Gibbs M A, Kunze K L, Howald W N, Thummel K E
Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle 98195, USA.
Drug Metab Dispos. 1999 May;27(5):596-9.
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 microg/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 Ki 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 r2 improved 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 derived Ki value.
本研究旨在评估相互游离抑制剂和游离酶消耗对三种抗真菌细胞色素P-450（CYP）3A抑制剂效力的影响，这三种抑制剂的酶亲和力范围超过1000倍。在人肝微粒体蛋白浓度从25至1000μg/ml变化的条件下进行孵育。使用咪达唑仑作为CYP3A探针评估每种抑制剂的作用。发现克霉唑是CYP3A的紧密结合抑制剂，其Ki为250 pM。对所用抑制剂和酶浓度矩阵的抑制百分比数据进行逐步线性回归分析表明，蛋白浓度可预测克霉唑的抑制百分比（r2 = 0.60，p <.001）。当将克霉唑浓度添加到模型中时，r2提高到0.81，p =.003。单独的克霉唑浓度不是抑制百分比的显著预测指标（r2 = 0.21，p =.08）。对于酮康唑，蛋白浓度对抑制百分比的预测较弱（r2 = 0.39，p =.006）。相反，单独的酮康唑浓度是抑制百分比的良好预测指标（r2 = 0.55，p <.001）。与克霉唑和酮康唑的结果相反，氟康唑的抑制百分比不依赖于蛋白浓度（r2 = 0.06，p =.39）。我们得出结论，微粒体抑制效力可能受孵育条件影响，这些条件会消耗酶可利用的游离抑制剂浓度。这可能会给基于体外获得的Ki值对体内药物 - 药物相互作用进行定量预测带来严重误差。
