Abstract

Bergamottin (BG) and 6′,7′-dihydroxybergamottin (DHB) are the most abundant furanocoumarins present in grapefruit juice and have been proposed as major intestinal CYP3A4 inhibitors contributing to grapefruit juice-drug interactions. The relative contribution of BG versus DHB to the interaction potential is unclear, in part due to inconsistencies in the literature regarding inhibitory potency. To resolve these inconsistencies, the inhibitory kinetics of each furanocoumarin toward CYP3A4 catalytic activity were systematically characterized using representative probes from two distinct CYP3A4 substrate subgroups (testosterone and midazolam). With human intestinal microsomes, DHB was a substrate-independent reversible (K_i, ∼0.8 μM) and mechanism-based (K_I, ∼3 μM; kinact, 0.3-0.4 min^-1) inhibitor of CYP3A4. In contrast, BG was a substrate-dependent reversible inhibitor, with a K_i (13 μM) using midazolam that was 8-fold greater than that using testosterone, but a substrate-independent mechanism-based inhibitor (K_I, ∼25 μM; kinact, ∼0.35 min^-1). Similar trends resulted with cDNA-expressed CYP3A4, only the K_I values for BG were ∼10-fold lower than with microsomes. This seemed to reflect a much greater degree of microsomal protein binding by BG compared with DHB. Differential inhibition kinetics and binding properties between BG and DHB could account in part for the apparent in vitro inconsistencies in the literature. Results also emphasize the importance of appropriate substrate selection when designing inhibition studies involving dietary constituents.