Roman Řemínek

On‐line drug metabolites generation and their subsequent target analysis by capillary zone electrophoresis with UV‐absorption detection

This study presents the in‐capillary enzymatic biotransformation of dextromethorphan, an antitusive drug and opioid receptor antagonist, and subsequent electrophoretic separation of its products. The study includes the optimization of separation parameters to fulfill the requirements of an online microreaction. The analyses were performed in a bare fused‐silica capillary using 100 mM sodium tetraborate (pH 10.0) mixed with linear polyacrylamide (20%, v/v) and 2‐propanol (10%, v/v). This BGE was suitable for monitoring both off‐line and in‐capillary incubations. The partial filling technique enabled the enzymatic reaction to be carried out in its optimal environment (20 mM sodium phosphate, pH 7.4). Finally, in‐capillary microreaction in the presence of cytochrome P450 3A4 gave satisfactory outcomes.

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.

New Capillary Electrophoretic Method for On-line Screenings of Drug Metabolism Mediated by Cytochrome P450 Enzymes

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 2C9s 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.

Study of atypical kinetic behaviour of cytochrome P450 2C9 isoform with diclofenac at low substrate concentrations by sweeping-MEKC combination

In view of the fact that several studies have shown that diclofenac hydroxylation by cytochrome P450 2C9 deviated from Michaelis-Menten kinetics at low substrate concentrations, sweeping combined with MEKC was applied for the kinetic study of this pharmacologically important reaction. A 50  μm fused silica capillary (56  cm effective length) was used to carry out all separations. 70  mM SDS in 20  mM phosphate 20  mM tetraborate buffer, pH 8.6, was used as the BGE. Injection was accomplished by the application of 50  mbar (5  kPa) pressure to the sample vial for 52  s. Separation was performed at 22  kV (positive polarity), with a capillary temperature of 25°C and detection at 200  nm. The higher sensitivity of the sweeping-MEKC combination compared with the simple MEKC method enabled this reaction to be fitted to a Hill kinetic model and confirmed the findings of other authors. A Michaelis constant of 2.91±0.10  μM, maximum reaction velocity of 9.16±0.16  nmol/min/nmol and Hill coefficient of 1.66±0.08 were determined. This value of Hill coefficient confirms the presence of a positive cooperativity at low diclofenac concentrations and supports the hypothesis of two substrates binding at or near the active site.

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

A new CE‐MS method with enzymatic reaction inside the capillary was developed for the study of drug metabolism by cytochromes P450. This automated method, based on the transverse diffusion of laminar flow profiles methodology, is comprised of the injection of substrates and enzyme, their mixing, incubation, and separation of the reaction products, all performed by CE, and their detection, identification, and quantification by MS. The developed and validated method was finally used to conduct a kinetic study of cytochrome P450 isoform 2C9 or human liver microsomes with diclofenac in order to demonstrate its practical functionality. All the estimated kinetic values—apparent Michaelis constants and apparent maximum reaction velocities were in agreement with literature data obtained using other techniques. In addition, the consumption of reactants was in the tens of nL per analysis. The methods usability was further demonstrated on tolbutamide, the other probe substrate of cytochrome P450 isoform 2C9. As a result, the method is conceptually applicable for the screening of any other cytochrome P450 isoform and its substrates and inhibitors after adapting the incubation and separation conditions.

Simulation and experimental study of enzyme and reactant mixing in capillary electrophoresis based on-line methods

The establishment of an efficient reaction mixture represents a crucial part of capillary electrophoresis based on-line enzymatic assays. For ketamine N-demethylation to norketamine mediated by the cytochrome P450 3A4 enzyme, mixing of enzyme and reactants in the incubation buffer at physiological pH was studied by computer simulation. A dynamic electrophoretic simulator that encompasses Taylor-Aris diffusivity which accounts for dispersion due to the parabolic flow profile associated with pressure driven flow was utilized. The simulator in the diffusion mode was used to predict transverse diffusional reactant mixing occurring during hydrodynamic plug injection of configurations featuring four and seven plugs. The same simulator in the electrophoretic mode was applied to study electrophoretic reactant mixing caused by voltage application in absence of buffer flow. Resulting conclusions were experimentally verified with enantioselective analysis of norketamine in a background electrolyte at low pH. Furthermore, simulations visualize buffer changes that occur upon power application between incubation buffer and background electrolyte and have an influence on the reaction mixture.

Development and comprehensive comparison of two on-line capillary electrophoretic methods for β-secretase inhibitor screening☆

Alzheimers disease is the most common cause of dementia, afflicting over 34 million patients worldwide. Since β-secretase is a rate-limiting enzyme of the production of neurotoxic β-amyloid peptide oligomers abnormally accumulated in the affected brain tissue, its specific inhibition appears to be a promising approach to slowing down or even stopping the progression of the disease. Hence two on-line capillary electrophoretic methods for studies of β-secretase activity based on the principles of transverse diffusion of laminar flow profiles and electrophoretically mediated microanalysis were developed, both using a simple unlabeled peptide substrate and UV detection. The optimized procedures were thoroughly validated and applied for determining the enzymes kinetic parameters and the inhibition characteristics of two potent probe inhibitors. The resulting values were found to be comparable to literature data obtained with other analytical techniques. The suitability of the employed methodologies for different experimental designs is discussed on the basis of a statistical evaluation of the experimental data. The presented methods constitute a miniaturized and fully automated tool, which should be suitable for kinetic and inhibition studies of β-secretase as a target for Alzheimers disease drug discovery in the early stages of the development of a new drug.