Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam.

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p-hydroxydiazepam [p-OH-DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p-OH-DZP was significantly less in the horse. Inhibition assays with a CYP-specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos-7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.

Partial cloning of CYP2C23a genes and hepatic protein expression in eight representative avian species.

Large interspecies differences in avian xenobiotic metabolism have been revealed by microsome-based studies, but specific enzyme isoforms in different bird species have not yet been compared. We have previously shown that CYP2C23 genes are the most induced CYP isoforms in chicken liver. In this study, we collected partial CYP2C23a gene sequences from eight avian species (ostrich, blue-eared pheasant, snowy owl, great-horned owl, Chilean flamingo, peregrin falcon, Humboldt penguin, and black-crowned night heron) selected to cover the whole avian lineage: Paleognathae, Galloanserae, and Neoaves. Genetic analysis showed that CYP2C23 genes of Galloanserae species (chicken and blue-eared pheasant) had unique characteristics. We found some duplicated genes (CYP2C23a and CYP2C23b) and two missing amino acid residues in Galloanserae compared to the other two lineages. The genes have lower homology than in other avian lineages, which suggests Galloanserae-specific rapid evolutionary changes. These genetic features suggested that the Galloanserae are not the most representative avian species, considering that the Neoaves comprise more than 95% of birds. Moreover, we succeeded in synthesizing an antipeptide polyclonal antibody against the region of CYP2C23 protein conserved in avians. However, comparative quantitation of CYP2C23 proteins in livers from six species showed that expression levels of these proteins differed no more than fourfold. Further study is needed to clarify the function of avian CYP2C23 proteins.