug metabolism because of its evolutionary closeness to humans. Despite their importance, regional distribution of cytochrome P450 (CYP) enzymes including CYP3As in the liver and small intestine, the major sites of drug metabolism, has not been fully investigated in cynomolgus monkeys. In this study, we measured mRNA expression levels of 14 CYPs in the CYP1, 2, and 3 subfamilies, including orthologs of human CYP3A4 and CYP3A5, in the liver and small intestine of cynomolgus monkeys. Expression levels of each CYP mRNA in various regions of the liver were quantified and comparisons were made between the right lobe, quadrate lobe, left medial lobe, left lateral lobe, and caudate lobe and with four different sections of the right lobe. In the small intestine, the same mRNAs were measured in the duodenum and six different sections from the proximal jejunum to the distal ileum. Expression levels of the CYP mRNAs were not substantially different between liver samples, but varied between the different sections of the small intestine, including CYP3A4. These results suggest that analysis of distinct sections is required for a better understanding of cynomolgus monkey CYPs in the small intestine.

RNA interference is a powerful tool for target-specific knockdown of gene expression. However, efficient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ, which is essential for therapeutic applications, has not been established. In this study we used alpha-tocopherol (vitamin E), which has its own physiological transport pathway to most of the organs, as a carrier molecule of siRNA in vivo. The alpha-tocopherol was covalently bound to the antisense strand of 27/29-mer siRNA at the 5'-end (Toc-siRNA). The 27/29-mer Toc-siRNA was designed to be cleaved by Dicer, producing a mature form of 21/21-mer siRNA after releasing alpha-tocopherol. The C6 hydroxyl group of alpha-tocopherol, associated with antioxidant activity, was abolished. Using this new vector, intravenous injection of 2 mg/kg of Toc-siRNA, targeting apolipoprotein B (apoB), achieved efficient reduction of endogenous apoB messenger RNA (mRNA) in the liver. The downregulation of apoB mRNA was confirmed by the accumulation of lipid droplets in the liver as a phenotype. Neither induction of interferons (IFNs) nor other overt side effects were revealed by biochemical and pathological analyses. These findings indicate that Toc-siRNA is effective and safe for RNA interference-mediated gene silencing in vivo.

The liver, a major organ for drug metabolism, is physiologically similar between monkeys and humans. However, the paucity of identified genes has hampered a deep understanding of drug metabolism in monkeys. To provide such a genetic resource, 28655 expressed sequence tags (ESTs) were generated from a cynomolgus monkey liver full-length enriched cDNA library, which contained 23 unique ESTs homologous to human drug-metabolizing enzymes. Our comparative genomics approach identified nine lineage-specific candidate ESTs, including three drug-metabolizing enzymes, which could be important for understanding the physiological differences between monkeys and humans.

Monkeys are widely used as a primate model to study drug metabolism because they generally show a metabolic pattern similar to humans. However, the paucity of information on cytochrome P450 (P450) genes has hampered a deep understanding of drug metabolism in the monkey. In this study, we report identification of the CYP2C76 cDNA newly identified in cynomolgus monkey and characterization of this CYP2C along with cynomolgus CYP2C20, CYP2C43, and CYP2C75. The CYP2C76 cDNA contains the open reading frame encoding a protein of 489 amino acids that are only approximately 80% identical to any human or monkey P450 cDNAs. Gene and protein expression of CYP2C76 was confirmed in the liver of cynomolgus and rhesus monkeys but not in humans or the great apes. Moreover, CYP2C76 is located at the end of the CYP2C gene cluster in the monkey genome, the region of which corresponds to the intergenic region adjacent to the CYP2C cluster in the human genome, strongly indicating that this gene does not have the ortholog in humans. Among the four CYP2C genes expressing predominantly in the liver, the expression level of CYP2C76 was the greatest, suggesting that CYP2C76 is a major CYP2C in the monkey liver. Assays for the capacity of CYP2C76 to metabolize drugs using several substrates typical for human CYP2Cs revealed that CYP2C76 showed unique metabolic activity. These results suggest that CYP2C76 contributes to overall drug-metabolizing activity in the monkey liver and might account for species difference occasionally seen in drug metabolism between monkeys and humans.

A collaborative study was conducted to evaluate whether a replicative DNA synthesis (RDS) test using the rat liver can detect nongenotoxic (Ames-negative) hepatocarcinogens with three or seven daily administrations at dose-levels effective in long-term bioassays. The assay methods were well-validated by the 14 participants. Of six compounds tested, carbon tetrachloride (50 and 100 mg/kg), clofibrate (125 and 250 mg/kg), diethylstilbestrol (0.125 and 0.25 mg/kg) and urethane (100 mg/kg) gave positive results, methyl carbamate (200 and 400 mg/kg) exerted equivocal effects, and D,L-ethionine (125 mg/kg) failed to elevate RDS. These findings suggest that the RDS test can detect many nongenotoxic rat hepatocarcinogens with short-term administration at dose-levels used in long-term bioassays.

To assess the efficacy of the in vivo-in vitro hepatocyte replicative DNA synthesis (RDS) test as a short-term assay, 41 putative nongenotoxic (Ames-negative) mouse hepatocarcinogens, as well as 31 noncarcinogens, were examined using male 8-week-old B6C3F1 mice and an in vitro [methyl-3]thymidine-incorporation technique. Animals were exposed to the maximum tolerated dose (MTD) and 1/2 MTD of each chemical by gavage and after 24, 39 or 48 h, hepatocytes were prepared with a collagenase-perfusion technique. Assessment of the distribution of spontaneous RDS in a total of 337 control mice gave an average incidence of 0.15 +/- 0.08% within the range of 0 to 0.39% (mean +/- 3 x SE) with a 99.7% probability. Values of 0.4% or more for RDS incidences induced by test samples were therefore judged as indicating a positive response in our RDS test. Under the experimental conditions applied, 32 of 41 putative nongenotoxic hepatocarcinogens gave clear positive responses (positive sensitivity: 78%), and of 31 noncarcinogens 25 samples gave negative responses (negative specificity: 81%), thus giving an overall concordance for the RDS test with long-term findings of 79%.

To establish a standard protocol for an in vivo-in vitro hepatocyte replicative DNA synthesis (RDS) test using male F344 rats for screening nongenotoxic (the Ames-negative) hepatocarcinogens, experimental conditions were examined. After treatment with three model hepatocarcinogens, isolated hepatocytes showed highest RDS incidences when plated at a density of 5 x 10(4) cells/ml. Spontaneous RDS incidences in hepatocytes from rats aged 9 weeks or older showed a constant value. The use of hepatocytes from 9-week-old rats at the 5 x 10(4) cells/ml plating density was therefore determined as the standard. Based on the distribution of mean spontaneous RDS incidences over 105 additional experiments (0.4 +/- 0.18%, with SEM), an RDS incidence of over 1% was adopted as the criterion for a positive response in our rat liver RDS test.