Novel Cytochrome P450 2C94 Functionally Metabolizes Diclofenac and Omeprazole in Dogs

Abstract

Cytochromes P450 (P450s or CYPs) are important drug-metabolizing enzymes. Because dogs are frequently used in drug metabolism studies, knowledge of dog CYP2C enzymes is essential because, in humans, these enzymes are abundant and play major roles in liver and intestine. The present study identified and characterized novel dog CYP2C94 along with previously identified dog CYP2C21 and CYP2C41. Dog CYP2C21, CYP2C41, and CYP2C94 cDNAs, respectively, contained open reading frames of 490, 489, and 496 amino acids and shared high sequence identities (70%, 75%, and 58%) with human CYP2Cs. Dog CYP2C94 mRNA was preferentially expressed in liver, just as dog CYP2C21 and CYP2C41 mRNAs were. In dog liver, CYP2C21 mRNA was the most abundant, followed by CYP2C94 and CYP2C41 mRNAs. Moreover, the hepatic expressions of all three dog CYP2C mRNAs varied in four individual dogs, two of which did not express CYP2C41 mRNA. The three dog CYP2C genes had similar gene structures, and CYP2C94, although located on the same chromosome, was in a genomic region far from the gene cluster containing CYP2C21 and CYP2C41.Metabolic assays with recombinant proteins showed that dog CYP2C94, along with CYP2C21 and CYP2C41, efficiently catalyzed oxidations of diclofenac, warfarin, and/or omeprazole, indicating that dog CYP2C94 is a functional enzyme. Novel dog CYP2C94 is expressed abundantly in liver and encodes a functional enzyme that metabolizes human CYP2C substrates; it is, therefore, likely responsible for drug clearances in dogs.

Novel Cytochrome P450 2C119 Enzymes in Cynomolgus and Rhesus Macaques Metabolize Progesterone, Diclofenac, and Omeprazole

“…Progesterone, paclitaxel, diclofenac, R,S-warfarin, R,S-omeprazole, and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Preparation of recombinant human and cynomolgus P450 2C8, 2C9, and 2C19 and marmoset P450 2C119 proteins in bacterial membranes was carried out as described previously (Uno et al, 2011b;Uehara et al, 2015;Uehara et al, 2016;Nakanishi et al, 2018a;Uno et al, 2023).…”

“…Progesterone 16-, 17-, and 21-hydroxylation; paclitaxel 6-hydroxylation; diclofenac 4′-hydroxylation; R,S-warfarin 7-hydroxylation; and R,S-omeprazole 5-hydroxylation and sulfoxidation activities of recombinant CYP2C proteins were analyzed as described previously (Uno et al, 2011b;Uehara et al, 2015;Uehara et al, 2016;Nakanishi et al, 2018a;Uno et al, 2023). In brief, typical reaction mixtures (0.20 mL) contained recombinant protein (25 pmol/mL), an NADPH-generating system, and substrate (10 or 100 M) in 50 mM potassium phosphate buffer (pH 7.4); the lower substrate concentrations were generally corresponded to those of reported Michaels-Menten constants for typical human CYP2C-depedent drug oxidations (Uno et al, 2011b;Uehara et al, 2015;Uehara et al, 2016;Nakanishi et al, 2018a;Uno et al, 2023). Reaction mixtures were incubated at 37°C for 15 or 30 min and then terminated.…”

“…Reaction mixtures were incubated at 37°C for 15 or 30 min and then terminated. Incubates were centrifuged at 900  g for 5 min, and supernatants were analyzed by reverse phase high-performance liquid chromatography with a fluorescence detector or UV detector and an analytical column (C 18 ) as described (Uehara et al, 2015;Uehara et al, 2016;Nakanishi et al, 2018a;Nakanishi et al, 2018b;Uno et al, 2023).…”