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.

Novel Tree Shrew Cytochrome P450 2Ds (CYP2D8a and CYP2D8b) Are Functional Drug-Metabolizing Enzymes that Metabolize Bufuralol and Dextromethorphan.

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 CYP2Ds are 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 STATEMENT: Novel 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.

Investigation of functional cytochrome P450 4A enzymes in liver and kidney of pigs, cats, tree shrews, and dogs in comparison with the metabolic capacity of human P450 4A11.

Pigs are sometimes utilized in preclinical drug metabolism studies, with growing interest, and so 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.

In Vivo and In Vitro Induction of Cytochrome P450 3A4 by Thalidomide in Humanized-Liver Mice and Experimental Human Hepatocyte HepaSH cells.

Autoinduction of cytochrome P450 (P450) 3A4-mediated metabolism of thalidomide was investigated in humanized-liver mice and human hepatocyte-derived HepaSH cells. The mean plasma ratios of 5-hydroxythalidomide and glutathione adducts to thalidomide were significantly induced (3.5- and 6.0-fold, respectively) by thalidomide treatment daily at 1000 mg/kg for 3 days and measured at 2 h after the fourth administration (on day 4). 5-Hydroxythalidomide was metabolically activated by P450 3A4 in HepaSH cells pretreated with 300 and 1000 µM thalidomide, and 5,6-dihydroxythalidomide was detected. Significant induction of P450 3A4 mRNA expression (4.1-fold) in the livers of thalidomide-treated mice occurred. Thalidomide exerts a variety of actions through multiple mechanisms following bioactivation by induced human P450 3A enzymes.

Modeled Hepatic/Plasma Exposures of Omeprazole Prescribed Alone in Cytochrome P450 2C19 Poor Metabolizers Are Likely Associated with Hepatic Toxicity Reported in a Japanese Adverse Event Database.

Omeprazole, a gastric acid pump inhibitor, is repeatedly administered and is oxidatively metabolized mainly by polymorphic cytochrome P450 2C19. The prescribed dosage of omeprazole was discontinued or reduced in 47 of the 135 patients who received omeprazole alone in this survey, as recorded in the Japanese Adverse Drug Event Report database. The days to onset of omeprazole-related disorders were 3-4 d (median) and 16 d for intravenous 20-40 mg and oral 20 mg daily doses, respectively, in 34 patients for whom relevant data were available. The maximum plasma concentration of omeprazole was pharmacokinetically modeled after a single oral 40-mg dose in P450 2C19-defective poor metabolizers and was 2.4-fold higher than that in extensive metabolizers. The modeled area under the hepatic concentration curves of omeprazole in P450 2C19 poor metabolizers after virtual daily 40-mg doses for 7 d was 5.2-fold higher than that in the extensive metabolizers. Omeprazole-induced P450 2C19 (approx. 2-fold), resulting in increased hepatic intrinsic clearance in repeated doses, was considered after the second day. Virtual plasma/hepatic exposure estimated using pharmacokinetic modeling in subjects with P450 2C19 poor metabolizers indicated that these exposure levels virtually estimated could be one of causal factors for unexpected hepatic disorders induced by prescribed omeprazole, such as those resulting from drug interactions with repeatedly co-administered medicines.

HepaSH cells: Experimental human hepatocytes with lesser inter-individual variation and more sustainable availability than primary human hepatocytes.

Primary human hepatocytes (PHHs) have been commonly used as the gold standard in many drug metabolism studies, regardless of having large inter-individual variation. These inter-individual variations in PHHs arise primarily from genetic polymorphisms, as well as from donor health conditions and storage conditions prior to cell processing. To equalize the effects of the latter two factors, PHHs were transplanted to quality-controlled mice providing human hepatocyte proliferation niches, and engrafted livers were generated. Cells that were harvested from engrafted livers, call this as experimental human hepatocytes (EHH; termed HepaSH cells), were stably and reproducibly produced from 1014 chimeric mice produced by using 17 different PHHs. Expression levels of acute phase reactant (APR) genes as indicators of a systemic reaction to the environmental/inflammatory insults of liver donors varied widely among PHHs. In contrast to PHHs, the expression of APR genes in HepaSH cells was found to converge within a narrower range than in donor PHHs. Further, large individual differences in the expression levels of drug metabolism-related genes (28 genes) observed in PHHs were greatly reduced among HepaSH cells produced in a unified in vivo environment, and none deviated from the range of gene expression levels in the PHHs. The HepaSH cells displayed a similar level of drug-metabolizing enzyme activity and gene expression as the average PHHs but retained their characteristics for drug-metabolizing enzyme gene polymorphisms. Furthermore, long-term 2D culture was possible and HBV infection was confirmed. These results suggest that the stably and reproducibly providable HepaSH cells with lesser inter-individual differences in drug-metabolizing properties, may have a potential to substitution for PHH as practical standardized human hepatocytes in drug discovery research.

