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 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 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 μM), similar to that in dog liver microsomes (K_m = 2.3 μM). CYP2D15 was also capable of metabolizing bufuralol (1′-hydroxylation), with a K_m of 3.9 μM, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16α-, 2α/2β-, and 6β-positions. Selectivity of CYP3A12 was observed toward testosterone 6β-(K_m = 83 μM) and 2α/2β-hydroxylations (K_m = 154 μM). However, the 16α-hydroxylation of testosterone was catalyzed by CYP2C21 also (K_m = 6.4 μM for CYP2C21). Therefore, the 6β- and 16α-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.