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

Briana D. Hamamoto-Hardman, Russell W. Baden, Dan S. McKemie, Heather K. Knych

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)763-772
Number of pages10
JournalVeterinary Anaesthesia and Analgesia
Volume47
Issue number6
DOIs
StatePublished - Nov 2020

Keywords

  • drug
  • equine
  • glucuronide
  • metabolism
  • morphine
  • uridine diphospho-glucuronosyltransferase

ASJC Scopus subject areas

  • veterinary(all)

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