A Comprehensive Investigation of Dog Cytochrome P450 3A (CYP3A) Reveals a Functional Role of Newly Identified CYP3A98 in Small Intestine.

Dogs are frequently used in drug metabolism studies, and their important drug-metabolizing enzymes, including cytochromes P450 (P450), have been analyzed. In humans, CYP3A4 is an especially important P450 due to its abundance and major roles in liver and intestine. In the present study, dog CYP3A98 and CYP3A99 were identified and characterized, along with previously identified CYP3A12 and CYP3A26. The dog CYP3A cDNAs contained open reading frames of 503 amino acids and shared high sequence identity (78%-80%) with human CYP3As. Among the dog CYP3A mRNAs, CYP3A98 mRNA was expressed most abundantly in small intestine. In contrast, dog CYP3A12 and CYP3A26 mRNAs were expressed in liver, where CYP3A12 mRNA was the most abundant. The four CYP3A genes had similar gene structures and formed a gene cluster in the dog and human genomes. Metabolic assays of dog CYP3A proteins heterologously expressed in Escherichia coli indicated that the dog CYP3As tested were functional enzymes with respect to typical human CYP3A4 substrates. Dog CYP3A98 efficiently catalyzed oxidations of nifedipine, alprazolam, and midazolam, indicating major roles of CYP3A98 in the small intestine. Dog CYP3A12 and CYP3A26 metabolizing nifedipine and/or midazolam would play roles in these reactions in the liver. In contrast, dog CYP3A99 showed minimal mRNA expression and minimal metabolic activity, and its contribution to overall drug metabolism is, therefore, negligible. These results indicated that newly identified dog CYP3A98, a testosterone 6 ß - and estradiol 16 α -hydroxylase, was abundantly expressed in the small intestine and is likely the major CYP3A in the small intestine in combination with liver-specific CYP3A12. SIGNIFICANCE STATEMENT: Novel dog cytochromes P450 3A98 (CYP3A98) and CYP3A99 were identified and characterized to be functional and highly identical to human CYP3A4. Known CYP3A12 and new CYP3A98 efficiently catalyzed estradiol 16α-hydroxylation and midazolam 1'-hydroxylation. CYP3A98 mRNA was expressed in small intestine, whereas CYP3A12 mRNA was predominant in liver. Dog hepatic CYP3A12 and intestinal CYP3A98 are the enzymes likely responsible for the metabolic clearances of orally administered drugs, unlike human CYP3A4/5, which are in both the liver and intestine.

Novel Cytochrome P450 2C94 Functionally Metabolizes Diclofenac and Omeprazole in Dogs.

Cytochromes P450 (P450s or CYPs) are important drug-metabolizing enzymes. Because dogs are frequently used in drug metabolism studies, knowledge of dog CYP2C enzymes is essential because in humans these enzymes are abundant and play major roles in liver and intestine. The present study identified and characterized novel dog CYP2C94 along with previously identified dog CYP2C21 and CYP2C41. Dog CYP2C21, CYP2C41, and CYP2C94 cDNAs, respectively, contained open reading frames of 490, 489, and 496 amino acids and shared high-sequence identities (70%, 75%, and 58%) with human CYP2Cs. Dog CYP2C94 mRNA was preferentially expressed in liver, just as dog CYP2C21 and CYP2C41 mRNAs were. In dog liver, CYP2C21 mRNA was the most abundant, followed by CYP2C94 and CYP2C41 mRNAs. Moreover, the hepatic expressions of all three dog CYP2C mRNAs varied in four individual dogs, two of which did not express CYP2C41 mRNA. The three dog CYP2C genes had similar gene structures, and CYP2C94, although located on the same chromosome, was in a genomic region far from the gene cluster containing CYP2C21 and CYP2C41 Metabolic assays with recombinant proteins showed that dog CYP2C94, along with CYP2C21 and CYP2C41, efficiently catalyzed oxidations of diclofenac, warfarin, and/or omeprazole, indicating that dog CYP2C94 is a functional enzyme. Novel dog CYP2C94 is expressed abundantly in liver and encodes a functional enzyme that metabolizes human CYP2C substrates; it is, therefore, likely responsible for drug clearances in dogs. SIGNIFICANCE STATEMENT: Novel dog cytochrome P450 2C94 (CYP2C94) was identified and characterized along with dog CYP2C21 and CYP2C41. Dog CYP2C94, isolated from liver, had 58% sequence identity and a close phylogenetic relationship with its human homologs and was expressed in liver at the mRNA level. Dog CYP2C94 (and CYP2C21 and CYP2C41) catalyzed oxidations of diclofenac and omeprazole, human CYP2C9 and CYP2C19 substrates, respectively, but CYP2C41 also hydroxylated warfarin. CYP2C94 is therefore a functional drug-metabolizing enzyme likely responsible for drug clearances in dogs.

