Enantioselective capillary electrophoresis : fundamental aspects and application to the "in vitro" assessment of CYP3A4 mediated ketamine N-demethylation

A large number of pharmaceutical substances are administered as racemates, consisting of two enantiomers with typically different pharmacodynamic and pharmacokinetic profiles. Thus, stereoselective analysis is important in drug development, therapeutic drug monitoring and research. Chiral separation by chromatographic methods such as HPLC and GC require expensive stationary phases. In contrast, in capillary electrophoresis (CE), enantioselective separation can be obtained using one or several chiral selectors which are added to the background electrolyte. Separation of enantiomers is based on different binding affinities with the enantiomers and/or different migration velocities of the formed analyte-selector complexes. High resolution can be achieved by varying type and concentration of chiral selector as well as buffer properties (pH, ionic strength, other additives etc.). Typical chiral selectors are neutral or charged cyclodextrins, proteins and bile acids. The simplicity of the technique makes enantioselective CE an attractive, promising and economic methodology for drug and metabolite analysis in pharmaceutical preparations, body fluids, tissues and microsomal preparations.
In the first part of this thesis, fundamental aspects of enantioselective CE were investigated using computer simulation. Dynamic computer simulators provide insight into the buffer system and improve understanding of the electrophoretic separation process. Simulation allows to predict proper separation and detection conditions for analytes prior to experiments. Using an extended version of the dynamic computer simulator GENTRANS, the interaction of methadone and its main metabolite EDDP with neutral chiral selectors were simulated. Experimentally determined complexation constants and mobilites of the formed complexes were employed as additional input parameters. Simulated electropherograms were qualitatively in good agreement with the experimental results.
In the second and third parts of the thesis, enantioselective CE was applied to study ketamine metabolism in an off-line study. Ketamine is a chiral phencyclidine derivative used in anesthesia. In vitro and also in vivo studies showed a higher affinity for the NMDA-receptors, higher anesthetic potency and shorter recovery time for S-ketamine compared to R-ketamine. The aims in this project were to characterize the kinetics of CYP 3A4 mediated ketamine N-demethylation in vitro including KM, Vmax and to investigate the stereoselective metabolism of this pathway. Furthermore, the inhibition kinetics of this pathway by ketoconazole, a potent CYP3A4 inhibitor, was investigated. Results showed a higher formation rate for S-norketamine after incubation of racemic ketamine as well as incubation of the single enantiomers. Data obtained in the absence of ketoconazole revealed that the N-demethylation occurred stereoselectively. Inhibition kinetics by ketoconazole fitted best to a one-site competitive model and no stereoselectivity could be demonstrated.
In the forth and final part of this thesis, an on-line method was developed to investigate the in vitro N-demethylation of ketamine via CYP3A4, with the incubation performed in-capillary with subsequent electrophoretic separation and detection of the ketamine enantiomers. Kinetic parameters obtained compared well with those of the off-line study mentioned above and the metabolic step was stereoselective, confirming the results of the off-line assay. After additional improvements, the in-capillary method should be widely applicable to assess enzymatic activity in a fast, low-cost and automated way.