Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: Application to fluoxetine metabolism by CYP2D6

Abstract

In this work, a capillary electrophoretic methodology for the enantioselective in vitro evaluation of drugs metabolism is applied to the evaluation of fluoxetine (FLX) metabolism by cytochrome 2D6 (CYP2D6). This methodology comprises the in-capillary enzymatic reaction and the chiral separation of FLX and its major metabolite, norfluoxetine enantiomers employing highly sulfated β-CD and the partial filling technique. The methodology employed in this work is a fast way to obtain a first approach of the enantioselective in vitro metabolism of racemic drugs, with the additional advantage of an extremely low consumption of enzymes, CDs and all the reagents involved in the process. Michaelis-Menten kinetic parameters (Km and Vmax ) for the metabolism of FLX enantiomers by CYP2D6 have been estimated by nonlinear fitting of experimental data to the Michaelis-Menten equation. Km values have been found to be 30 ± 3 μM for S-FLX and 39 ± 5 μM for R-FLX. Vmax estimations were 28.6 ± 1.2 and 34 ± 2 pmol·min(-1) ·(pmol CYP)(-1) for S- and R-FLX, respectively. Similar results were obtained using a single enantiomer (R-FLX), indicating that the use of the racemate is a good option for obtaining enantioselective estimations. The results obtained show a slight enantioselectivity in favor of R-FLX.

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…Mixing by longitudinal diffusion through the interface between the plugs is also called "at-inlet reaction" due to the fact that enzyme and substrate plugs are placed at the inlet of the capillary. This simple approach has been applied successfully, for example, in the case of l-tyrosinase [76], protein kinase C [77], CYP enzymes [66,78,79] and α-glucosidase [80]. However, longitudinal diffusion as mixing mode is highly inefficient when relatively long plugs or more than two plugs have to be mixed because longitudinal diffusion is a rather slow process and, moreover, the interface between the plugs is relatively small.…”

“…This has been realized in a series of papers on the stereoselective CYP-mediated metabolism of the drugs fluoxetine [78], verapamil [79] and ketamine [86,92]. The partial filling mode was applied in all cases.…”

“…The background electrolyte contained either highly sulfated β-cyclodextrin (β-CD) or highly sulfated γ-CD as chiral selector. Mixing of enzyme and substrate/cofactor plugs was achieved either electrophoretically [92] or by longitudinal diffusion [78,79] and TDLFP [86], respectively. In the latter case, seven consecutive short plugs of enzyme and drug/cofactor solutions were efficiently mixed.…”

Advances in enzyme substrate analysis with capillary electrophoresis

“…Incubation times can be extended for low substrate concentrations to generate sufficient product for detection as long as the generation of product is low enough that the rate is not impacted by the presence of product. Ultraviolet absorbance is utilized to measure the K M values in the μM to mM range, as shown in Table 1 for K M values in the range of 30 μM [20,32] to 4.6 mM [20]. While most analytes inherently absorb light in the ultraviolet region and do not require modification for detection, labeling with a chromophore can be utilized for analytes like carbohydrates that do not absorb light.…”

“…Most of the on-line incubations described in the paper either use the same pH [13,18,23,24,34,43,56–62], or a pH close to enzyme reaction [26,32,33,41,55]. In some instances a wide difference of pH existed between the enzyme incubation buffer and the separation buffer [31,63].…”