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.

Oxidation of Naringenin, Apigenin, and Genistein by Human Family 1 Cytochrome P450 Enzymes and Comparison of Interaction of Apigenin with Human P450 1B1.1 and Scutellaria P450 82D.1.

Naringenin, an initial synthesized flavanone in various plant species, is further utilized for production of many biologically active flavonoids, e.g., apigenin, eriodictyol, and genistein, by various plant enzymes including cytochrome P450s (P450s or CYPs). We examined how these flavonoids are oxidized by human P450 family 1 and 2A enzymes. Naringenin was principally oxidized at the 3'-position to form eriodictyol by CYP1 enzymes more efficiently than by CYP2A enzymes, and the resulting eriodictyol was further oxidized to two penta-hydroxylated products. In contrast to plant P450 enzymes, these human P450s did not mediate the desaturation of naringenin and eriodictyol to give apigenin and luteolin, respectively. Apigenin was oxidized at the C3' and C6 positions to form luteolin and scutellarein by these P450s. CYP1B1.1 and 1B1.3 had high activities in apigenin 6-hydroxylation with a homotropic cooperative manner, as has been observed previously in chrysin 6-hydroxylation (Nagayoshi et al., Chem. Res. Toxicol. 2019, 32, 1268-1280). Molecular docking analysis suggested that CYP1B1 had two apigenin binding sites and showed similarities in substrate recognition sites to plant CYP82D.1, one of the enzymes in catalyzing apigenin and chrysin 6-hydroxylations in Scutellaria baicalensis. The present results suggest that human CYP1 enzymes and CYP2A13 in some reactions have important roles in the oxidation of naringenin, eriodictyol, apigenin, and genistein and that human CYP1B1 and Scutellaria CYP82D.1 have similarities in their SRS regions, catalyzing 6-hydroxylation of both apigenin and chrysin.

Plasma and Hepatic Exposures of Celecoxib and Diclofenac Prescribed Alone in Patients with Cytochrome P450 2C9*3 Modeled after Virtual Oral Administrations and Likely Associated with Adverse Drug Events Reported in a Japanese Database.

The impacts of polymorphic cytochrome P450 (P450 or CYP) 2C9 on drug interactions and the pharmacokinetics of cyclooxygenase inhibitors have attracted considerable attention. In this survey, the prescribed dosage was reduced or discontinued in 150 and 56 patients, respectively, receiving celecoxib and diclofenac prescribed alone, as recorded in a Japanese database of adverse drug events. Among the factors underlying adverse events, intrinsic drug clearance rates may be a contributing factor. The pharmacokinetically modeled plasma concentrations of celecoxib after an oral 200-mg dose increased in CYP2C9*3 homozygotes: the area under the plasma concentration curve was 4.7-fold higher than that in CYP2C9*1 homozygotes. In patients with CYP2C9*3/*3, the virtual hepatic concentrations of diclofenac after three daily 25-mg doses for a week were 11-fold higher than the plasma concentrations in subjects with CYP2C9*1/*1. The in vivo and in vitro fractions of the victim drug metabolized by a specific polymorphic P450 form is an important determining factor for estimating drug-drug interactions. Virtual hepatic and plasma exposures estimated by pharmacokinetic modeling in patients harboring the impaired CYP2C9*3 allele could represent a causal factor for adverse events induced by celecoxib or diclofenac in a manner similar to that for drug interactions.

2-Oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole, a contributor to the odour of boar-tainted pork meat, mediated by porcine liver microsomal cytochromes P450 1A2, 2A19, 2E1, and 3A22.

