Abstract

Grapefruit juice elevates blood levels of some drugs taken orally, primarily by inhibiting intestinal CYP3A4-mediated first-pass metabolism. Two prominent furanocoumarins in the juice, 6′,7′-dihydroxybergamottin (DHB) and bergamottin (BG), have been demonstrated as important contributors to grapefruit juice-drug interactions. Using CYP3A4-expressing Caco-2 cells and representative probes from distinct CYP3A4 substrate subgroups (midazolam, testosterone), we compared the time-dependent inhibitory properties of DHB and BG. DHB rapidly inhibited CYP3A4 activity in a substrate-independent fashion with maximal inhibition (≥85%) generally occurring within 30 min. In contrast, BG had a slower onset and exhibited substrate-dependent inhibition. Whereas testosterone 6β-hydroxylation was inhibited by >50% with all exposure times (0.5-3 h), midazolam 1′-hydroxylation was unaffected, or even activated, with short exposure times (<1 h). After a 3-h exposure, however, BG had begun to “catch up” with DHB, causing ≥70% inhibition, independent of substrate. Likewise, loss of CYP3A4 protein, believed to reflect rapid intracellular degradation of the enzyme following mechanism-based inactivation, was comparable between the furanocoumarins (40-50%). The time course of BG-mediated inhibition was similar after just a 30-min exposure, indicating that the short exposure presumed to occur after juice ingestion is sufficient to initiate the events required to cause substantial inhibition (≥50%). These results suggest that after ingestion of a glass of grapefruit juice, CYP3A4 is maximally inhibited by DHB before BG has the opportunity to act. However, foods containing BG but not DHB (e.g., lime juice) could produce a substrate-dependent interaction with drugs consumed concomitantly, but a substrate-independent interaction with drugs taken several hours after food consumption.