Inactivation of Cytochrome P450 3A4 by Bergamottin, a Component of Grapefruit Juice

Abstract

Grapefruit juice has been found to significantly increase oral bioavailability 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 acetate extracts from grapefruit juice revealed the presence of several furanocoumarins of which bergamottin (BG) is the major one. BG was shown to inactivate P450 3A4 in a reconstituted system consisting of purified P450 3A4, NADPH-cytochrome P450 reductase, cytochrome b5, and phospholipids. Inactivation was time- and concentration-dependent and required metabolism of BG. The loss of catalytic activity exhibited pseudo-first-order kinetics. The values of kinactivation and KI calculated from the inactivation studies were 0.3 min-1 and 7.7 microM, respectively. While approximately 70% of the erythromycin N-demethylation activity was lost during incubation with BG in the reconstituted system, P450 3A4 retained more than 90% of the heme as determined either by UV-visible spectroscopy or by HPLC. However, approximately 50% of the apoP450 in the BG-inactivated P450 3A4 incubation mixture could not be recovered from a reverse-phase HPLC column when compared with the -NADPH control. The mechanism of the inactivation appears to involve modification 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 mechanism-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 liver microsomes.

Detection of Covalent Adducts to Cytochrome P450 3A4 Using Liquid Chromatography Mass Spectrometry

“…For L-754,394, it has been previously shown that it labels a protein corresponding to P450 3A4 using radioactive compound and HPLC with radiometric detection of the labeled protein (26,31). For DHB, it is postulated that this compound is a mechanismbased inhibitor of P450 3A4, although no direct experimental evidence of this is available (no radiolabeled compound was available) (27,29,32).…”

“…The exact mechanism of inactivation has not been elucidated, although adduction to heme has been ruled out and indirect evidence suggests that inactivation is through modification of the apoenzyme (27,32). LC-MS analysis of P450 3A4 after incubation with DHB resulted in the deconvoluted spectrum shown in Figure 6.…”

Food–drug interaction: grapefruit juice augments drug bioavailability—mechanism, extent and relevance

“…The major furanocoumarin present in grapefruit is bergamottin, which demonstrates a time-and concentration-dependent inactivation of CYP enzymes in vitro (He et al, 1998), as well Food-drug interaction A Dahan and H Altman as its metabolite 6 0 ,7 0 -dihydroxybergamottin (DHB) and a number of other furanocoumarin derivatives (Ohnishi et al, 2000;Mohri & Uesawa, 2001). DHB, along with four newly isolated furanocoumarins have reproduced roughly the inhibitory potency of grapefruit juice when mixed together, while the omission of any of the components resulted in decreased potency, suggesting that all major furanocoumarins contribute to the properties of grapefruit juice .…”

A Comparison Between Lignans from Creosote Bush and Flaxseed and Their Potential to Inhibit Cytochrome P450 Enzyme Activity

“…In particular, bioactivation, or the metabolic activation of a compound to an electrophilic reactive intermediate, which subsequently undergoes covalent binding to critical cellular macromolecules and interferes with their www.intechopen.com function, has long-standing recognition as a biochemical mechanism of organ toxicity (Miller & Miller 1947;Mitchell et al, 1973;Masubuchi et al, 2007). Several examples exist of natural products undergoing P450-mediated bioactivation and irreversible P450 enzyme inhibition and hepatotoxicity (Johnson et al, 2003;Zhou et al, 2004;Surh & Lee 1995;He et al, 1998;Kent et al, 2002). Furthermore, natural products are known to result in significant interactions with coadministered therapeutic agents resulting in adverse effects or therapeutic failures (Dietz & Bolton 2007;Yuan et al, 2004;Kupiec & Raj 2005).…”