GRAPEFRUIT JUICE-DRUG-INTERACTION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SPECIAL GRANT
• Reporting Frequency: Annual
• Accession No.: 0199426
• Grant No.: 2004-34507-14451
• Proposal No.: 2004-06089
• Project Start Date: Jul 1, 2004
• Project End Date: Jun 30, 2006
• Grant Year: 2004
• Program Code: [VP]- (N/A)

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

Performing Department
(N/A)

Non Technical Summary
Purchased by 21% of all households in the United States, 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 a variety of 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 a new marker compound (talinolol) males 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

pharmacokinetics

flavonoids

pharmacology

pharmaceuticals

drug therapy

coumarin

enzymes

enzyme activity

glycoproteins

nutrient function

toxicology

antibiotics

enzyme inhibitors

mechanism of action

intestines

risk

adverse reactions

human health

Goals / Objectives
Drug interactions with grapefruit juice (GFJ) have received much attention in recent years. The serum concentrations of a number of drugs are increased when taken with grapefruit juice. Most notable are the effects on cyclosporine, some 1,4-dihydropyridine calcium antagonists, and some 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. In the case of some drugs, these increased drug concentrations have been associated with an increased frequency of dose-dependent adverse effects. The predominant mechanism for this interaction is the inhibition of cytochrome P-450 3A4 (CYP450 3A4) in the small intestine, resulting in a significant reduction of drug presystemic metabolism. An additional mechanism for this 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. Although few data suggested an activation of P-gp efflux by grapefruit juice, most investigators observed an inhibition of the transporter activity. Recently, the β-blocker talinolol, which is a non-CYP450 substrate, yet a P-gp substrate, showed an increase in bioavailability when concomitantly taken with GFJ in both in vitro and in vivo models. Therefore, the modulation of P-gp function by components of GFJ may be an important factor in modifying the pharmacokinetics of drugs. In the last decade, investigations with GFJ focused on the medications with which it interacts, and the mechanism of action. Data about active constituents of GFJ are discussed controversially and at this stage it is hard to draw definite conclusions. Many compounds have been proposed to be the active ingredients in GFJ. These include both flavonoids (eg. naringenin, naringin, quercetin and kaempferol) and nonflavonoids (eg. bergamottin, 6',7'-dihydroxybergamottin). All inhibit CYP3A4 in vitro; however, in vivo studies have shown modest if any effects. Many of these constituents of GFJ are present as a mixture of chiral isomers that vary markedly in proportion and concentration, depending on the maturity of the fruit and the method of juice extraction and purification. It is therefore likely that no single compound is responsible for the interactions, but probably a combination of the effects of many constituents. Furthermore, the influence of different GFJ compounds on P-glycoprotein is still unclear, particularly regarding the in vivo relevance of this potential interaction mechanism. The objective of this application is to systematically investigate in vitro the potential drug interaction between selected ingredients of GFJ -alone and in combination- and the P-glycoprotein transporter system which controls many barriers in the body. Once the active compound(s) is/are identified, these in vitro studies will then be followed by in vivo studies. GFC fractions, single compounds and combinations of those will be tested in all experiments according to their amount in the juice and in a dose dependent manner. Using this strategy will help to accomplish the major goal of evaluating interactions of GFJ with synthetic drugs.

Project Methods
The working hypothesis is that besides the known interaction with the CYP 3A4 enzyme system, grapefruit juice (GFJ) modifies the P-gp transporter activity. Although some data suggested an activation of P-gp efflux by GFJ, most investigators observed an inhibition of the transporter activity. Thus, the extent to which GFJ modifies P-gp transporter activity still remains unclear. Recently, the β-blocker talinolol, which is a non-CYP substrate, yet a P-gp substrate, showed an increase in bioavailability when concomitantly taken with GFJ in both in vitro and in vivo models. It is proposed to test the central hypothesis and accomplish the objective of this application by pursuing the following specific aims: (1) Assess the extent to which GFJ acts on P-gp transporter activity, and (2) Assess the extent to which isolated GFJ fraction, single compounds and compounds in combination act on P-gp transporter activity. In the proposed experiments we will study the effects of GFJ on P-gp transporter activity using a human colon carcinoma cell line, Caco-2 cells, as a model of intestinal absorption. Thus, it is one aim of the proposed study to systematically investigate in vitro the potential drug interaction between selected ingredients of GFJ - alone and in combination- and the P-glycoprotein transporter system which controls many barriers in the body. Once the active compound(s) is/are identified, these in vitro studies will then be followed by in vivo studies. It is proposed to perform bioavailability studies in rats after administration of the active GFJ compound in presence or absence of talinolol. GFC fractions, single compounds and combinations of those will be tested in all experiments according to their amount in the juice and in a dose dependent manner.

