Abstract

The IC₅₀ values of 14 drugs were determined in recombinantly expressed CYP2C9 (rCYP2C9) and human hepatocytes and the data used to simulate clinical area under the plasma concentration-time curve (AUC) changes upon coadministration with prototypic CYP2C9 substrates. There was an excellent correlation between IC_{50, apparent} values determined using diclofenac and naproxen as CYP2C9 substrates (r² = 0.82, p < 0.0001), with values being generally higher in the naproxen assay. After correcting for nonspecific binding, the IC_{50, unbound} values were similar between the assays, for the majority of compounds. Two compounds, amiodarone and benzbromarone, demonstrated substrate-specific differences, activating naproxen O-demethylase to ∼250% of control activity at 1 mM and 1 μM, respectively, while inhibiting diclofenac 4′-hydroxylation with IC_{50, apparent} values of 3 μM and 0.04 μM, respectively. CYP2C9 IC_{50, apparent} values generated in human hepatocytes were systematically higher than those determined with rCYP2C9. After correcting for nonspecific binding, there was an excellent correlation of IC_{50, unbound} values generated in the different milieu (r² = 0.88, p < 0.0001). The ratio of inhibitor concentration at the entrance to the liver to the inhibition constant ([I]_in/K_i) was used to simulate clinical δAUC changes and compared with that observed in vivo. Where [I]_{in, total}/K_{i, apparent} was used, there were zero false negatives (observed δAUC ≥2, predicted δAUC <2), eight correct assignations, and seven false positives (observed δAUC ≤2, predicted δAUC >2. Where [I]_{in, unbound}/K_{i, unbound} was used, there was one false negative, 14 correct assignations, and zero false positives. In summary, the data presented here suggest that for CYP2C9 interactions, the use of total liver inhibitor concentrations may indeed avoid false negatives, but more realistic predictions may be achieved using unbound liver inhibitor concentrations and unbound in vitro inhibition parameters.