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This article is available online at http://dmd.aspetjournals.org ABSTRACT: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 K m enzyme, 1 M). CYP2B11 and CYP3A12 effectively catalyzed the N 1 -demethylation and C 3 -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 M) were achieved, suggesting a high K m for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low K m process (K m ‫؍‬ 0.7 M), similar to that in dog liver microsomes (K m ‫؍‬ 2.3 M). CYP2D15 was also capable of metabolizing bufuralol (1-hydroxylation), with a K m of 3.9 M, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16␣-, 2␣/2␤-, and 6␤-positions. Selectivity of CYP3A12 was observed toward testosterone 6␤-(K m ‫؍‬ 83 M) and 2␣/2␤-hydroxylations (K m ‫؍‬ 154 M). However, the 16␣-hydroxylation of testosterone was catalyzed by CYP2C21 also (K m ‫؍‬ 6.4 M for CYP2C21). Therefore, the 6␤-and 16␣-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.

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