Accelerated Communication MECHANISMS OF ENHANCED ORAL AVAILABILITY OF CYP3A4 SUBSTRATES BY GRAPEFRUIT CONSTITUENTS Decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins

Grapefruit juice increases the oral availability of a variety of CYP3A4 substrates. It has been shown that recurrent grapefruit juice ingestion results in a loss of CYP3A4 from the small bowel epithelium. We now show that the reduction in intestinal CYP3A4 concentration is rapid; a 47% decrease occurred in a healthy volunteer within 4 hr after consuming grapefruit juice. To identify the specific components of the juice responsible for this effect, we used a recently developed Caco-2 cell culture model of human intestinal epithelium that expresses catalytically active CYP3A4. We found that grapefruit oil and two furanocoumarin constituents (6*,7*-dihydroxybergamottin and a closely related dimer) caused a dose-dependent fall in CYP3A4 catalytic activity and immunoreactive CYP3A4 concentration. The effect was selective in that concentrations of CYP1A1 and CYP2D6 did not fall, consistent with previous results obtained in vivo. Assays of various juices confirmed that 6*,7*-dihydroxybergamottin is the major furanocoumarin present and, although its concentration varies significantly among types and brands of grapefruit juice, it is consistently present in concentrations exceeding the IC50 (1 mM) for loss of midazolam 1*-hydroxylase activity determined in the Caco-2 cells. Studies with recombinant CYP3A4 revealed that 6*,7*-dihydroxybergamottin is a mechanism-based inactivator, which supports the idea that loss of CYP3A4 results from accelerated degradation of the enzyme. We conclude that the effect of grapefruit juice on oral availability of CYP3A4 substrates can be largely accounted for by the presence of 6*,7*-dihydroxybergamottin although other furanocoumarins probably also contribute. Grapefruit juice has been reported to increase the oral availability of a variety of CYP3A4 substrates including cyclosporin A (1), felodipine (2), midazolam (3), terfenadine (4), verapamil (5), saquinavir (6), and ethinyl estradiol (7). Grapefruit juice has little effect on intravenously administered drugs (1,3), however, and the primary effect on orally administered drugs is to increase their peak serum concentration (Cmax) with little change in the subsequent rate of elimination as measured by half-life (2, 8). Therefore, it appears that the grapefruit juice effect is at the level of the intestine and not the liver. The grapefruit juice effect has been postulated to be the result of competitive inhibition of CYP3A4 in the enterocytes lining the small intestine (1, 2) because grapefruit juice has been shown to contain substances capable of competitively inhibiting this enzyme (9–11). However, in a recent study performed in healthy volunteers, consumption of grapefruit juice for 6 days resulted in a mean 62% decrease in enterocyte CYP3A4 immunoreactive protein concentration with no change in CYP3A4 mRNA concentration (12). The magnitude of the decrease in immunoreactive CYP3A4 correlated with the magnitude of increase in felodipine AUC when the drug was taken with grapefruit juice instead of water (12). It was suggested that the effect may be a result of a mechanism-based (“suicide”) inactivation of the enzyme, followed by its rapid intracellular degradation (12). Flavonoids, which are major constituents of grapefruit juice, are not known to be mechanism-based inactivators. Naringin, the major flavonoid present in grapefruit juice, and quercetin do not reproduce the grapefruit juice effect when administered orally, also suggesting that flavonoids are not the active compounds (8, 13). Edwards et al. recently reported that 69,79-dihydroxybergamottin (DHB), a furanoThis work was supported by the NIGMS (GM 38149, P.B.W.), the NCRR (MO1 RR00042, University of Michigan General Clinical Research Center), and the NIEHS (1-P30-ES06639, D.J.E.). Send reprint requests to: Paul B. Watkins, M.D., University of Michigan Medical Center, Room A7119 University Hospital, Box 0108, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0108. 1 Abbreviations used are: DHB, 69,79-dihydroxybergamottin; FC726, furanocoumarin of M.W. 726. 0090-9556/97/2511-1228–1233$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 25, No. 11 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 1228 at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from coumarin present in grapefruit juice, is a potent inhibitor of CYP3A enzymes in rat liver microsomes (14). Furanocoumarins are known mechanism-based inactivators of cytochromes P450 (15, 16) and might be responsible for the loss of intestinal CYP3A4 protein following grapefruit juice ingestion. In this report, we show that the fall in CYP3A4 immunoreactive protein in human enterocytes in vivo after grapefruit juice ingestion occurs rapidly. A human intestinal epithelial cell line also exhibited a rapid fall in CYP3A4 concentration when exposed to grapefruit oil. We therefore used this culture model to test the hypothesis that furanocoumarins are the