The 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole (a contributor to boar taint) mediated by minipig liver microsomes and recombinant P450 enzymes expressed in bacterial membranes were investigated.At low substrate concentrations of 10 µM, the formation rates of indole-3-carbinol, 6-hydroxyskatole, and the sum of 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole in male minipig liver microsomes were significantly lower than those in female minipig liver microsomes.Compensatory 3-methyloxindole and indole-3-carbinol formation in minipig liver microsomes, which lack 6-hydroxyskatole formation in males, was mediated partly by liver microsomal P450 1A2 and P450 1A2/2E1, respectively. These enzymes were suppressed by typical P450 inhibitors in female minipig liver microsomes.Among the 14 pig P450 forms evaluated, P450 2A19 was the dominant form mediating 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole formation from skatole at substrate concentrations of 100 µM. Positive cooperativity was observed in 3-methyloxindole formation from skatole mediated by male minipig liver microsomes and by pig P450 3A22 with Hill coefficients of 1.2-1.5.These results suggest high skatole 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation activities of pig P450 2A19 and compensatory skatole oxidations mediated by pig P450 1A2, 2E1, or 3A22 in male minipig liver microsomes.

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.

Tree shrew cytochrome P450 2E1 is a functional enzyme that metabolises chlorzoxazone and p-nitrophenol.

Cytochromes P450 (CYPs or P450s) are important enzymes for drug metabolism. Tree shrews are non-primate animal species used in various fields of biomedical research, including infection (especially hepatitis viruses), depression, and myopia. A recent tree shrew genome analysis indicated that the sequences and the numbers of P450 genes are similar to those of humans; however, P450s have not been adequately identified and analysed in this species.In this study, a novel CYP2E1 was isolated from tree shrew liver and was characterised in comparison with human, dog, and pig CYP2E1. Tree shrew CYP2E1 and human CYP2E1 showed high amino acid sequence identity (83%) and were closely related in a phylogenetic tree.Gene and genome structures of CYP2E1 were generally similar in humans, dogs, pigs, and tree shrews. Tissue expression patterns showed that tree shrew CYP2E1 mRNA was predominantly expressed in liver, just as for dog and pig CYP2E1 mRNAs. In tree shrews, recombinant CYP2E1 protein and liver microsomes metabolised chlorzoxazone and p-nitrophenol, probe substrates of human CYP2E1, just as they do in dogs and pigs.These results suggest that tree shrew CYP2E1 encodes a functional drug-metabolising enzyme that plays a role in the liver, similar to human CYP2E1.

Cytochrome P450 1A2 and 2C enzymes autoinduced by omeprazole in dog hepatocytes and human HepaRG and HepaSH cells are involved in omeprazole 5-hydroxylation and sulfoxidation.

The induction assay for the cytochromes P450 (P450s) is an important tool in drug discovery and development. The inductions of dog P450 1A2 and 3A12 by omeprazole and rifampicin were functionally characterised in dog hepatocytes and were compared with induction in human HepaRG and HepaSH cells.P450 1A2-dependent ethoxyresorufin O-deethylation was induced by R,S-omeprazole and P450 3 A-dependent midazolam 1'-hydroxylation was induced by rifampicin, and both reactions were significantly enhanced in cultured dog hepatocytes and human HepaRG and HepaSH cells.Recombinant dog P450 1A2 exhibited activities towards R- and S-omeprazole 5-hydroxylation with low Km values of 23-28 µM, whereas dog P450 2C21 and 3A12 efficiently mediated S-omeprazole 5-hydroxylation and sulfoxidation, respectively, with high Vmax values of 12-17 min-1.Although omeprazole 5-hydroxylation by human P450 2C19 (and sulfoxidation by P450 3A4) in human HepaSH cells were slightly (∼2-fold) induced by R,S-omeprazole, dog P450 1A2 was autoinduced by omeprazole in dog hepatocytes and showed enhanced R-omeprazole 5-hydroxylation activity (∼5-fold).These results indicate that omeprazole can be an autoinducer of P450 1A2 in hepatocytes, and this enzyme was found to be involved in omeprazole 5-hydroxylation and sulfoxidation in dog hepatocytes and human HepaRG and HepaSH cells.

Molecular and Functional Characterization of N-Acetyltransferases in Common Marmosets and Pigs.

