In vitro investigations on phase I biotransformation of ketamine in horses

Schmitz, Andrea (2013). In vitro investigations on phase I biotransformation of ketamine in horses. (Dissertation, Department of Clinical Research and Veterinary Public Health, Veterinär-Pharmakologie und -Toxikologie Name Mevissen, Meike, Vetsuisse Fakultät Bern)

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Introduction: Ketamine is an anesthetic and analgesic drug widely used in human and veterinary medicine. Pharmacokinetics and metabolism of ketamine are not well researched in many animal species. In the context of an interdisciplinary project on ketamine pharmacokinetics and pharmacodynamics in horses, in vitro studies were performed using horse liver microsomes and single equine cytochrome P450 enzymes. Single equine cytochrome P450 enzymes were assigned in the horse genome and recombinantly expressed in our laboratory since they are not commercially available.
Methods: Metabolite formation in incubations of ketamine with equine liver microsomes was monitored using enantioselective capillary electrophoresis, High-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC-MS). Metabolite formation in incubations with liver microsomes from dogs and humans was compared to the results obtained with horse liver microsomes. In vitro pharmacokinetics of ketamine
N-demethylation were calculated using the Michaelis-Menten equation. Results were compared between liver microsomes from horses, dogs and humans and between racemic ketamine and the single enantiomers.
The CYP3A gene cluster within the horse genome was assigned by comparison with the human CYP3A4 sequence and the deduced equine CYP3A genes were amplified from cDNA of a horse liver. The same procedure was performed to find the equine orthologs of human CYP2B6 and CYP2C19. Names for the equine orthologs were retrieved and sequencing of the coding regions and detection of polymorphisms was followed by cloning of equine CYP3A94, CYP2B6 and CYP2C113 and heterologous expression in V79 hamster lung fibroblasts. Activity of the expressed CYPs for ketamine N-demethylation was monitored in transfected V79 cell cultures with enantioselective capillary electrophoresis.
Results: Ketamine N-demethylation to norketamine is the most important metabolic reaction in all species. Norketamine is then further metabolized to several hydroxynorketamine metabolites and dehydronorketamine in liver microsomes of all tested species. Differences in proportions and enantioselectivity of metabolites were discovered. In vitro pharmacokinetics of ketamine N-demethylation were well characterized with a Michaelis-Menten model.
Vmax, Kd and intrinisic clearance of the single enantiomers and the racemic mixture differed between species and might explain differences in pharmacodynamics of the ketamine enantiomers in the different species. In the horse genome the CYP2B, CYP2C and CYP3A gene clusters contain more members than the respective human gene clusters. Coding sequences of six out of seven genes of the equine CYP3A gene cluster could be amplified from horse liver cDNA together with equine CYP2B6 and CYP2C113. Single nucleotide polymorphisms and one 6-pb deletion were found in all sequences except for CYP2C113. Ketamine was metabolized to norketamine by CYP3A94 and CYP2B6 but not by CYP2C113. CYP2C113 showed very low metabolism of mephenytoin to 4-hydroxymephenytoin and omeprazole to 5-hydroxyomeprazole.
Conclusions: The work presented here suggests that enantioselective in vivo pharmacokinetics in horses as observed in other studies can be possibly explained by the enantioselective N-demethylation of ketamine in the equine liver. Number of CYPs and substrate specificity of equine CYPs within a subfamily are not the same as in the orthologous human CYP subfamilies supporting the hypothesis that metabolism studies have to be carried out for each species individually. The coding sequences of all investigated CYPs are highly polymorphic compared to the reference sequence and further research will likely find links between genotype and pharmacological phenotype. Knowledge of CYPs responsible for ketamine metabolism will help to define drug-drug interactions between ketamine and other coadministered drugs in horses.

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