Abstract
Small intestinal microsomes of cynomolgus monkeys were found to catalyze hydroxylation and dealkylation of an H1-antihistamine prodrug, ebastine. To identify the main enzyme responsible for ebastine hydroxylation, which has been hitherto unknown, we purified two cytochrome P450 isoforms, named P450 MI-2 and P450 MI-3, from the intestinal microsomes on the basis of the hydroxylation activity. P450 MI-2 and P450 MI-3 showed the respective apparent molecular weights of 56,000 and 53,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The internal amino acid sequence of P450 MI-2 had high similarity with those of human CYP4F2, CYP4F3, and CYP4F8. The first 27 amino acid residues of P450 MI-3 were highly homologous with those of monkey CYP3A8 and human CYP3A4/5/7. Furthermore, P450 MI-2 and P450 MI-3 were recognized by anti-CYP4F and anti-CYP3A antibodies, respectively, in immunoblot analysis and catalyzed leukotriene B4 ω-hydroxylation and testosterone 6β-hydroxylation, which are known to be mediated by CYP4F and CYP3A, respectively. Although both enzymes had ebastine hydroxylation activity, the V max value of P450 MI-2 was much higher than that of P450 MI-3 (37.0 versus 0.406 nmol/min/nmol of P450), and the former K M (5.1 μM) was smaller than the latter K M (10 μM). Anti-CYP4F antibody inhibited the hydroxylation in small intestinal microsomes strongly (70%), but anti-CYP3A antibody did not. These results indicate that P450 MI-2 belongs to the CYP4F subfamily and is mainly responsible for hydroxylation of ebastine in monkey small intestinal microsomes. This suggests that the small intestinal CYP4F enzyme, P450 MI-2, can play an important role in the metabolism of drugs given orally.
Footnotes
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Send reprint requests to: Takanori Hashizume, Dept. of Pharmacokinetics, Developmental Research Laboratories, Dainippon Pharmaceutical Co., Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan. E-mail: takanori-hashidume{at}dainippon-pharm.co.jp
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This work was supported in part by a grant from the Japan Health Science Foundation.
- Abbreviations used are::
- P450
- cytochrome P450
- LTB4
- leukotriene B4
- PMSF
- phenylmethylsulfonyl fluoride
- DTT
- dithiothreitol
- DLPC
- dilauroyl-l-3-phosphatidylcholine
- PAGE
- polyacrylamide gel electrophoresis
- HPLC
- high-performance liquid chromatography
- Received October 23, 2000.
- Accepted February 5, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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