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

Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

doi:10.1124/dmd.115.067561

Cited by 10 publications

(8 citation statements)

References 20 publications

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“…CYP protein levels varied by 2–10 folds among microsomal samples from individual monkeys 33 , 34 . Several studies also characterized intestinal CYP expression in the common marmosets, another species of non-human primate 35 , 36 , 37 .…”

Section: Expression Of Drug-metabolizing Cyps In the Intestinementioning

confidence: 99%

An update on the role of intestinal cytochrome P450 enzymes in drug disposition

Xie

Ding

Zhang

2016

Acta Pharmaceutica Sinica B

116

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Oral administration is the most commonly used route for drug treatment. Intestinal cytochrome P450 (CYP)-mediated metabolism can eliminate a large proportion of some orally administered drugs before they reach systemic circulation, while leaving the passage of other drugs unimpeded. A better understanding of the ability of intestinal P450 enzymes to metabolize various clinical drugs in both humans and preclinical animal species, including the identification of the CYP enzymes expressed, their regulation, and the relative importance of intestinal metabolism compared to hepatic metabolism, is important for improving bioavailability of current drugs and new drugs in development. Here, we briefly review the expression of drug-metabolizing P450 enzymes in the small intestine of humans and several preclinical animal species, and provide an update of the various factors or events that regulate intestinal P450 expression, including a cross talk between the liver and the intestine. We further compare various clinical and preclinical approaches for assessing the impact of intestinal drug metabolism on bioavailability, and discuss the utility of the intestinal epithelium–specific NADPH-cytochrome P450 reductase-null (IECN) mouse as a useful model for studying in vivo roles of intestinal P450 in the disposition of orally administered drugs.

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Section: Expression Of Drug-metabolizing Cyps In the Intestinementioning

confidence: 99%

An update on the role of intestinal cytochrome P450 enzymes in drug disposition

Xie

Ding

Zhang

2016

Acta Pharmaceutica Sinica B

116

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“…To prepare the recombinant proteins, expression plasmids of marmoset and human P450 4F forms were constructed using pCW vectors containing a cDNA of human NADPH-P450 reductase as described previously (Uehara et al, 2016c). The N-terminal modification was performed by PCR using the forward and reverse primers containing the restriction sites of the NdeI and XbaI restriction enzymes (underlined), respectively, including cjCYP4F2 (5exp1bov) 59-CATATGG-CTCTGTTATTAGCAGTTTTTCTGGGCCTCGGGCCG-39 and cjCYP4F2 (3exp2) 59-TCTAGATTAGCTCAGGGGCTCCA-39 for marmoset P450 4F2, cjCYP4F2 (5exp1bov) 59-CATATGGCTCTGTTATTAGCAGTTTTTCTG-GGCCTCGGGCCG-39 and cjCYP4F3B (3exp2) 59-TCTAGATTAGCT-CCGGGGCTCCA-39 for marmoset P450 4F3B, cjCYP4F11 (5exp2bov) 59-CATATGGCTCTGTTATTAGCAGTTTTTCTGGGCCTCGGGCCAGTG-39 and cjCYP4F11 (3exp2) 59-TCTAGATTACTGTGGGTTTGCACCC-39 for marmoset P450 4F11, cjCYP4F12 (5exp1bov) 59-CATATGGCTCTGTTATT-AGCAGTTTTTCTGGGCTTCGGGCCAG-39 and cjCYP4F12 (3exp2) 59-TCTAGATTACTGTGAGCCCGCGT-39 for marmoset P450 4F12, hCYP4F2 (5exp1bov) 59-CATATGGCTCTGTTATTAGCAGTTTTTCTGGGCCTCTG-GCCAGTG-39 and hCYP4F2 (3exp1) 59-TCTAGATTAGCTCAGGGGCTC-CACCCGC-39 for human P450 4F2, hCYP4F3B (5exp1bov) 59-CATA-TGGCTCTGTTATTAGCAGTTTTTCTGGGCCTTTGGCCAATG-39 and hCYP4F2 (3exp1) 59-TCTAGATTAGCTCAGGGGCTCCACCCGC-39 for human P450 4F3B, cjCYP4F11 (5exp2bov) 59-CATATGGCTCTGTTAT-TAGCAGTTTTTCTGGGCCTCGGGCCAGTG-39 and hCYP4F11 (3exp1) 59-TCTAGATTACTGTGAGTTCGCACCCAGG-39 for human P450 4F11, hCYP4F12 (5exp1bov) 59-CATATGGCTCTGTTATTAGCAGTTTTTCT-GGGCCTCAGACCGGTG-39 and hCYP4F12 (3exp1) 59-TCTAGAT-TACTGCAAGCTTACATTCAGG-39 for human P450 4F12, and Human 4F2 93 91 84 81 4F3B 90 93 82 81 4F11 86 86 89 82 4F12 82 82 81 87 Cynomolgus monkey 4F2 91 90 84 82 4F3B 90 91 83 81 4F11 83 85 85 79 4F12 81 81 80 88 Marmoset 4F2 91 84 81 4F3B 83 81 4F11 80 Fig.…”

