INACTIVATION OF CYTOCHROME-P450 3A4 BY BERGAMOTTIN, A COMPONENT OF GRAPEFRUIT JUICE

Grapefruit juice has been found to significantly increase oral bioavai lability of several drugs metabolized by cytochrome P450 3A4 (P450 3A4 ) through inhibiting the enzymatic activity and decreasing the content of intestinal P450 3A4. HPLC/MS/MS and HPLC/UV analyses of ethyl acet ate extracts from grapefruit juice revealed the presence of several fu ranocoumarins of which bergamottin (BG) is the major one. BG was shown to inactivate P450 3A4 in a reconstituted system consisting of purifi ed P450 3A4, NADPH-cytochrome P450 reductase, cytochrome bs, and phosp holipids. Inactivation was time-and concentration-dependent and requir ed metabolism of BG. The loss of catalytic activity exhibited pseudo-f irst-order kinetics. The values of k(inactivation) and K-I calculated from the inactivation studies were 0.3 min(-1) and 7.7 mu M, respectiv ely. While approximately 70% of the erythromycin N-demethylation activ ity was lost during incubation with BG in the reconstituted system, P4 50 3A4 retained more than 90% of the heme as determined either by UV-v isible spectroscopy or by HPLC. However, approximately 50% of the apoP 450 in the BG-inactivated P450 3A3 incubation mixture could not be rec overed fi om a reverse-phase HPLC column when compared with the -NADPH control. The mechanism of the inactivation appears to involve modific ation of the apoP450 in the active site of the enzyme instead of heme adduct formation or heme fragmentation. These results indicate that BG , the primary furanocoumarin extracted from grapefruit juice, is a mec hanism-based inactivator of P450 3A4. BG was also found to inhibit the activities of P450s 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 in human l iver microsomes.