Identification and characterization of the enzymes responsible for the metabolism of the non-steroidal anti-inflammatory drugs, flunixin meglumine and phenylbutazone, in horses - PubMed (original) (raw)

Identification and characterization of the enzymes responsible for the metabolism of the non-steroidal anti-inflammatory drugs, flunixin meglumine and phenylbutazone, in horses

Heather K Knych et al. J Vet Pharmacol Ther. 2021 Jan.

Abstract

The in vivo metabolism and pharmacokinetics of flunixin meglumine and phenylbutazone have been extensively characterized; however, there are no published reports describing the in vitro metabolism, specifically the enzymes responsible for the biotransformation of these compounds in horses. Due to their widespread use and, therefore, increased potential for drug-drug interactions and widespread differences in drug disposition, this study aims to build on the limited current knowledge regarding P450-mediated metabolism in horses. Drugs were incubated with equine liver microsomes and a panel of recombinant equine P450s. Incubation of phenylbutazone in microsomes generated oxyphenbutazone and gamma-hydroxy phenylbutazone. Microsomal incubations with flunixin meglumine generated 5-OH flunixin, with a kinetic profile suggestive of substrate inhibition. In recombinant P450 assays, equine CYP3A97 was the only enzyme capable of generating oxyphenbutazone while several members of the equine CYP3A family and CYP1A1 were capable of catalyzing the biotransformation of flunixin to 5-OH flunixin. Flunixin meglumine metabolism by CYP1A1 and CYP3A93 showed a profile characteristic of biphasic kinetics, suggesting two substrate binding sites. The current study identifies specific enzymes responsible for the metabolism of two NSAIDs in horses and provides the basis for future study of drug-drug interactions and identification of reasons for varying pharmacokinetics between horses.

Keywords: cytochrome P450; equine; flunixin meglumine; metabolism; phenylbutazone.

© 2020 John Wiley & Sons Ltd.

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Conflict of interest statement

CONFLICT OF INTEREST

All authors declare no conflicts of interest.

Figures

FIGURE 1

FIGURE 1

Metabolic pathway for flunixin in the horse

FIGURE 2

FIGURE 2

Metabolic pathway for phenylbutazone in the horse

FIGURE 3

FIGURE 3

Kinetic plots for the determination of the apparent _K_m and _V_max values for flunixin metabolism to 5-OH flunixin by equine liver microsomes. Plots (left and right) represent incubations in liver microsomes collected from two different horses. Incubations from each horse were run concurrently and carried out in triplicate

FIGURE 4

FIGURE 4

Kinetic plots for the determination of the apparent _K_m and _V_max values for metabolism of phenylbutazone to oxyphenbutazone (a) and gamma-hydroxy phenylbutazone (b) by equine liver microsomes. Plots (left and right) represent incubations in liver microsomes collected from two different horses. Incubations from each horse were run concurrently and carried out in triplicate

FIGURE 5

FIGURE 5

Kinetic plots of substrate concentrations versus the rate of the reaction for generation of 5-OH flunixin by baculovirus-expressed equine CYP450 enzymes following incubation with flunixin meglumine. Triplicate incubations were performed concurrently at each substrate concentrations

FIGURE 6

FIGURE 6

Kinetic plots of substrate concentrations versus the rate of the reaction for generation of oxyphenbutazone by baculovirus-expressed equine CYP3A97 enzymes following incubation with phenylbutazone. Triplicate incubations were performed concurrently at each substrate concentration

FIGURE 7

FIGURE 7

Generation of gamma-hydroxy phenylbutazone and oxyphenbutazone in equine liver microsomal incubations containing known inhibitors of human CYP450 enzymes. Metabolite production is expressed as a percentage compared to control (no inhibitor) incubations

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