Arylamine N-acetyltransferases (NATs) are drug-metabolizing enzymes that are essential for the metabolism of endogenous substrates and xenobiotics. The molecular characteristics of NATs have been extensively investigated in humans but remain to be investigated in common marmosets and pigs, animal species that are often used in drug metabolism studies. In this study, marmoset NAT1 and pig NAT1 cDNAs were isolated from liver samples and were characterized by molecular analyses and drug-metabolism assays. These NAT genes were intronless and formed gene clusters with one other NAT gene in the genome, just as human NAT genes do. Marmoset NAT1 and pig NAT1 amino acid sequences showed high sequence identities (94% and 85%, respectively) to human NAT1. Phylogenetic analysis indicated that marmoset NAT1 and pig NAT1 were more closely clustered with human NATs than with rat or mouse NATs. Marmoset NAT1 and pig NAT1 mRNAs were expressed in all the tissue types analyzed, with the expression levels being highest in the small intestine. Metabolic assays using recombinant proteins found that marmoset NAT1 and pig NAT1 metabolized human NAT substrates p-aminobenzoic acid, 2-aminofluorene, sulfamethazine, and isoniazid. Marmoset NAT1 and pig NAT1 substantially acetylated p-aminobenzoic acid and 2-aminofluorene relevant human NAT1, but their activities were lower toward sulfamethazine and isoniazid than those of the relevant human NAT2. Therefore, marmoset and pig NATs are functional enzymes with molecular similarities to human NAT1, but their substrate specificities, while similar to human NAT1, differ somewhat from human NAT2. SIGNIFICANCE STATEMENT: Marmoset N-acetyltransferase NAT1 and pig NAT1 were identified and showed high sequence identities to human NAT1. These NAT mRNAs were expressed in various tissues. Marmoset and pig NAT1s acetylated typical human NAT substrates, although their substrate specificities differed somewhat from human NAT2. Marmoset NAT1 and pig NAT1 have similarities with human NAT1 in terms of molecular and enzymatic characteristics.

Cytochrome P450 2J Genes Are Expressed in Dogs, Cats, and Pigs, and Encode Functional Drug-Metabolizing Enzymes.

Cytochrome P450s (P450s) have been identified and analyzed in dogs and pigs, species that are often used in preclinical drug studies. Moreover, P450s are clinically important for drug therapy not only in humans, but also in species under veterinary care, including dogs and cats. In the present study, seven P450s homologous to human CYP2J2, namely, dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J35, CYP2J91, and CYP2J93, were newly identified and characterized, along with pig CYP2J34 previously identified. The cDNAs of these CYP2Js contain open reading frames of 502 amino acids, except for CYP2J35 (498 amino acids), and share high sequence identity (77%-80%) with human CYP2J2. Phylogenetic analysis revealed that dog and cat CYP2J2 were closely related, whereas pig CYP2Js formed a cluster. All seven CYP2J genes contain nine coding exons and are located in corresponding genomic regions, with the pig CYP2J genes forming a gene cluster. These CYP2J2 mRNAs were predominantly expressed in the small intestine with additional expression in the kidney and brain for dog CYP2J2 and pig CYP2J91 mRNAs, respectively. All seven CYP2Js metabolized human CYP2J2 substrates terfenadine, ebastine, and astemizole, indicating that they are functional enzymes. Dog CYP2J2 and pig CYP2J34 and CYP2J35 efficiently catalyzed ebastine primary hydroxylation and secondary carebastine formation at low substrate concentrations, just as human CYP2J2 does. Velocity-versus-substate plots exhibited sigmoidal relationships for dog CYP2J2, cat CYP2J2, and pig CYP2J33, indicating allosteric interactions. These results suggest that dog, cat, and pig CYP2Js have similar functional characteristics to human CYP2J2, with slight differences in ebastine and astemizole oxidations. SIGNIFICANCE STATEMENT: Dog CYP2J2; cat CYP2J2; and pig CYP2J33, CYP2J34, CYP2J35, CYP2J91, and CYP2J93, homologous to human CYP2J2, were identified and characterized by sequence, phylogenetic, and genomic structure analyses. Intestinal expression patterns of CYP2J mRNAs were characteristic in dogs, cats, and pigs. Dog, cat, and pig CYP2Js likely play roles as drug-metabolizing enzymes in the small intestine, similar to human CYP2J2.

Low cerebrospinal fluid-to-plasma ratios of orally administered lenalidomide mediated by its low cell membrane permeability in patients with hematologic malignancies.

