Utilization of In-capillary Nanoreactor: Inhibition Study of Cytochrome P450 2C9 Isoform Reaction with Diclofenac and Sulfaphenazole by Means of Capillary Electrophoresis
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| Year of publication | 2012 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | A new methodology for creation of nanoscale bioreactor inside the separation capillary in capillary electrophoresis was developed. In principle, solutions of reactants are injected by relatively low pressure as a series of consecutive narrow plugs with slightly parabolic profiles due to laminar nature of the hydrodynamic flow inside the capillary. Reaction mixture is then mixed by combination of longitudinal and transverse diffusion within minute period. This procedure provides homogenous reaction mixture with easily changeable composition. Kinetic and inhibition studies of cytochrome P450 2C9 (CYP2C9) reaction with diclofenac as a probe substrate and sulfaphenazole as a probe inhibitor were performed in order to prove the applicability of proposed methodology for on-line screenings of CYP2C9 inhibitors in early stages of a new drug development. As a result apparent Michaelis constant of 2.66 +- 0.18 uM, apparent maximum reaction velocity of 7.91 +- 0.22 nmol/min/nmol, Hill coefficient of 1.59 +- 0.16, 50 % inhibitory concentration of 0.94 +- 0.04 uM and apparent inhibition constant of 0.39 +- 0.07 uM were determined. All these values are in agreement with literature data obtained by different techniques. What is more, developed methodology enables reduction of CYP2C9 solution consumption to 0.6 nl per analysis. |
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