Phenacetin Pharmacokinetics in CYP1A2-Deficient Beagle Dogs

Abstract

ABSTRACT:Phenacetin is widely used as an in vitro probe to measure CYP1A2 activity across species. To investigate whether phenacetin can be used as an in vivo probe substrate to phenotype CYP1A2 activity in dogs, beagle dogs previously genotyped for a single nucleotide polymorphism that yields an inactive CYP1A2 protein were selected and placed into one of three groups: CC (wild-type), CT (heterozygous), or TT (homozygous mutants). The dogs were dosed with phenacetin orally at 5 and 15 mg/kg and intravenously at 15 mg/kg. Plasma samples were analyzed by liquid chromatography-tandem mass spectrometry, and phenacetin and its primary metabolite, acetaminophen, were monitored. After intravenous dosing, all groups showed similar exposure of phenacetin irrespective of genotype. After oral dosing at 15 mg/kg, the exposure of phenacetin in CC and CT dogs was similar, but phenacetin exposure was 2-fold greater in TT dogs. Exposure of the metabolite, acetaminophen, was similar in all groups; however, the mean acetaminophen/phenacetin ratio in TT dogs was 1.7 times less than that observed in CC dogs. Similar trends between the groups of dogs with respect to phenacetin exposure were also observed after a lower 5 mg/kg p.o. dose of phenacetin; however, a proportionally greater amount of acetaminophen was generated. Although oral exposure of phenacetin was 2-fold higher and acetaminophen exposure was 2-fold lower in CYP1A2-deficient (TT) dogs, these results were considered modest and suggest that phenacetin is not a selective or robust in vivo probe to measure CYP1A2 enzyme activity in the dog. IntroductionCytochrome P450 (P450) enzymes play a significant role in the oxidative metabolism and elimination of xenobiotics and endogenous substances. 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).The CYP1A2 enzyme is expressed in all mammals with few species differences in function or regulation, and all have very similar amino acid sequences (Parkinson and Ogilvie, 2008). In humans, CYP1A2 polymorphisms in the coding region are rare and only occasionally appear to decrease enzyme activity (Faber et al., 2005). A CYP1A2 polymorphism in beagle dogs has been reported (Tenmizu et al., 2004). The mutation is a single nucleotide polymorphism, CYP1A2 1117 CϾT, which results in an amino acid change from arginine to an early stop codon at position 373 (truncated inactive protein). Pharmacokinetic variability was observed in dogs after administration of 5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-2-oxo-1,2-d...

Identification and Characterization of Psoralen and Isopsoralen as Potent CYP1A2 Reversible and Time-Dependent Inhibitors in Human and Rat Preclinical Studies

“…The CYP1A2 enzyme is known to be expressed in all mammalian livers, with few species differences in function or regulation and high amino acid sequence homology (Parkinson and Ogilvie, 2008). Phenacetin is a substrate widely used as an in vitro and in vivo probe (except for in beagle dogs; Whiterock et al, 2012) to measure CYP1A2 activity because the metabolism of phenacetin to acetaminophen is thought to be a selective CYP1A2-mediated reaction (Distlerath et al, 1985;Tassaneeyakul et al, 1993). Hence, in the present study, the inhibitory effects of PRN and IPRN on CYP1A2-mediated metabolism were assessed in vitro and in vivo in rats and in vitro in humans using phenacetin as the probe substrate.…”

Canine Cytochrome P-450 Pharmacogenetics

“…However, other (human selective) CYP1A2 substrates including caffeine and melatonin were unaffected by the deficiency, implying that dog CYP1A2 does not selectively metabolize these latter drugs (unlike human CYP1A2). A recent study of phenacetin pharmacokinetics following oral and intravenous administration to CYP1A2 genotype Beagles 15 showed about 2-fold higher phenacetin exposure (based on area under the plasma concentration time curve; AUC) after oral administration in CYP1A2 deficient dogs, but there were much smaller (non-significant) differences in phenacetin levels after intravenous exposure. The authors concluded that phenacetin was not a selective or robust in vivo probe for CYP1A2 probably because of metabolism of phenacetin by other enzymes (such as canine CYP1A1 and/or canine CYP2A13).…”

Population variability in animal health: Influence on dose–exposure–response relationships: Part I: Drug metabolism and transporter systems

“…The following provides an update on research studies that have been newly identified since those reviews were published. No enzyme protein or activity Mise, Hashizume, and Komur (2008), Mise et al (2004), Tenmizu, Endo, Noguchi, and Kamimura (2004), Kamimura (2006); , Tenmizu, Noguchi, Kamimura, Ohtani, and Sawada (2006) About twofold increase in phenacetin plasma concentrations Whiterock, Morgan, Lentz, Orcutt, and Sinz (2012) Up to 17-fold increase in plasma concentrations of a test compound in homozygous deficient dogs , Tenmizu, Noguchi, Kamimura, Ohtani, et al (2006) Highest allele frequencies in Irish Wolfhounds 2000; Sams, Muir, Detra, & Robinson, 1985;Zoran, Riedesel, & Dyer, 1993). Two CYP2B11 polymorphisms were reported as part of a doctoral thesis (Wenker, 2009).…”