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

Abstract

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.

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

“…. The obtained values of K m ′ = 2.66 ± 0.18 μM, V max ′ = 7.91 ± 0.22 nmol min −1 nmol ‑1 and n = 1.59 ± 0.16 are in a good agreement with literature data determined by other techniques .…”

“…The IC 50 and K i ′ values calculated from the obtained data were 0.94 ± 0.04 μM ( R 2 = 0.998) and 0.39 ± 0.07 μM ( R 2 = 0.985), respectively. These values are of the same order of magnitude as the data found in the literature and thus prove the applicability of the proposed method for reactants mixing inside the capillary for on‐line inhibition studies of drug‐metabolizing enzymes. Additionally, less than 30 nL of CYP2C9 solution per analysis was consumed in these kinetic and inhibition studies.…”

Micellar electrokinetic chromatography: A review of methodological and instrumental innovations focusing on practical aspects

“…Although sweeping MEKC is recognized as an attractive, powerful online sample concentration method, it was scarcely used in MEKC‐based determinations in the reviewed period; by contrast, as shown in Section , its combination with other on‐ and off‐line sample concentration approaches has grown significantly by virtue of the increased enhancement factors potentially obtained. In most cases, the sweeping protocol uses SDS in an alkaline BGE ; however, an alternative choice suppressing the EOF with a low‐pH BGE provided by 45 mM phosphoric acid was also reported . In general, up to 250‐fold improvements in concentration sensitivity have been achieved in the determination of various analytes such as aflatoxins (B1, B2, G1, and G2) in rice , and six steroids including two epimer pairs and melamine in food .…”

Cooperative effects for CYP2E1 differ between styrene and its metabolites

“…Nevertheless, it has become increasingly clear that P450 metabolism of compounds can be more complex involving homo- and heterotropic cooperative (allosteric) interactions. In fact, these non-Michaelis–Menten mechanisms may be more of the rule than the exception for many P450s especially CYP1A2 (Isin et al, 2008; Miller & Guengerich, 2001), CYP2C9 (Liu et al, 2005; Řemínek & Glatz, 2010) and CYP3A4 (Kapelyukh et al, 2008; Roberts et al, 2011; Woods et al, 2011), as extensively reviewed elsewhere (Atkins, 2006; Denisov et al, 2009; Niwa 2008). Consequently, identification of the underlying mechanisms for these processes is necessary for establishing a biochemical explanation for their biological impacts on drug and pollutant metabolism.…”