Optimized On-Line Capillary Electrophoretic Method for CYP3A4 Mediated Enantioselective N-Demethylation of Ketamine
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| Year of publication | 2014 |
| Type | Conference abstract |
| MU Faculty or unit | |
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| Description | Enantioselective discrimination is a significant phenomenon influencing biological activity of chiral drugs in humans and animals. Rational drug discovery thus requires an early appraisal of this characteristic impacting on the likely success of a drug candidate in the subsequent clinical testing. Capillary electrophoresis (CE) is a promising technique in this field due to simple implementation of chiral separations, high separation efficiency, minuscule sample and reagent consumption, high throughput by automation and possibility of coupling with mass spectrometric detection. Furthermore, a fused-silica capillary can be used not only as a separation column but also as a nanoscale reaction chamber corresponding to the current trend of miniaturization and throughput enhancement of screening assays in early stages of the development of a new drug. After initial attempts [1], an improved on-line method for determination of enantioselective metabolism of anesthetic ketamine mediated by cytochrome P450 3A4 (CYP3A4) was developed. As mixing inside the capillary based on diffusion is generic, robust and rapid, the principle of transverse diffusion of laminar flow profiles was adopted [2]. Conceptually, solutions of CYP3A4 and mixture of ketamine and NADPH were injected by hydrodynamic pressure as a series of consecutive plugs with parabolic profiles allowing rapid mixing of the reactants by transverse diffusion [3]. Optimized conditions providing the highest yields of the reaction products and method repeatability were elucidated. All experiments were carried out on the ProteomeLab P800 CE system. Three plugs of 400 nM CYP3A4 solution (each plug was injected for 4 s by pressure of 0.5 psi) and four plugs of ketamine at a certain concentration and 4 mM NADPH solution (each plug was injected for 3 s by pressure of -0.5 psi) were alternately inserted into the 50 µm i.d. capillary of 64 cm total length that was thermostated at 37 °C. The reaction was terminated after 10 minutes and reaction products were separated by application of -20 kV. A 50 mM TRIS-phosphate buffer (pH 2.5) containing 3 % (w/v) of highly sulfated ?-cyclodextrin was used as BGE. Performed validation showed a good intraday and interday repeatability (RSD < 5 % and 8.5 %, respectively) for both norketamine enantiomers and the optimized method is suitable for on-line study of ketamine metabolism by CYP3A4. The mixing of reactants based on diffusion ensures the versatile applicability of the method for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450 enzymes. |
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