major active ingredients in grapefruit juice. Materials and Methods Grapefruit, grapefruit juice, and frozen concentrated grapefruit juice were obtained from local markets. DHB was synthesized from bergamottin (Indofine Chemical Company, Somerville, NJ) as described (14). One sample of grapefruit oil was obtained from Berjé (Bloomfield, NJ). A second sample of grapefruit oil (Spectrum Chemical Manufacturing Corp., Garden, CA), R(1)and S(-)-limonene, decanal, and ketoconazole were gifts from Dr. Vincent Wacher (AvMax, Berkeley, CA). Midazolam was received as a gift from Dr. Bruce Mico (Roche Laboratories, Nutley, NJ). Isopropyl b-D-thiogalactoside was purchased from Calbiochem Corp. (La Jolla, CA). NADPH, L-a-dilauroyland L-a-dioleyl-sn-glycero-3-phosphocholines, phosphatidyl serine, testosterone, 6band 11b-hydroxytestosterone, catalase, glutathione, and daminolevulinic acid hydrochloride and other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). NADPH-cytochrome P450 reductase and cytochrome b5 were purified from liver microsomes of phenobarbital treated Long-Evans rats as previously described (17, 18). Microsomes were prepared as described (19) from a human liver (HL4; medically unsuitable for organ transplantation) that was obtained under the auspices of the Washington Regional Transplant Consortium (Washington, DC). Grapefruit Juice Fractionation. Grapefruit juice frozen concentrate (354 ml) was reconstituted with water to a final volume of 1 liter. The grapefruit juice was then extracted with ten 100-ml portions of hexane, the organic layer was dried with sodium sulfate, and the hexane was removed in vacuo. The resulting residue was dissolved in ethanol, and the ethanol solution was separated into fractions by a Hewlett-Packard (Wilmington, DE) 1090 HPLC system. Grapefruit juice components were separated on a 4.6 x 150 mm M-80 C18 column (YMC, Wilmington, NC) that was eluted at 1 ml/min with a water and acetonitrile gradient as follows: (time:% acetonitrile; 0:10; 5:10; 30:80; 40:80; 45:10; 50:10). The eluate was monitored at 260 nm and UV absorption data were collected from 200–600 nm. All HPLC fractions were dried with a Savant SpeedVac system (Farmington, NY) and stored at 280°C until used in microsomal incubations. Instrumental Analysis of Grapefruit Juice Fractions. Isolated grapefruit juice fractions were analyzed further by LC/MS with a Hewlett-Packard 1050 HPLC system coupled to a Finnigan TSQ-7000 mass spectrometer operating in the positive ion electrospray ionization (ESI) mode with the following operating conditions: ESI spray voltage, 4.5 kV; capillary temperature, 275°C; auxiliary and sheath gas pressures, 5 and 65 psi, respectively. Conditions for LC/MS/MS analyses operating in the daughter ion scan mode were as noted and with a collision induced dissociation (CID) voltage of 260 eV and an argon CID pressure of 1 mTorr. HPLC conditions for LC/MS analyses were as noted previously except a 2.0 3 150 mm YMC M-80 C18 column was substituted for the standard column and the flow rate was reduced to 0.2 ml/min. Microsomal Incubations with Grapefruit Juice Fractions. Each of the HPLC fraction residues was dissolved in 40 ml of ethanol, and 10 ml of these solutions were added to 990 ml of human hepatic microsomal suspensions (0.5 mg/ml) that contained an NADPH regenerating system. Control incubations contained the same concentration of ethanol but without grapefruit juice fractions. The microsomes were incubated with 10 mM taxol for 30 min. Taxol metabolites 6a-hydroxytaxol, a marker activity for CYP2C8, and taxol metabolite B, a marker activity for CYP3A4, were quantitated as described (19,20). Fraction 39 was subsequently studied more extensively. Using its mass obtained from HPLC fractionation and its molecular weight based on LC/MS/MS, microsomal incubations were prepared that contained either 0, 0.1, 0.5, 1, 5, or 10 mM fraction 39 and either 10, 20, or 50 mM taxol. After 30 min incubations, 6a-hydroxytaxol and taxol metabolite B were quantitated. Quantitation of Bergamottin, DHB, 6*-Epoxybergamottin, and FC726 in Grapefruit, Grapefruit Juice, and Grapefruit Oil (data in table 1). White grapefruit juice frozen concentrate (10 ml; Kroger) was diluted with water to a final volume of 100 ml, 1 ml of 500 mM bergapten internal standard was added, and the mixture was extracted with six 100-ml portions of chloroform. The chloroform was removed in vacuo, and the resulting residue was dissolved in methanol and analyzed with a Waters 600E HPLC system. One white Florida grapefruit was halved, the fruit (juice vesicles only, not the membranes surrounding the sections) was removed and homogenized in a blender, and 1 ml of 500 mM bergapten internal standard was added. The homogenate was extracted with six 100-ml portions of chloroform. The chloroform was removed in vacuo, and the resulting residue was dissolved in methanol and analyzed with a Waters 600E HPLC system. Samples of cold-pressed grapefruit oil obtained from two sources were each diluted 1/100 with methanol, and the resulting solutions were analyzed by