MECHANISMS OF GRAPEFRUIT JUICE INTERACTIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SPECIAL GRANT
• Reporting Frequency: Annual
• Accession No.: 0203101
• Grant No.: 2005-34507-15690
• Proposal No.: 2005-06090
• Project Start Date: Jul 1, 2005
• Project End Date: Jun 30, 2007
• Grant Year: 2005
• Program Code: [VP]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
118 NEWINS-ZEIGLER HALL
GAINESVILLE,FL 32611

Performing Department
(N/A)

Non Technical Summary
Grapefruit juice (GFJ) carries the American Heart Associations's healthy "heart check" and contains compounds that might both reduce atherosclerotic plaque formation and inhibit cancer cell proliferation. However, unlike other citrus fruit juices, GFJ interacts with some prescription medications, raising the potential for concern. This is particularly worrying in that juice and medications are commonly consumed together at breakfast. Due to alarmist news reports many patients are concerned about these potential interactions and have stopped drinking grapefruit juice. It is the purpose of the proposed research to shed light on the mechanism of these interactions to be able to make future predictions about their potential adverse or beneficial significance. In particular, the interaction of components of grapefruit juice with intestinal drug transport systems will be studied. Initial research has resulted in conflicting data, but it is known that for some drugs (such as cyclosporine, a drug that prevents organ rejection after transplant surgery) grapefruit juice can increase the serum concentrations significantly. However, for cyclosporin and most drugs it is difficult to sort out the different potential interaction mechanisms. The availability of new markers (talinolol, fexofenadine) makes it now possible to pinpoint down the exact mechanism of these events. The proposed research will allow a rational assessment and risk management of the use of these drugs with grapefruit juice.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	0910	1010	10%
702	0910	1150	10%
702	0910	1180	80%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
0910 - Grapefruit;

Field Of Science
1180 - Pharmacology; 1010 - Nutrition and metabolism; 1150 - Toxicology;

Keywords

grapefruit juice

interactions

pharmacokinetics

flavonoids

pharmaceuticals

human health

glycoproteins

enzymes

enzyme activity

furano terpenoids

coumarin

mechanism of action

toxicology

nutrient function

enzyme inhibitors

cytochromes

intestines

liver

enzyme substrates

availability

markers

cell physiology

Goals / Objectives
Grapefruit juice (GFJ)contains compounds with a wide range of beneficial properties including anti-inflammatory and anti-tumor activity, inhibition of blood clots and strong antioxidant activity. However, unlike other citrus fruit juices, grapefruit juice interacts with a variety of prescription medications, raising the potential for concern. The mechanism of this effect is presumed to involve inhibition of metabolism rather than improved absorption, since many of the drugs affected appear to be well absorbed when taken in the absence of grapefruit juice. Most of the drugs affected by grapefruit juice are known to be primarily metabolized by cytochrome P450 (CYP) 3A4, the major CYP isoform expressed in adult human small intestine and liver. Because grapefruit juice does not generally affect the systemic clearance of affected drugs, it appears that grapefruit juice selectively reduces intestinal CYP3A4 activity while having little effect on liver CYP3A4. An additional mechanism for the GFJ interaction is the inhibition of P-glycoprotein (P-gp), a transporter that carries drug from the enterocyte back to the gut lumen, resulting in a further increase in the fraction of drug absorbed. P-glycoprotein (P-gp) is localized on the luminal membranes of the intestinal epithelial cells and functions as an efflux pump to limit the absorption of xenobiotics. We could show recently that GFJ inhibits the function of P-gp in vitro. Furanocoumarins such as bergamottin and 6,7-dihydroxybergamottin have been identified as probable active constituents. However, it has been reported recently that GFJ reduced the bioavailability of an antihistamine, fexofenadine, and a β1-receptor antagonist, celiprolol. While fexofenadine and celiprolol are substrates for P-gp and not for the CYPs, P-gp is not likely to play a role in this interaction, because the inhibition of P-gp may not decrease but increase the plasma concentration of P-gp substrate. A possible explanation for the observed interaction is the inhibition of uptake transporter(s) expressed on the luminal membranes of intestinal epithelial cells. The inhibition may cause a decrease in the instestinal absorption of substrate drugs. The organic anion transporting polypeptide (OATP) family is a group of membrane solute carriers that mediate the uptake of various anionic compounds. The inhibition of drug uptake mediated by OATPs in the intestine may decrease the plasma concentration of a substrate for OATPs. The objective of this application is to A) systematically investigate in vivo the potential drug interaction between GFJ, selected fractions and ingredients of GFJ- on the OATP transporter system and B) investigate the interplay between intestinal CYP3A4 activity and P-gp mediated efflux transport of GFJ and selected fractions and ingredients in vitro using a modified cell culture system. Our data would help to understand why GFJ influences the bioavailability of certain drugs. These findings would also enhance our understanding of the complex nature of food-drug interactions, and their possible influence on the clinical effects of medications.

