The multiple depletion curves method provides accurate estimates of intrinsic clearance (CLint), maximum velocity of the metabolic reaction (Vmax), and Michaelis constant (Km): accuracy and robustness evaluated through experimental data and Monte Carlo simulations.

The use of multiple depletion curves for the estimation of maximum velocity of the metabolic reaction (V(max)), the Michaelis constant (K(m)), and intrinsic clearance (CL(int)) was thoroughly evaluated by means of experimental data and through a series of Monte Carlo simulations. The enzyme kinetics of seven compounds were determined using the multiple depletion curves method (MDCM), the traditional initial formation rate of metabolite method (IFRMM), and the "in vitro t(1/2)" method, and the parameter estimates that were derived from the three methods were compared. The impact of a change in enzyme activity during the incubation period on the parameter estimates and the possibility to correct for this were also investigated. The MDCM was in good overall agreement with the IFRMM. Correction for a change in enzyme activity was possible and resulted in a better concordance in CL(int) estimates. The robustness of the method in coping with different rates of substrate turnover and variable starting concentrations were also demonstrated through Monte Carlo simulations. Furthermore, the limitations imposed by assumptions inherent in the in vitro t(1/2) method were demonstrated both experimentally and by simulations. This study demonstrates that the MDCM is a robust and efficient method for estimating enzyme kinetic variables with high accuracy and precision. The method may potentially be used in a wide range of applications, from pure enzyme kinetics to in vitro-based predictions of the pharmacokinetics of compounds with multiple and/or unknown metabolic pathways.