Section: Methodsmentioning

confidence: 99%

A New Marmoset P450 4F12 Enzyme Expressed in Small Intestines and Livers Efficiently Metabolizes Antihistaminic Drug Ebastine

Uehara

Uno

Yuki

et al. 2016

Drug Metabolism and Disposition

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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 v-hydroxylation of leukotriene B 4 . 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.

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“…These results indicated that P450 2E1 enzymes had similar substrate specificities and catalytic activities among humans, cynomolgus monkeys, and marmosets. Our separated studies collectively indicated that other drug‐metabolizing forms of marmoset P450 enzymes effectively metabolized typical human P450 probe substrates: oxidations of ethoxyresorufin, ethoxycoumarin, and phenacetin by marmoset P450 1A2; S ‐warfarin, flurbiprofen, and omeprazole by marmoset P450 2C19; metoprolol, bufuralol, and dextromethorphan by marmoset P450 2D6; and midazolam, nifedipine, and testosterone by marmoset P450 3A4 (Uehara, Uno, Inoue, Sasaki, & Yamazaki, ; Uehara, Uno, Inoue, et al, ; Uehara, Uno, Hagihira, et al, ; Uehara et al, ). Taken together, marmosets may be good animal models for evaluating P450‐dependent drug oxidation in preclinical testing.…”

Section: Resultsmentioning

confidence: 74%

Functional characterization and tissue expression of marmoset cytochrome P450 2E1

Uehara

Uno

Tomioka

et al. 2017

Biopharm & Drug Disp

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Common marmosets (Callithrix jacchus) have attracted increasing attention as a useful small non-human primate model in preclinical research. However, studies on marmoset cytochrome P450 (P450) 2E enzyme have scarcely been conducted. In this study, the full-length cDNA encoding P450 2E1 enzyme was isolated from marmoset livers by reverse transcription (RT)-polymerase chain reaction (PCR). Marmoset P450 2E1 amino acid sequences were highly identical (>88%) to those of cynomolgus monkey and human P450 2E1 enzymes. Phylogenetic analysis indicated a close evolutionary relationship among marmoset, cynomolgus monkey, and human P450 2E1 enzymes. The tissue expression pattern analyzed by real-time RT-PCR and immunoblotting demonstrated that marmoset P450 2E1 mRNA and proteins were predominantly expressed in livers. Marmoset P450 2E1 enzyme heterologously expressed in Escherichia coli catalyzed the hydroxylation of p-nitrophenol, chlorzoxazone, and theophylline, similar to cynomolgus monkey and human P450 2E1 enzymes. By kinetic analyses, those P450 2E1 enzymes catalyzed p-nitrophenol hydroxylation with similar affinities and relatively high intrinsic clearance efficiencies. These results indicated that tissue distribution and enzyme-substrate specificity of marmoset P450 2E1 were similar to cynomolgus monkey and human P450 2E1 enzymes, suggesting that marmosets are a suitable primate model for P450 2E1-dependent drug and xenobiotic metabolism.

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

Cited by 10 publications

References 20 publications

An update on the role of intestinal cytochrome P450 enzymes in drug disposition

An update on the role of intestinal cytochrome P450 enzymes in drug disposition

A New Marmoset P450 4F12 Enzyme Expressed in Small Intestines and Livers Efficiently Metabolizes Antihistaminic Drug Ebastine

Functional characterization and tissue expression of marmoset cytochrome P450 2E1

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