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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 CYP4Agenes 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 STATEMENTCytochrome 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.

Tree shrews are a nonprimate species used in a range of biomedical studies. Recent genome analysis of tree shrews found that the sequence identities and the numbers of genes of cytochrome P450 (CYP or P450), an important family of drug-metabolizing enzymes, are similar to those of humans. However, tree shrew P450s have not yet been sufficiently identified and analyzed. In this study, novel CYP2D8a and CYP2D8b cDNAs were isolated from tree shrew liver and were characterized, along with human CYP2D6, dog CYP2D15, and pig CYP2D25. The amino acid sequences of these tree shrew CYP2Ds were 75%–78% identical to human CYP2D6, and phylogenetic analysis showed that they were more closely related to human CYP2D6 than rat CYP2Ds, similar to dog and pig CYP2Ds. For tree shrew CYP2D8b, two additional transcripts were isolated that contained different patterns of deletion. The gene and genome structures of CYP2Dsare generally similar in dogs, humans, pigs, and tree shrews. Tree shrew CYP2D8a mRNA was most abundantly expressed in liver, among the tissue types analyzed, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Tree shrew CYP2D8b mRNA was also expressed in liver, but at a level 7.3-fold lower than CYP2D8a mRNA. Liver microsomes and recombinant protein of both tree shrew CYP2Ds metabolized bufuralol and dextromethorphan, selective substrates of human CYP2D6, but the activity level of CYP2D8a greatly exceeded that of CYP2D8b. These results suggest that tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver.SIGNIFICANCE STATEMENTNovel tree shrew CYP2D8a and CYP2D8b cDNAs were isolated from liver. Their amino acid sequences were 75%–78% identical to human CYP2D6. For CYP2D8b, two additional transcripts contained different patterns of deletion. Tree shrew CYP2D8a mRNA was abundantly expressed in liver, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Recombinant tree shrew CYP2Ds catalyzed the oxidation of bufuralol and dextromethorphan. Tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver.

Flavin-containing monooxygenases (FMOs) are a family of enzymes that are involved in the oxygenation of heteroatom-containing molecules. In humans, FMO3 is the major hepatic form, whereas FMO1 is predominant in the kidneys. FMO1 and FMO3 have also been identified in monkeys, dogs, and pigs. The predicted contribution of human FMO3 to drug candidate N-oxygenation could be estimated using the classic base dissociation constants of the N-containing moiety. A basic quinuclidine moiety was found in natural quinine and medicinal products. Consequently, N-oxygenation of quinuclidine was evaluated using liver and kidney microsomes from humans, monkeys, dogs, and pigs as well as recombinant FMO1, FMO3, and FMO5 enzymes. Experiments using simple reversed-phase liquid chromatography with fluorescence monitoring revealed that recombinant FMO1 mediated quinuclidine N-oxygenation with a high capacity in humans. Moreover, recombinant FMO1, FMO3, and/or FMO5 in monkeys, dogs, and pigs exhibited relatively broad substrate specificity toward quinuclidine N-oxygenation. Kinetic analysis showed that human FMO1 efficiently, and pig FMO1 moderately, mediated quinuclidine N-oxygenation with high capacity, which is consistent with the reported findings for larger substrates readily accepted by pig FMO1 but excluded by human FMO1. In contrast, human FMO3-mediated quinuclidine N-oxygenation was slower than that of the typical FMO3 substrate trimethylamine. These results suggest that some species differences exist in terms of FMO-mediated quinuclidine N-oxygenation in humans and some animal models (monkeys, dogs, and minipigs); however, the potential for quinuclidine, which has a simple chemical structure, to be inhibited clinically by co-administered drugs should be relatively low, especially in human livers.SIGNIFICANCE STATEMENTThe high capacity of human flavin-containing monooxygenase (FMO) 1 to mediate quinuclidine N-oxygenation, a basic moiety in natural products and medicines, was demonstrated by simple reversed-phase liquid chromatography using fluorescence monitoring. The substrate specificity of FMO1 and FMO3 toward quinuclidine N-oxygenation in monkeys, dogs, and pigs was suggested to be relatively broad. Human FMO3-mediated quinuclidine N-oxygenation was slower than trimethylamine N-oxygenation. The likelihood of quinuclidine, with its simple chemical structure, being clinically inhibited by co-administered drugs is relatively low.

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, CYP2Cgenes, 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 CYP2C119gene, expressed in macaque liver, encodes a functional enzyme that metabolizes human CYP2C substrates and is likely responsible for drug clearances.SIGNIFICANCE STATEMENTCytochrome 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 CYP2C119gene expressed in the macaque liver encodes a functional enzyme that metabolizes human CYP2C substrates.