Different Roles of Canine Cytochrome P450 2C and 3A Enzymes Involved in the Oxidative Metabolism of Phenobarbital, a Key Anticonvulsant for Dogs: Comparison With Human P450 Enzymes

Phenobarbital is a key anticonvulsant used in dogs. Cytochrome P450 (P450) 2C19 is reportedly involved in the oxidative metabolism of phenobarbital in humans; however, the hepatic P450 enzymes responsible for phenobarbital p ‐hydroxylation in dogs have not yet been identified. In the present study, we investigated the roles of a range of dog P450 enzymes in phenobarbital p ‐hydroxylation activity using dog liver microsomes and recombinant proteins. Recombinant dog P450 2C21, 2C41, 3A12, and 3A98 enzymes and four liver microsomal preparations from four different dogs were used to investigate the biphasic P450‐dependent phenobarbital p ‐hydroxylation activity. Human P450 2C19 is the predominant enzyme for the p ‐hydroxylation of phenobarbital, but not exclusively, and a similar process was anticipated in dogs. Phenobarbital p ‐hydroxylation activities in four dog liver microsomal preparations at substrate concentrations of 5.0 and 50 μM were significantly correlated with P450 2C immunochemical band intensities, but activities were not correlated at a substrate concentration of 500 μM. Liver microsomes from dog 3 had a monophasic capacity ( V max , 0.35 pmol/min/mg protein) for phenobarbital p ‐hydroxylation ( K m , 4.9 μM), whereas liver microsomes from dog 6 had biphasic low ( V max1 , 0.11 pmol/min/mg protein) and high ( V max2 , 0.22 pmol/min/mg protein) capacities with high ( K m1 , 1.6 μM) and low ( K m2 , 72 μM) affinities, respectively. The primary P450 2C21‐dependent and secondary P450 3A12‐dependent phenobarbital p ‐hydroxylation activities of dog liver microsomes showed approximately two‐fold differences among the four individual dogs. This information could help develop an individualized approach for controlling epilepsy in dogs treated with phenobarbital.