Abstract

In the present study, we conducted a retrospective analysis of 343 in vitro experiments to ascertain whether observed (experimentally determined) values of K_i for reversible cytochrome P450 (P450) inhibition could be reliably predicted by dividing the corresponding IC₅₀ values by two, based on the relationship (for competitive inhibition) in which K_i = IC₅₀/2 when [S] (substrate concentration) = K_m (Michaelis-Menten constant). Values of K_i and IC₅₀ were determined under the following conditions: 1) the concentration of P450 marker substrate, [S], was equal to K_m (for IC₅₀ determinations) and spanned K_m (for K_i determinations); 2) the substrate incubation time was short (5 minutes) to minimize metabolism-dependent inhibition and inhibitor depletion; and 3) the concentration of human liver microsomes was low (0.1 mg/ml or less) to maximize the unbound fraction of inhibitor. Under these conditions, predicted K_i values, based on IC₅₀/2, correlated strongly with experimentally observed K_i determinations [r = 0.940; average fold error (AFE) = 1.10]. Of the 343 predicted K_i values, 316 (92%) were within a factor of 2 of the experimentally determined K_i values, and only one value fell outside a 3-fold range. In the case of noncompetitive inhibitors, K_i values predicted from IC₅₀/2 values were overestimated by a factor of nearly 2 (AFE = 1.85; n = 13), which is to be expected because, for noncompetitive inhibition, K_i = IC₅₀ (not IC₅₀/2). The results suggest that, under appropriate experimental conditions with the substrate concentration equal to K_m, values of K_i for direct, reversible inhibition can be reliably estimated from values of IC₅₀/2.