New capillary electrophoretic method for on‐line screenings of drug metabolism mediated by cytochrome P450 enzymes

Abstract

A new method for the determination of kinetic and inhibition parameters of cytochromes P450 reactions by means of on-line CE was developed. It is based on transverse diffusion of laminar flow profiles methodology introduced by Krylov et al. that injection procedure was modified. The solutions of an enzyme and its substrates are injected by hydrodynamic pressure as a series of repeated consecutive plugs. Proposed injection of three plugs of enzyme surrounded with plugs of substrates represents a certain trade-off to obtain the reaction mixture with the satisfying homogeneity by the short-injection procedure as possible. Mathematical modeling confirmed the assumption of a consistent distribution of reactants in the final reaction mixture. Kinetic and inhibition studies of cytochrome P450 isoform 2C9's reaction with diclofenac as a probe substrate and sulfaphenazole as a probe inhibitor were conducted in order to prove the practical applicability of the proposed method for on-line screenings of drug metabolism mediated by cytochrome P450 enzymes. As a result, an apparent Michaelis constant of 2.66 ± 0.18 μM, apparent maximum reaction velocity of 7.91 ± 0.22 nmol min(-1) nmol(-1) , Hill coefficient of 1.59 ± 0.16, half maximal inhibitory concentration of 0.94 ± 0.04 μM and apparent inhibition constant of 0.39 ± 0.07 μM were determined. All these values are in agreement with literature data obtained using different techniques. In addition, less than 30 nL of cytochrome P450 2C9 solution was consumed per analysis in the kinetic and inhibition studies using this method.

Combination of on‐line CE assay with MS detection for the study of drug metabolism by cytochromes P450

“…After the method optimisation, the validation was performed under the conditions In contrast with the former CE-UV method [17], a new CE-MS one achieved LOD 40 times and LOQ 30 times lower, and also slightly better repeatability of migration times. The RSD of the repeatability of peak areas published by Wang et al who performed the in-capillary reaction of matrix metalloproteinase using the EMMA methodology combined with MS detection were better  3.2 % for classic and 4.8 % for pressure-mediated EMMA [19].…”

“…As was mentioned above, the main aim was not only to increase the sensitivity of the previously developed TDLFP-based method [17] by introducing MS detection, but also to extend its application potential in terms of metabolite identification and characterization.…”

“…Based on the results of the IB composition effect evaluation on activity CYP2C9 performed in our previous study [17] 50 mM and 25 mM phosphate buffers (pH 7.4) were tested as the IB. The injection procedure was simplified to a single zone, 15 mbar for 6 s of the sample containing the substrate (20 µM DC), product (10 µM HDC) and cofactor (1 mM NADPH) Electrophoresis…”

“…The solutions of substrates DC or TB and cofactor NADPH -S and enzyme CYP2C9 or HLM -E prepared in the IB (25 mM phosphate buffer (pH 7.4)) were maintained in a cooled tray at 4 °C and transferred to CE sample tray before the start of the analysis as described in the previous published work [17].…”

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…Multiple injection plugs of enzyme, substrate and inhibitor can be effectively mixed by TDLFP. In recent years, TDLFP has been successfully applied to study β-N-acetylhexosaminidase [35], or CYP enzymes [85,86]. The group of Nehme reported the development of generalized in-capillary assays for the analysis of kinases based on the conversion of ATP to ADP in the presence of suitable substrates [62,[87][88][89].…”