Project Methods
The antihistamine fexofenadine was identified as a substrate for P-glycoprotein-mediated efflux with use of polarized human intestinal cells and mice that lacked mdr1a-encoded P-glycoprotein. Cellular fexofenadine uptake was also found to be mediated by human OATP and rat oatp1 and oatp2 with use of a recombinant vaccinia expression system. Because the metabolism of fexofenadine is negligible in humans, P-glycoprotein and OATP transporters may be important determinants of fexofenadine disposition. Thus, if grapefruit juice inhibited only P-gp, then bioavailability of fexofenadine, should have increased rather than decreased. Because fexofenadine is also a substrate for OATP, our hypothesis is that the decrease in fexofendaine oral bioavailability by fruit juices is a result of greater inhibition of OATP over P-gp. This proposal seeks to advance this research by investigating the interaction between grapefruit juice, single components, and OATP in an in vivo system in a dose dependent manner to assess the potential of clinically relevant drug interactions when taken with grapefruit juice. Interactions between drugs and grapefruit juice are unique, in that, when the juice is consumed in usual volumes, inhibition occurs exclusively in the small intestine. Modes of intestinal CYP3A4 inhibition by grapefruit juice include both reversible and mechanism-based, as well as destruction of the CYP3A4 protein. However, most of the in vitro studies on CYP3A4 inhibition have been performed using microsomes or recombinant enzymes. The major limitation of microsomes is that they express phase I activities, but only part of phase II activities, and can only be used for short incubation times. In addition, the problem with these in vitro systems is that the furanocoumarin mediated loss of enterocyte CYP3A4 protein does not occur in isolated enzyme systems. Thus, the addition of extrachromosomal CYP3A4 to Caco-2 cells would provide an intact cell model in which to study metabolic processes in vitro. Furthermore, the endogenous expression of efflux transporters such as P-gp, MRP1, and MRP2 in Caco-2 cells makes it an appropriate model to study intestinal absorption as well. It is the another aim of our proposed study to use transfected Caco-2 cells which mimic the situation in the human intestinal mucosa and give valid information about the dynamic interplay between intestinal P-glycoprotein and CYP3A4 in GFJ interactions.

