Expression and Characterization of Dog Cytochrome P450 2A13 and 2A25 in Baculovirus-Infected Insect Cells

Abstract

ABSTRACT:Dog CYP2A13 and CYP2A25 were coexpressed with dog NADPHcytochrome P450 reductase (OR) in baculovirus-infected Sf9 insect cells. CYP2A13 effectively catalyzed 7-ethoxycoumarin (7EC) deethylation and coumarin hydroxylation with apparent K m values of 4.8 and 2.1 M, respectively, similar to those observed using dog liver microsomes (7.5 and 0.75 M, respectively). CYP2A25 exhibited much lower affinity toward 7EC, with an apparent K m value of 150 M, which indicates that CYP2A13 plays a more significant role in the metabolism of these CYP2A substrates. Similar to the dog CYP1A2 enzyme, CYP2A13 efficiently catalyzed phenacetin deethylation with a K m value of 3.9 M, which suggests that phenacetin is not a selective probe for dog CYP1A2 activity. Both dog CYP2A13 and CYP2A25 exhibited little or no catalytic activity toward other common cytochrome P450 probe substrates, including bupropion, amodiaquine, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, midazolam, and testosterone. These results provided additional information about the selectivity of these commonly used probe substrates.

Phenacetin Pharmacokinetics in CYP1A2-Deficient Beagle Dogs

“…In summary, in vivo studies were conducted to assess the metabolic disposition of phenacetin and formation of acetaminophen in dogs having normal CYP1A2 activity versus those deficient in CYP1A2 activity. Data obtained in CYP1A2-deficient dogs supports recent in vitro data by Zhou et al, 2010 and strengthens the conclusion that the phenacetin to acetaminophen pathway is not selective for CYP1A2 activity in the dog. These results, particularly at the 5 mg/kg phenacetin dose, show that CYP1A2 is a significant enzyme responsible for the formation of acetaminophen from phenacetin; however, it is not likely to be the only enzyme or pathway responsible for the elimination of phenacetin or generation of acetaminophen.…”

“…Recent studies suggest that dog CYP2A13, which is predominantly present in the liver, catalyzes phenacetin O-deethylation to acetaminophen. The phenacetin to acetaminophen intrinsic clearance was found to be similar for dog CYP2A13 (0.92 l ⅐ min Ϫ1 ⅐ pmol Ϫ1 ) and CYP1A2 (1.1 l ⅐ min Ϫ1 ⅐ pmol Ϫ1 ) (Zhou et al, 2010). Although the intrinsic clearance of phenacetin through the CYP1A2 and CYP2A13 pathways is similar, the overall in vivo metabolic disposition of phenacetin to acetaminophen will also be governed by the relative amounts of each enzyme present in the liver, which are unknown at this time.…”

“…P450 enzymes have been extensively studied in humans and rodents; however, dogs are commonly used for pharmacological and absorption, distribution, metabolism, and excretion/toxicology studies of new drug candidates, and studies of their P450 enzymes and polymorphisms are few (Tenmizu et al, 2006;Zhou et al, 2010). Although genetic polymorphisms have been reported in the dog, our understanding of their contribution to the interindividual variability in drug pharmacokinetics related to pharmaceutical drug development and veterinary practice is limited (Mise et al, 2004a).…”

“…1). However, recent in vitro studies by Zhou et al (2010) have found that the dog CYP2A13 enzyme has a similar in vitro intrinsic clearance to catalyze phenacetin to acetaminophen compared with CYP1A2, suggesting that phenacetin may not be a selective probe for dog CYP1A2 enzyme activity in vitro. To investigate the effects of the CYP1A2 genotypic deficiency and the potential contribution of CYP2A13 on the in vivo metabolism of phenacetin; previously genotyped dogs (Whiterock et al, 2007) were dosed orally and intravenously with phenacetin.…”

Canine Cytochrome P-450 Pharmacogenetics

“…For example midazolam is metabolized exclusively by human CYP3A4 and CYP3A5 (but not by human CYP2B6) while dog CYP2B11 (and not CYP3A12) primarily metabolizes midazolam 6 . Similarly, both dog CYP1A2 and dog CYP2A13 metabolize phenacetin 7, 8 , while only human CYP1A2 (and not human CYP2A6) metabolizes this drug. Although most dog CYPs have unique names, 3 of the drug metabolizing CYPs (CYP1A1, CYP1A2, and CYP2E1) have identical names to those found in other mammalian species, in part because they have relatively conserved gene sequences between species, and in part because their naming preceded the convention to give unique names to the drug metabolizing CYPs in different species.…”