Abstract
Metabolism of the prototype human CYP2D6 substrates debrisoquine and bufuralol proceeds at a much slower rate in mice; therefore, the mouse has been proposed as an animal model for the human CYP2D6 genetic deficiency. To interpret the molecular mechanism of this deficiency, a cDNA belonging to the CYP2D gene subfamily (Cyp2d22) has been cloned and sequenced from a mouse mammary tumor-derived cell line. In the current study, Cyp2d22 enzyme was overexpressed and purified from insect cells using a baculovirus-mediated system. The activity of this purified enzyme was directly compared with purified human CYP2D6 toward codeine, dextromethorphan, and methadone as substrates. Purified Cyp2d22 was found to catalyze the O-demethylation of dextromethorphan with significantly higher Km values (250 μM) than that (4.2 μM) exhibited by purified human CYP2D6. The Km for dextromethorphan N-demethylation by Cyp2d22 was found to be 418 μM, much lower than that observed with human CYP2D6 and near the Km for dextromethorphan N-demethylation catalyzed by CYP3A4. CYP2D6 catalyzed codeine O-demethylation, whereas Cyp2d22 and CYP3A4 mediated codeine N-demethylation. Furthermore, methadone, a known CYP3A4 substrate and CYP2D6 inhibitor, was N-demethylated by Cyp2d22 with a Km of 517 μM and Vmax of 4.9 pmol/pmol/min. Quinidine and ketoconazole, potent inhibitors to CYP2D6 and CYP3A4, respectively, did not show strong inhibition toward Cyp2d22-mediated dextromethorphan O- or N-demethylation. These results suggest that mouse Cyp2d22 has its own substrate specificity beyond CYP2D6-like-deficient activity.
Footnotes
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This work was supported in part by Grant ES09894 from the National Institute of Environmental Health Sciences.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.008870.
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ABBREVIATIONS: P450, cytochrome P450; ESI/LC-MS, electrospray ionization/liquid chromatography mass spectrometry; EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; PMSF, phenylmethylsulfonyl fluoride; HPLC, high-performance liquid chromatography; HT, hexahistidine-tagged.
- Received December 7, 2005.
- Accepted March 29, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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