The Impact of Study Design on the Estimation of Parameters Describing Mechanism-Based Enzyme Inhibition

Determination of the kinetics of mechanism-based enzyme inactivation (mbi) involves a pre-incubation followed by an incubation stage. to minimise inhibition during the second stage it is recommended to dilute the concentration of inhibitor and to use a high concentration of probe substrate (e.g. 10 fold > km). the aim of this study was to quantify the impact of changing the study design. two different dilutions of the pre-incubation samples (1.25 and 4 times) and different concentrations of the probe substrate dextromethorphan (5, 10 and 20 |nm) were used to determine the kinetic constants describing the mbi of cyp2d6 by methylenedioxymetham-phetamine. changing the concentration of dextromethorphan had no significant effect on either ki or kinact values (p> 0.05). this is expected when the substrate concentration is well in excess of its km. ki and kinact values increased as expected (3.81 to 12.92 (mu)m (p< 0.01) and 0.22 to 0.29 min-1 (p< 0.02), respectively) as the dilution was increased. these data provide evidence that study design is important in characterising mbi.