Progress 07/01/05 to 06/30/07

Outputs
Grapefruit juice (GFJ) has been demonstrated to inhibit the drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 specifically located in the small intestine. As a result, the oral bioavailability of a variety of medications is significantly augmented including some calcium channel blockers (felodipine, nicardipine, nisoldipine), 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (lovastatin, simvastatin), and benzodiazepines (midazolam, diazepam). Additional efforts revealed the involvement of GFJ on the permeability glycoprotein (P-gp) and organic anion transporting polypeptide (OATP) activities. These intestinal transporters have been shown to alter the absorption of drugs from different therapeutic groups. In this study, we systematically investigated the effect of two varieties of GFJ normal strength (white and ruby red; WNS, RR) and its components naringin (NAR), naringenin (NAG), bergamottin (BG) on the carrier mediated transport of talinolol, a highly selective β1-adrenoceptor antagonist, in male Sprague-Dawley rats. The pure compounds used in this work were tested according to their quantitative amounts in the white juice normal strength and concentrate, respectively Talinolol, a non-metabolized substrate, was used as a marker compound. When co-administered with talinolol, verapamil (VER) (4mg/kg), a known inhibitor of P-gp, promoted a slight but not significant increase on the drug Cmax and AUC(0-∞) by 1.4- and 1.7-fold, correspondingly. Additionally, the absorption (tmax) was delayed from 2 to 2.7 hours when compared to the control group. The elimination half-life (t1/2) remained practically unchanged among all treatments. According to our results the WNS-GFJ provided a slight increase on Cmax and AUC(0-∞) of talinolol, but without statistical significance when compared to the control. Among the GFJ components, the furanocoumarin BG (0.22 mg/kg) modified significantly the talinolol disposition, augmenting the Cmax and AUC(0-∞) by 2.4- and 1.8-fold, respectively, but no effect was observed at the lower dose of BG. Compared to the control group, the flavonoids NAG (0.7 mg/kg) and NAR (2.4 and 9.4 mg/kg) increased the talinolol Cmax and AUC(0-∞) by 1.5- to 1.8-fold, respectively, whereas RR-GFJ decreased the same pharmacokinetic parameters by half. In conclusion, we demonstrated that outcomes obtained during this GFJ-drug interaction study seem to be dependent on the variety of the juice and the concentration of the GFJ component ingested. It further showed that flavonoids (NAR and NAG) and furanocoumarin (BG) present in GFJ can selectively modulate the activity of intestinal efflux transporters rather than the uptake carriers. When performing food-drug interactions studies one should be aware that the overall result may not be a simply additive effect of all components present in the diet. In fact, synergism or antagonism can not be excluded.

Impacts
During the last years, many reports about interactions between various drugs and grapefruit juice have been published. It has become clear that several different mechanisms are involved in these interactions involving both drug metabolism and drug transporter systems. It is of critical importance to fully understand these mechanisms in order to manage and avoid these potential interactions. The research in this project contributes to a better understanding of these interaction mechanisms. It will allow to develop a rational approach to evaluate magnitude and relevance of the interaction and make recommendations for specific drugs. For example, for some drugs it has been shown that a grapefruit-juice induced elevation of drug concentrations is not of clinical consequence and hence insignificant. For other drugs, where the interaction with grapefruit juice may be of clinical concern, alternative drugs from the same class may be recommended which provide the same therapeutic benefit but do not interact significantly with grapefruit juice. Results from our research demonstrate that polyphenolic components from grapefruit do have an inhibitory effect on the efflux-transporter P-gp. Overall, the research in this project provides valuable information regarding citrus compounds which have a potential to interact with drug-transport and also helps to develop and test alternative methods of juice production and breeding procedures with the goal of designing products with lower drug interaction potential.

