Inhibitory properties of grapefruit juice components on human cytochrome P450 3A5 activity

Purpose Grapefruit juice (GFJ) evokes food-drug interactions (FDIs) via mechanism-based inhibition (MBI) of gastrointestinal cytochrome P450 (CYP) 3A4. Bergamottin (BG) and 6',7'-dihydroxybergamottin (DHB) are the primary constituents responsible for this inhibition. Although CYP3A4 and CYP3A5 share overlapping substrate specificity, their inhibitor sensitivity can differ. While BG is a known mechanism-based inhibitor of CYP3A5, the inhibitory property of DHB on the CYP3A5 remains uncharacterized. This study aimed to characterize the inhibitory kinetics of DHB and BG on CYP3A5 metabolic activity in vitro . Methods Recombinant CYP3A5 activity was determined by 1’-hydroxylation of midazolam (MDZ). The concentration of 1’-hydroxymidazolam (1’-OH MDZ) was quantified using LC-MS/MS. The residual intestinal CYP3A5 activity following the ingestion of 200 mL GFJ was estimated using the obtained MBI parameters, k inact,max and K I , and the concentrations of DHB and BG in GFJ. Results and Discussion: Both DHB and BG inhibited MDZ 1'-hydroxylation in a concentration- and preincubation time-dependent manner, confirming MBI of CYP3A5. DHB exhibited more potent inhibition than BG, with a k inact,max of 0.20 min -1 and K I of 2.4 μM. These findings suggest that GFJ-mediated FDIs may involve the concurrent inhibition of CYP3A5 alongside CYP3A4, primarily by DHB.