Abstract

The purpose of this work was to evaluate the effect of mutual unbound inhibitor and unbound enzyme depletion on the potency of three antifungal cytochrome P-450 (CYP)3A inhibitors with over 1000-fold range in enzyme affinity. Incubations were performed with human liver microsomal protein concentrations that varied from 25 to 1000 μg/ml. The effect of each inhibitor was evaluated using midazolam as a CYP3A probe. Clotrimazole was found to be a tight binding inhibitor of CYP3A with a K_i of 250 pM. Analysis of percent inhibition data by stepwise linear regression for the matrix of inhibitor and enzyme concentrations used showed that protein concentrations predicted the percent inhibition by clotrimazole (r² = 0.60, p < .001). When clotrimazole concentrations were added to the model, the r²improved to 0.81, p = .003. Clotrimazole concentrations alone were not a significant predictor of percent inhibition (r² = 0.21, p = .08). For ketoconazole, protein concentrations provided a weak prediction of the percent inhibition (r² = 0.39, p = .006). Conversely, ketoconazole concentrations alone were a good predictor of percent inhibition (r² = 0.55,p < .001). In contrast to results with clotrimazole and ketoconazole, percent inhibition by fluconazole was not dependent on protein concentrations (r² = 0.06,p = .39). We conclude that microsomal inhibitory potency can be affected by incubation conditions that deplete the unbound concentration of inhibitor available to the enzyme. This may introduce serious error into a quantitative prediction of an in vivo drug-drug interaction based on an in vitro derivedK_i value.