Effect of α₂-receptor agonists on the ketamine metabolism in different species assessed by enantioselective capillary electrophoresis

Medication around and during surgery includes a broad range of different drugs. Effects like anesthesia, analgesia, sedation and muscle relaxation are strived. Other drugs can be added in emergency cases or for controlling vital signs. For a safe surgery episode, however, polymedication is necessary. Side effects and the risk of pharmacokinetic and pharmacodynamic drug drug interactions have to be considered when multiple drugs are administered. For that reason it is important to know as much as possible about metabolism, pharmacokinetics and drug drug interactions of the used drugs. In this dissertation the combination of the racemic drug ketamine with anesthetic, analgesic and antidepressive properties and various sedative α2-receptor agonists was investigated in vitro and in vivo in different species using enantioselective capillary electrophoresis (CE). CE is a high-resolution separation technique that permits the separation and analysis of the stereoisomers of drugs and metabolites in the same run and can thus be used to determine enzyme kinetics, pharmacokinetics and drug drug interactions. Three different assays were developed and/or optimized during this dissertation to describe the interactions between ketamine and the α2-receptor agonists medetomidine, its active enantiomer dexmedetomidine, detomidine, xylazine and romifidine. The effect of medetomidine and its active enantiomer dexmedetomidine on the N-demethylation of ketamine to norketamine was analyzed in vitro with canine liver microsomes, human liver microsomes and the single cytochrome P450 enzymes CYP3A12 (canine) and CYP3A4 (human). Inhibition of norketamine formation in presence of medetomidine or dexmedetomidine was observed in most of the performed in vitro experiments and described with the inhibition parameter Ki and IC50. Decreased norketamine formation under medetomidine comedication was also seen in an in vivo study with Beagle dogs. One group received racemic or single S-ketamine under sevoflurane anesthesia and another after medetomidine sedation. For analyzing the blood samples which were collected between 0 and 900 min after ketamine injection an enantioselective CE microassay was developed and validated. Besides the advantage that only 50 µL of serum or plasma are needed for analysis it quantifies not only the enantiomers of ketamine and norketamine but also the stereoisomers of 6-hydroxynorketamine (6HNK) and dehydronorketamine (DHNK). Stereoselectivities were detected for 6HNK and DHNK. With the obtained plasma levels the pharmacokinetics of these substances could be described by using two compartment models for ketamine and norketamine enantiomers and single compartment models for 6HNK and DHNK stereoisomers. The impact of the four α2-receptor agonists medetomidine, detomidine, xylazine and romifidine on the ketamine metabolism was assessed in vitro with equine liver microsomes. Medetomidine was found to be the strongest inhibitor, followed by detomidine. The incubation time was extended and 6HNK and DHNK were determined as well. In these experiments the four α2-receptor agonists exhibited an effect on the formation of all metabolites. To have a closer look at HNK a new assay was developed which permits the separation of the stereoisomers of four hydroxylated norketamine metabolites and DHNK. HNK and DHNK stereoisomers are reported to be responsible for antidepressive effects of ketamine. A mixture of sulfated β-cyclodextrin and highly sulfated γ-cyclodextrin was found to be an effective chiral selector for that task. This assay was applied to analyze in vitro and in vivo samples and data obtained revealed differences in the ketamine metabolism of dogs and horses that could hitherto not be assessed.

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