Publications

• Variation of Flavonoids and Furanocumarins in Grapefruit Juices DE CASTRO, W.V., MERTENS-TALCOTT, S., RUBNER, A., BUTTERWECK, V., DERENDORF, H. J Agricul Food Chem 54(1):249-55 (2006)
• Grapefruit-drug interactions: can interactions with drugs be avoided? MERTENS-TALCOTT, S., ZADEZENSKY, I., DE CASTRO, W.V., DERENDORF, H., BUTTERWECK, V. Clin Pharmacol. 46 (12):1390-416 (2006)
• Grapefruit Juice Drug Interactions: grapefruit juice and its components inhibit P-glycoprotein mediated transport of talinolol in Caco-2 Cells DE CASTRO, W.V., MERTENS-TALCOTT, S.U., DERENDORF, H., BUTTERWECK, V. Lecture: 54th Annual Meeting of the Society of Medicinal Plant Research, Helsinki, Finland, August 29st - September 2nd, 2006
• Grapefruit Juice Drug Interactions: grapefruit juice and its components inhibit P-glycoprotein mediated transport of talinolol in Caco-2 Cells DE CASTRO, W.V., MERTENS-TALCOTT, S.U., DERENDORF, H., BUTTERWECK, V. Poster Presentation on the 47th Annual Meeting of the American Society of Pharmacognosy, Arlington, VA, USA, August 5th - 9th, 2006
• Effects of grapefruit juice and its components on the activity of P-glycoprotein DE CASTRO, V.W., TALCOTT, S., BUTTERWECK, V., DERENDORF, H. Posterpresentation on the AAPS Annual Meeting, San Antonio, October 2006
• Citrus flavonoids in the inhibition of P-glycoprotein in vitro TALCOTT, S., DE CASTRO, V.W., DERENDORF, H., BUTTERWECK, V. Poster Presentation on the AAPS Annual Meeting, San Antonio, October 2006 Drug-Interactions of Grapefruit- and Other Citrus-Polyphenolics - What have we learned?
• MERTENS-TALCOTT, S.U., ZDROJEWSKI. I., DE CASTRO, W.V., BUTTERWECK, V., DERENDORF, H. In: FRANCIS LAM, Y.W., HUANG, S.M., HALT, S.D., Herbal Supplement-Drug Interactions Taylor and Francis., New York, pp 147-190 (2006)

Progress 07/01/05 to 06/30/06

Outputs
Grapefruit juice (GFJ) previously has been demonstrated to interact with a variety of prescription medications, raising the potential for concern regarding the concomitant consumption with drugs. The major mechanism for grapefruit-drug interaction involves the inhibition of the drug-metabolizing enzyme cytochrome P-450 3A4 (CYP450 3A4) in the small intestine. An additional mechanism is the interaction with P-glycoprotein (P-gp), a energy-dependent membrane efflux-transporter that carries a wide range of substrates. Among the components of grapefruit juice, the flavonoids (naringin and naringenin) and the furanocoumarins (bergamottin and 6',7'-dihydroxybergamottin) derivatives have been suggested to contribute to grapefruit juice-drug interaction. Their contents in different commercially available and fresh-squeezed GJF as well as in different tissues of grapefruits were analyzed by HPLC. Naringenin was not detected in any sample tested. Great brand-to-brand variability of naringin (174 to 1492 μMol/L), bergamottin (1 to 37 μMol/L) and 6',7'-dihydroxybergamottin (0.2 to 52.5 μMol/L) was observed. The overall great differences in the polyphenolic profile of different brands also could explain the high variability of grapefruit-induced effects on P-gp activity found by different research groups. In order to estimate the role of different components on the overall GFJ-mediated inhibition we investigated the effects of the isolated flavonoids (naringin, naringenin, eriodictyol, hesperetin), coumarins (bergaptol, limettin, geranosyloxycoumarin, 7-hydroxycoumarin), furanocoumarins (bergamottin, 6',7'-dihydroxybergamottin, epoxybergamottin), and polymethoxylated flavones (nobiletin, sinensetin, heptamethoxyflavone, tangeretin) on P-gp activity using the human colon carcinoma cell line Caco-2 using talinolol, a P-gp substrate, as a drug model. The Basolateral-Apical to Apical-Basolateral ratio of talinolol across the Caco-2 monolayers in absence of any inhibitor was higher than 3, suggesting the presence of polarized transport. The direct inhibitory data suggests that naringin and 6',7'-dihydroxybergamottin seems to have a significant inhibitory effect on P-gp activity when used at the same range of concentration as found in commercially available grapefruit juices. Since previous studies estimated that epoxybergamottin is present in GFJ at concentrations of 0.1 to 7.4 μMol/L it also would be sufficient to inhibit the P-gp activity noticeably. Although, the polymethoxyflavones are present in GFJ at very low levels they were shown to be potent inhibitors of P-gp and therefore, contributing for the overall effect of GFJ-P-gp interaction. Results from this study demonstrated that P-gp is selectively inhibited by GFJ components, where the clinical relevance of this inhibition remains to be determined. Results from in vivo experiments in rats with different grapefruit juices(ruby red, white, and pink) and single compounds (naringin and bergamottin) showed that only for naringin the AUC of talinolol was significantly increased (1.8-fold) compared to a water control after a concomitant administration.

