Equine uridine diphospho-glucuronosyltransferase 1A1, 2A1, 2B4, 2B31: cDNA cloning, expression and initial characterization of morphine metabolism.

OBJECTIVE: Uridine diphospho-glucuronosyltransferases (UGTs) are membrane-bound enzymes that catalyze the conjugation of glucuronic acid onto a diverse set of xenobiotics. Horses efficiently and extensively glucuronidate a number of xenobiotics, including opioids, making UGTs an important group of drug-metabolizing enzymes for the clearance of drugs. Recombinant enzymes have allowed researchers to characterize the metabolism of a variety of drugs. The primary objective was to clone, express and characterize equine UGTs using drugs characterized as UGT substrates in other species. A secondary objective was to characterize the in vitro metabolism of morphine in horses. STUDY DESIGN: In vitro drug metabolism study using liver microsomes and recombinant enzyme systems. ANIMALS: Liver microsomes and RNA from tissue collected from two Thoroughbred mares euthanized for other reasons. METHODS: Based on homology to the human UGT2B7, four equine UGT variants were expressed: UGT1A1, UGT2A1, UGT2B31 and UGT2B4. cDNA sequences were cloned and resulting protein expressed in a baculovirus expression system. Functionality of the enzymes was assessed using 4-methylumbelliferone, testosterone, diclofenac and ketoprofen. Recombinant enzyme, control cells, equine liver microsomes and human UGT2B7 supersomes were then incubated with morphine. Concentrations of metabolites were measured using liquid chromatography-tandem mass spectrometry and enzyme kinetics determined. RESULTS: 4-Methylumbelliferone was glucuronidated by all expressed equine UGTs. Testosterone glucuronide was not produced by any of the expressed enzymes, and diclofenac glucuronide and ketoprofen glucuronide were produced by UG2A1 and UGT1A1, respectively. UGT2B31 metabolized morphine to morphine-3-glucuronide and low concentrations of morphine-6-glucuronide. CONCLUSIONS AND CLINICAL RELEVANCE: This is the first successful expression of functional recombinant equine UGTs. UGT2B31 contributes to the glucuronidation of morphine; however, it is probably not the main metabolizing enzyme. These results warrant further investigation of equine UGTs, including expression of additional enzymes and further characterization of UGT2B31 as a contributor to morphine metabolism.

Pharmacokinetics and anti-inflammatory effects of flunixin meglumine as a sole agent and in combination with phenylbutazone in exercised Thoroughbred horses.

BACKGROUND: Flunixin meglumine (FM) and phenylbutazone (PBZ) are potent anti-inflammatory agents and as such their potential to mask injuries that would otherwise keep a horse from training or racing is concerning. A common practice in racetrack medicine in the USA is to administer the two drugs within close proximity (24 hours apart) of each other, raising the concern of pharmacokinetic interactions and enhanced anti-inflammatory effects. OBJECTIVES: Describe the pharmacokinetics and effects of PBZ on the clearance of FM when administered in close proximity as well as effects on inflammatory mediators. STUDY DESIGN: Two-way randomised balanced crossover experiment. METHODS: Twelve Thoroughbred exercised horses received 500 mg FM IV alone or in combination with 2 g of IV PBZ 24 hours later. Blood and urine samples were collected prior to and for up to 120 hours post-drug administration. Whole blood samples were collected at various times and challenged with lipopolysaccharide or calcium ionophore to induce ex vivo synthesis of eicosanoids. Concentrations of FM, PBZ and eicosanoids were measured using LC-MS/MS and noncompartmental pharmacokinetic analysis performed on concentration data. RESULTS: Flunixin meglumine clearance was significantly increased when horses received PBZ 24 hours post-administration (P = .03). No other differences in pharmacokinetic parameters were noted between groups. Thromboxane B2 was significantly suppressed, relative to baseline for 96 hours post-FM administration. Subsequent administration of PBZ prolonged the suppression. Prostaglandin E2 was decreased for 24 hours following administration of FM with subsequent administration of PBZ prolonging the suppression until 120 hours. PGF2alpha concentrations were decreased for up to 168 hours post-FM administration. FM administration significantly decreased 15-HETE. MAIN LIMITATIONS: Small sample size and lack of a phenylbutazone-only treatment group. CONCLUSIONS: Administration of PBZ post-FM administration increased FM clearance. The anti-inflammatory effects of FM appear to be prolonged when PBZ is administered 24 hours post-administration.

Characterization of the in vitro CYP450 mediated metabolism of the polymorphic CYP2D6 probe drug codeine in horses.

Despite their widespread popularity as sport and companion animals and published and anecdotal reports of vast difference in drug disposition and pharmacokinetics between individuals, studies describing equine drug metabolism are limited. It has been theorized that similar to humans, members of the CYP2D family in horses may be polymorphic in nature leading to differences in metabolism of substrates. This study aims to build on the limited current knowledge regarding P450 mediated metabolism in horses by describing the metabolism of the polymorphic CYP2D6 probe drug codeine in vitro. Codeine, at varying substrate concentrations, was incubated with equine liver microsomes (±UDPGA) and a panel of baculovirus expressed recombinant equine P450s. Parent drug and metabolite concentrations were determined using LC-MS/MS. Incubation of codeine in equine liver microsomes generated norcodeine, morphine, codeine glucuronide and morphine 3- and 6- glucuronide. In recombinant P450 assays, the newly described CYP2D82 was responsible for catalyzing the biotransformation of codeine to morphine (Km of 247.4 µM and a Vmax of 1.6 pmol/min/pmol P450). CYP2D82 is 80% homologous to the highly polymorphic CYP2D6 enzyme, which is responsible for biotransformation of codeine to morphine in humans. CYP3A95, which shares 79% sequence homology with human CYP3A4 and CYP2D50 catalyzed the conversion of codeine to norcodeine (Km of 104.1 and 526.9 µM, Vmax of 2.8 and 2.6 pmol/min/pmol P450). In addition to describing the P450 mediated metabolism of codeine, the current study offers a candidate probe drug that could be used in vivo to study the functional implications of polymorphisms in the CYP2D gene in horses.