Optimized on-line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes.
Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests. Hence, an optimized and economical capillary electrophoretic method for on-line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion-based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter-scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ-cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4-mediated N-demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost-effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450-mediated metabolic steps within early stages of the development of a new drug.
Department of Biochemistry, Faculty of Science and CEITEC-Central European Institute of Technology, Masaryk University, Brno, Czech Republic.|Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
Roman
R
Řemínek
Department of Biochemistry, Faculty of Science and CEITEC-Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
Zdeněk
Z
Glatz
Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern, Bern, Switzerland.
Wolfgang
W
Thormann
D065607:Cytochrome P-450 Enzyme Inhibitors / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D020927:Dexmedetomidine / Q000378:metabolism / Q000493:pharmacokinetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D007649:Ketamine / Q000378:metabolism / Q000493:pharmacokinetics; D007654:Ketoconazole / Q000378:metabolism / Q000493:pharmacokinetics; D013237:Stereoisomerism; D047408:gamma-Cyclodextrins / Q000378:metabolism / Q000493:pharmacokinetics
D065607:Cytochrome P-450 Enzyme Inhibitors; D047408:gamma-Cyclodextrins; D020927:Dexmedetomidine; D007649:Ketamine; D003577:Cytochrome P-450 Enzyme System; C023792:gamma-cyclodextrin; D007654:Ketoconazole
Capillary electrophoresis; Cytochrome P450 3A4; Dexmedetomidine; Enantioselective drug metabolism; Ketamine; Ketoconazole