Impacts
It has become clear that several different mechanisms are involved in drug-GFJ-interactions involving both drug metabolism and drug transporter systems. It is of critical importance to fully understand these mechanisms in order to manage and avoid these potential interactions. It will allow to develop a rational approach to evaluate magnitude and relevance of the interaction and make recommendations for specific drugs. For example, for some drugs it has been shown that a GFJ induced elevation of drug concentrations is not of clinical consequence and hence insignificant. For other drugs, where the interaction with GFJ may be of clinical concern, alternative drugs from the same class may be recommended which provide the same therapeutic benefit but do not interact significantly with GFJ. Results from our research demonstrate that polyphenolic components from grapefruit do have an inhibitory effect on the efflux-transporter Pg-P, where not only the major grapefruit components 6',7'-dihydroxybergamottin, bergamottin, naringin, and naringenin do show a significant inhibitory effects, but also other components such as 7'-epoxybergamottin, and the polymethoxylated flavones nobiletin, tangeretin, heptamethoxyflavone, and sinensetin. In general, the in vivo data in rats demonstrate that the efflux-transporter is likely to be affected by grapefruit juice and its components. Overall, the research in this project provides valuable information regarding citrus compounds and helps to demystify that GFJ has a potential to interact with any kind of drug.

Publications

• De Castro W.V., Mertens-Talcott, S.U, Rubner A., Butterweck V., Derendorf H. Variation of Flavonoids and Furanocoumarins in Grapefruit Juice: A Source of Variability in Grapefruit-Drug Interaction Studies. J Agric Food Chem. 2006 Jan 11;54(1):249-55.
• De Castro W.V., Mertens-Talcott, S.U., Rubner A., Butterweck V., Derendorf H. Analysis of flavonoids and coumarins in grapefruit juice. AAPS Annual Meeting, Nashville, TN, 2005. Abstract # T2104
• De Castro W.V., Mertens-Talcott, S.U., Rubner A., Butterweck V., Derendorf H. Great Variability of flavonoids and furanocoumarins in commercially available grapefruit juice: a source of inconsistency in grapefruit juice-drug interaction studies. 53rd Annual Congress GA, Florence, Italy, 2005, Poster# P546
• Mertens-Talcott S.U., Zdrojewski I., De Castro W.V., Butterweck V., Derendorf H. Drug-Interactions of Grapefruit- and Other Citrus- Polyphenolics - What have we learned? In: Herbal Supplements-Drug Interactions. Editors: Huang, Shaw, Publisher: Marcel Dekker (2006)
• De Castro W.V., Mertens-Talcott, S.U., Rubner A., Butterweck V., Derendorf H. Variation of Flavonoids and Furanocoumarins in Grapefruit Juice: A Source of Variability in Grapefruit-Drug Interaction Studies - 18th Annual Research Showcase - April 14, 2005 - College of Pharmacy - University of Florida
• De Castro W.V., Mertens-Talcott, S.U., Rubner A., Butterweck V., Derendorf H. Flavonoids and furanocoumarins in grapefruit juice: Analysis and effect of naringin and 6`,7`-dihydroxybergamottin on P-glycoprotein in vitro. ACCP 34th Annual meeting, Rockville, MD, 2005, J. Clin. Pharm., 45 (9), 1086
• Mertens-Talcott S.U. Food-Drug Interactions: An overview. Technical Session 104, Book of Abstracts, Institute of Food Technologists Annual Meeting 2005, New Orleans, LA, www.ift.org
• Derendorf H. Recent Advances in Food-Drug Interactions - How concerned should we be? Technical Session 104, Book of Abstracts, Institute of Food Technologists Annual Meeting 2005, New Orleans, LA, www.ift.org