Novel dog hepatic cytochrome P450 3A293 oxidizes endogenous testosterone and estradiol

Essential drug-metabolizing enzymes, such as the cytochromes P450 (P450s or CYPs), have been analyzed in dogs, which are frequently used during drug development. Dog CYP3A12, CYP3A26, CYP3A98, and CYP3A99 have been previously identified and analyzed, and in this study, novel dog CYP3A293 cDNA was isolated. CYP3A293 cDNA contains an open reading frame of 503 amino acids and shares 71-77 % and 86-96 % amino acid identity with human and dog CYP3As, respectively. Phylogenetic analysis revealed that dog CYP3A293 was most closely related to dog CYP3A99. Dog CYP3A293 is the fifth CYP3A form found in dogs and has a gene structure similar to human and other dog CYP3A genes, with 13 coding exons. Among the eight tissues analyzed, dog CYP3A293 mRNA was preferentially expressed in liver. By analyzing all five dog CYP3A mRNAs in liver samples from four dogs, CYP3A12 mRNA was the most abundant, followed by CYP3A26 and CYP3A293 mRNAs. Recombinant dog CYP3A293 mediated similar rates for the sum of testosterone 6(3-/16 alpha-hydroxylations and estradiol 2-/16 alpha- hydroxylations, but the rates of midazolam 1 '- and 4-hydroxylations were slow, similar to the slow testosterone 6(3-hydroxylation activity of dog CYP3A293. Dog CYP3A293 effectively mediated diclofenac 4 ' and 5-hydroxylations and caffeine N7-demethylation. Docking simulations supported the finding of regioselective oxidation of testosterone by dog CYP3A293. These results suggest that novel dog hepatic CYP3A293, the fifth known form of dog CYP3A among P450 enzymes, extensively oxidizes endogenous testosterone and estradiol and exogenous diclofenac and caffeine but does not effectively metabolize midazolam.