title: Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam.
abstract: 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.
authors.affiliation: Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.
authors.forename: S M M
authors.initials: SM
authors.lastname: Nakayama
authors.affiliation: Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.|Water Research Group, Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa.
authors.forename: Y
authors.initials: Y
authors.lastname: Ikenaka
authors.affiliation: Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Hayami
authors.affiliation: Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Mizukawa
authors.affiliation: Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.|Food Control Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
authors.forename: W S
authors.initials: WS
authors.lastname: Darwish
authors.affiliation: Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.
authors.forename: K P
authors.initials: KP
authors.lastname: Watanabe
authors.affiliation: Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.|Diagnostic Center for Animal Health and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan.
authors.forename: Y K
authors.initials: YK
authors.lastname: Kawai
authors.affiliation: Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Ishizuka
mesh_terms: D000818:Animals; D019556:COS Cells / Q000201:enzymology / Q000378:metabolism; D002522:Chlorocebus aethiops; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism*; D003975:Diazepam / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D006736:Horses / Q000378:metabolism*; D006993:Hypnotics and Sedatives / Q000493:pharmacokinetics*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D003708:Nordazepam / Q000493:pharmacokinetics; D010076:Oxazepam / Q000493:pharmacokinetics; D010802:Phylogeny; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013045:Species Specificity; D013693:Temazepam / Q000493:pharmacokinetics
chemical_list: D006993:Hypnotics and Sedatives; D020033:Protein Isoforms; C049480:4'-hydroxydiazepam; D003708:Nordazepam; D010076:Oxazepam; D013693:Temazepam; D051544:Cytochrome P-450 CYP3A; D003975:Diazepam