Application of sweeping - MEKC combination for monitoring of diclofenac reaction with cytochrome P450 2C9 isoform
| Authors | |
|---|---|
| Year of publication | 2009 |
| Type | Article in Proceedings |
| Conference | Book of Abstracts 15th American symposium on Biotechnology, Biomedical and Pharmaceutical and Industrial Application of CE and Microchip Technology |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | sweeping; MEKC; diclofenac; P450 |
| Description | During the last decades a large number of new drugs have been introduced into clinical practice. All these new entities had to undergo the complicated time and money consuming developing process. Besides adsorption, distribution, excretion and toxicological studies, this process also includes the study of their metabolism, the affinity to certain drug-metabolising enzymes and the investigation of drug-drug interactions. A number of analytical approaches have been applied for these purposes mostly based on radiometric, colorimetric, fluorescent and high-performance liquid chromatography/mass spectrometry. In addition to these well established methods capillary electrophoresis is gaining the position in this field. The major aim of this work was to demonstrate the possibility of using MEKC for kinetic study of cytochrome P450 2C9 (CYP2C9), one of the most important isoforms in human liver, with diclofenac as a probe substrate. In view of the fact that our recent study showed that diclofenac hydroxylation deviated slightly from typical Michaelis-Menten kinetics at low substrate concentration, the combination of sweeping with MEKC was applied in this study. The enzymatic reaction thus could be simply monitored by the analysis of the reaction mixture without any pre-treatment; moreover high sensitivity and repeatability of the separations were preserved. A 50 um fused silica capillary (56 cm effective length) was used to carry out all separations. 60 mM SDS in 20 mM phosphate 20 mM tetraborate buffer pH 8.3 was used as a background electrolyte. Injection was accomplished by an application of 50 mbar pressure to the sample vial for 48 s. Separation was performed at 24 kV (positive polarity), with the temperature of capillary 25 oC and detection at 200 nm. As the results Michaelis constant 4,62 uM, maximum reaction velocity 18,35 nmol.min-1.nmol-1 and Hill coefficient 1,22 were determined which were in agreement with the literature data. Value of Hill coefficient confirms a presence of weak positive cooperativity in low substrate concentration region. The explanation could be found in binding of two substrates in or near the active site of CYP2C9 as it was established in the case of CYP3A4 isoform. |
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