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0090-9556/03/3109-1161-1169$20.00
DMD 31:1161-1169, 2003

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SUBSTRATE SPECIFICITY AND KINETIC PROPERTIES OF SEVEN HETEROLOGOUSLY EXPRESSED DOG CYTOCHROMES P450

Magang Shou, Ryan Norcross, Grit Sandig, Ping Lu, Yinghe Li, Yuh Lin, Qin Mei, A. David Rodrigues, and Thomas H. Rushmore

Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania

Seven dog cytochromes P450 (P450s) were heterologously expressed in baculovirus-Sf21 insect cells. Of all enzymes examined, CYP1A1 exhibited high 7-ethoxyresorufin O-deethylase activity (low Km enzyme, 1 µM). CYP2B11 and CYP3A12 effectively catalyzed the N1-demethylation and C3-hydroxylation of diazepam (and its derivatives), whereas CYP3A12 and CYP2D15 catalyzed exclusively the N- and O-demethylation, respectively, of dextromethorphan. However, no saturation velocity curves for the N-demethylation of dextromethorphan (up to 500 µM) were achieved, suggesting a high Km for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low Km process (Km = 0.7 µM), similar to that in dog liver microsomes (Km = 2.3 µM). CYP2D15 was also capable of metabolizing bufuralol (1'-hydroxylation), with a Km of 3.9 µM, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16{alpha}-, 2{alpha}/2ß-, and 6ß-positions. Selectivity of CYP3A12 was observed toward testosterone 6ß-(Km = 83 µM) and 2{alpha}/2ß-hydroxylations (Km = 154 µM). However, the 16{alpha}-hydroxylation of testosterone was catalyzed by CYP2C21 also (Km = 6.4 µM for CYP2C21). Therefore, the 6ß- and 16{alpha}-hydroxylation of testosterone can potentially be employed as markers of CYP3A12 and CYP2C21 (at low concentration), respectively. CYP2C21 was also capable of catalyzing diclofenac 4'-hydroxylation, although some activity was detected with CYP2B11. Surprisingly, none of the P450s selectively metabolized (S)-mephenytoin 4'-hydroxylation. The results described herein are a first step toward the systematic evaluation of a panel of dog P450s and the development of dog P450 isoenzyme-selective marker substrates, as well as providing useful information on prediction and extrapolation of the results from in vitro to in vivo and from dog to human.

Address correspondence to: Dr. Magang Shou, Department of Drug Metabolism, WP75A-203, Merck Research Laboratories, West Point, PA 19486. E-mail: magang_shou{at}merck.com




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