UC Davis

Morphine is a potent, effective analgesic that is used widely in both human and veterinary medicine. Metabolism of morphine by UDP glucuronidation conjugation creates two metabolically active glucuronide metabolites: morphine-6 glucuronide and morphine-3 glucuronide. Morphine and its metabolites have been extensively studied in human medicine, however their pharmacokinetics and pharmacodynamics have yet to be explored to the same extent in horses. Such studies would help contribute to more effective opioid use in equine medicine. Our laboratory previously published a paper identifying M3G and M6G as the major metabolites of morphine in horses. This study also confirmed previous reports of increased motor activity, muscle fasciculation, and flared nostrils following intravenous administration of high doses of morphine (0.2 mg/kg and 0.5mg/kg). Additionally, horses in all dose groups (0.05, 0.1, 0.2 and 0.5 mg/kg) had decreased gastrointestinal activity, a common side effect associated with morphine use. Other side effects associated with morphine use in horses include increased motor activity, increased respiratory rate, increased blood pressure, and muscle fasciculation. The first aim of this work was to characterize and correlate the pharmacokinetics and selected pharmacodynamics of morphine administration in horses. Additional characterization of the pharmacokinetics and pharmacodynamic effects of morphine and its metabolites would add to existing data describing the relative concentrations of the metabolites of M3G and M6G. A total of ten horses were administered a single intravenous dose of morphine: 0.05, 0.1, 0.2, or 0.5 mg/kg, or saline control and blood samples were collected and analyzed for morphine and metabolites by LC/MS/MS to conduct pharmacokinetic analysis. Pharmacodynamic data in the forms of step count, heart rate and rhythm, gastrointestinal borborygmi, fecal output, packed cell volume, and total protein was also assessed. Morphine-3 glucuronide (M3G) was the predominant metabolite detected, with concentrations exceeding those of morphine-6 glucuronide (M6G) at all time point. The results also included decreased gastrointestinal motility and increased central nervous excitation with a correlation between increasing doses of morphine, increases in M3G concentrations and adverse effects Findings from this study supported additional studies of administration of purified M3G and M6G to horses to directly characterize pharmacodynamic activity of these metabolites. The next aim was to characterize and correlate the pharmacokinetics and selected pharmacodynamics of purified M6G in horses. In part one, 3 horses received a single intravenous administration of saline, 0.5 mg/kg M6G and 0.5 mg/kg morphine. Blood samples were collected up to 96-hours post administration, concentrations of drug and metabolites measured, and pharmacokinetics determined. Behavioral and physiologic effects were recorded. In part two of this study, two horses, scheduled to be euthanized for other reasons, were administered 0.5 mg/kg M6G. Blood, CSF and various tissue samples were collected post administration and concentrations of drug determined. The clearance of M6G was more rapid and the volume of distribution at steady state smaller for M6G compared to morphine. A reaction characterized by head shaking, pawing and slight ataxia was observed immediately following administration of both morphine and M6G horses. Following M6G administration, the behaviors subsided rapidly and was followed by a longer period of sedation. Following administration, M6G was detected in the kidney, liver, CSF and regions of the brain. Results of the current student encourage further investigation of M6G as an analgesic in horses. The final aim was to characterize the invitro metabolism of morphine by determining metabolic enzymes responsible for the formation of M3G and M6G. This was accomplished by expressing four equine UGT variants: UGT1A1, UGT2A1, UGT2B31 and UGT2B4 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. UGT2B31 metabolized morphine to morphine-3-glucuronide and low concentrations of morphine-6-glucuronide. While 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.