Lenalidomide is a synthetic analog of thalidomide formed by the removal of one keto group (plus the addition of an amino group); it has anti-tumor activities beneficial for the treatment of hematologic malignancies. However, lenalidomide distribution to brain in animal models is reportedly low compared with that of thalidomide. The aim of this study was to evaluate plasma and cerebrospinal fluid concentrations of lenalidomide in three patients with malignant hematologic malignancies. Lenalidomide was detected in plasma from the three Japanese patients 1.5 h following oral administration of 20 mg lenalidomide using liquid chromatography/mass spectrometry, despite the in vitro gastrointestinal permeability of lenalidomide being low. Clinically observed cerebrospinal fluid-to-plasma ratios of lenalidomide were low (1.3-2.4%). Observed influx permeability values for lenalidomide in monkey blood-brain barrier model and human placental cell systems were one order of magnitude lower than those of thalidomide and another second-generation drug, pomalidomide along with a positive permeability control, caffeine. Because of the low cell-barrier permeability of lenalidomide demonstrated in in vitro assays, clinically relevant pharmacokinetic profiles of lenalidomide resulted in low penetrability from plasma into cerebrospinal fluid in patients with hematologic malignancies. Lenalidomide is conclusively suggested to expert its favorable immunomodulatory effects via systemic exposures in the patients.

Comparison of mouse and human cytochrome P450 mediated-drug metabolising activities in hepatic and extrahepatic microsomes.

Despite the importance of mice as a preclinical species in drug testing, their hepatic and extrahepatic drug-metabolising characteristics are poorly understood. Here, we compared the P450-dependent drug oxidation activity in tissue microsomes and distribution patterns of P450 protein/mRNA between humans and mice.The activities of midazolam 1'-/4-hydroxylation in the liver and intestine and chlorzoxazone 6-hydroxylation in the liver were similar in humans and mice. The activities of coumarin 7-hydroxylation, flurbiprofen 4'-hydroxylation, and S-mephenytoin 4'-hydroxylation in the liver were higher in humans than in mice. The activities of 7-ethoxyresorufin O-deethylation in the liver, 7-pentoxyresorufin O-depentylation in the lung/liver/intestine, bufuralol 1'-hydroxylation in the liver/intestine, propafenone 4'-hydroxylation in liver/intestine, and diazepam N-demethylation in the liver/intestine were higher in mice than in humans.CYP1A2/2E1 mRNAs were mainly expressed in the livers of humans and mice. Cyp2b9/2b10 mRNAs were abundant in the mouse lung/liver/intestine, but CYP2B6 was mainly expressed in the human liver. CYP2C/2D/3A mRNAs were expressed in the liver and intestine, with the respective proteins detected in tissue microsomes of both humans and mice.These information on P450-dependent drug-metabolising characteristics in hepatic and extrahepatic tissues is useful to understand the similarities and differences between humans and mice in drug metabolism.

Effects of multi-kinase inhibitors on the activity of cytochrome P450 2J2.

1. Cytochrome P450 2J2 (CYP2J2) shows high expression in extrahepatic tissues, including the heart and kidney and in tumours. Inhibition of CYP2J2 has attracted attention for cancer treatment because it metabolises arachidonic acid (AA) to epoxyeicosatrienoic acid (EET), which inhibits apoptosis and promotes tumour growth. Multi-kinase inhibitor (MKI) is a molecular-targeted drug with antitumor activities. This study aimed to clarify the inhibitory effects of MKIs on CYP2J2 activity. We also investigated whether MKIs affected CYP2J2-catalysed EET formation from AA.2. Twenty MKIs showed different inhibitory potencies against astemizole O-demethylation in CYP2J2. In particular, apatinib, motesanib, and vatalanib strongly inhibited astemizole O-demethylation. These three MKIs exhibited competitive inhibition with inhibition constant (Ki) values of 9.3, 15.4, and 65.0 nM, respectively. Apatinib, motesanib, and vatalanib also inhibited CYP2J2-catalysed 14,15-EET formation from AA.3. In simulations of docking to CYP2J2, the U energy values of apatinib, motesanib, and vatalanib were low, and measured -84.5, -69.9, and -52.3 kcal/mol, respectively.4. In conclusion, apatinib, motesanib, and vatalanib strongly inhibited CYP2J2 activity, suggesting that the effects of a given CYP2J2 substrate may be altered upon the administration of these MKIs.