Abstract

Cryopreserved human hepatocytes suspended in human plasma (HHSHP) have previously provided accurate CYP3A drug-drug interaction (DDI) predictions from a single IC₅₀ that captures both reversible and time-dependent inhibition. The goal of this study was to compare the accuracy of DDI predictions by a protein-free human hepatocyte system combined with the fraction unbound in plasma for inhibitor(s) with those obtained with protein-containing incubations. Seventeen CYP3A, CYP2C9, or CYP2D6 inhibitors were incubated with hepatocytes in human plasma or hepatocyte maintenance medium (HMM) for 20 min over a range of concentrations after which midazolam 1′-hydroxylation, diclofenac 4′-hydroxylation or (R)-bufuralol 1′-hydroxylation were used to quantify the corresponding cytochrome P450 (P450) catalytic activities. Two methods were used to predict the human exposure ratio of the victim drug in the presence and absence of inhibitor. The HMM K_{i, app} values were combined with the free average systemic plasma concentration (“free [I] with HMM K_{i, app}”) and the plasma K_{i, app} values were combined with the total average systemic plasma concentration (“total [I] with plasma K_{i, app}”). Of 63 clinical DDI studies, the total [I] with plasma K_{i, app} method predicted 89% of cases within 2-fold of the reported interaction whereas the free [I] with HMM K_{i, app} method predicted only 59%. There was a general underprediction by the free [I] with HMM K_{i, app} method, which is consistent with an underestimation of in vitro inhibition potency in this system. In conclusion, the HHSHP system proved to be a simple, accurate predictor of DDIs for three major P450s and superior to the protein-free approach.