Progress 07/01/04 to 06/30/06

Outputs
Unlike other citrus fruit juices, 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. Most notable are its effects on cyclosporine, some 1,4-dihydropyridine calcium antagonists and some 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, leading to elevation of their serum concentrations from 1.5 to 15-fold after oral administration. The major mechanism involves the inhibition of the drug-metabolizing enzyme cytochrome P-450 3A4 (CYP450 3A4) in the small intestine, resulting in a significant reduction of the presystemic metabolism of drugs. An additional mechanism is the interaction with P-glycoprotein (P-gp), a energy-dependent membrane efflux-transporter that carries a wide range of substrates such as: immunosuppressive and antifungal agents, anti-HIV drugs, digoxin, talinolol, anti-cancer drugs, antibiotics and steroid hormones from the enterocyte back to the gut lumen. However, the modulation of P-glycoprotein activity (in vivo or in vitro) by grapefruit juice, the duration of such interaction after intake of juice as well as its clinical relevance is still controversial, since some authors have reported increased, others reduced, others no effect on drug permeability. The objective of our studies is screening the effect of such components, alone or in combination, on the activity of P-gp in vitro and in vivo. Their contents in different commercially available and fresh-squeezed GJF as well as in different tissues of grapefruits were analyzed by HPLC. 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 white grapefruit showed the highest concentration of naringin and furanocoumarins located in albedo and peel when compared with ruby red. 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, coumarins, furanocoumarins, and polymethoxylated flavones on P-gp activity using the human colon carcinoma cell line Caco-2 as a model for intestinal absorption and 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. None of the compounds tested changed the Apical to Basolateral permeability of talinolol (100 μMol/L). However, the Basolateral to Apical was significant decreased by naringin (IC50=2562 μMol/L); naringenin (IC50=233 μMol/L); epoxybergamottin (IC50=1 μMol/L); 6',7'-dihydroxybergamottin (IC50=32.6 μMol/L); nobiltein (IC50=3.2 μMol/L); tangeretin (IC50=3.2 μMol/L); sinensitin (IC50=3.9 μMol/L) and heptamethoxyflanone (IC50=3.8 μMol/L).

Impacts
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. 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 some drugs it has been shown that a GFJ induced elevation of drug concentrations is not of clinical consequence. 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 GFJ. Results from our research demonstrate that polyphenolic components from grapefruit do have an inhibitory effect on the efflux-transporter P-gp, 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 better understand drug-GFJ interactions.

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.
• 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. 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
• 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

Progress 10/01/04 to 09/30/05

Outputs
The major mechanism for grapefruit juice (GFJ)-drug interaction involves the inhibition of the drug-metabolizing enzyme cytochrome P-450 3A4 (CYP450 3A4) in the small intestine, resulting in a significant reduction of the presystemic metabolism of drugs. An additional mechanism is the interaction with P-glycoprotein (P-gp), a energy-dependent membrane efflux-transporter that carries a wide range of substrates from the enterocyte back to the gut lumen. However, the modulation of P-glycoprotein activity by grapefruit juice as well as its clinical relevance is still unclear. Flavonoids (naringin and naringenin) and furanocoumarins (bergamottin and 6',7'-dihydroxybergamottin) derivatives have been suggested to contribute to grapefruit juice-drug interaction. The objective of our studies is screening the effect of such components on the activity of P-gp in vitro and in vivo. Their contents in different commercially available and fresh-squeezed GJF as well as in different tissues of grapefruits were analyzed by HPLC. Great brand-to-brand variability of naringin (174-1492 μMol/L), bergamottin (1-37 μMol/L) and 6',7'-dihydroxybergamottin (0.2-52.5 μMol/L) was observed. The white grapefruit showed the highest concentration of naringin and furanocoumarins located in albedo and peel when compared with ruby red. 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 and talinolol, a P-gp substrate. The Basolateral-Apical (BA) to Apical-Basolateral (AB) ratio of talinolol across the Caco-2 monolayers in absence of any inhibitor was higher than 3, suggesting the presence of polarized transport. None of the compounds tested changed the AB permeability of talinolol (100 μMol/L). However, the BA was significant decreased by naringin (IC50=1252 μMol/L); naringenin (IC50=411 μMol/L); epoxybergamottin (IC50=1 μMol/L); 6',7'-dihydroxybergamottin (IC50=32.6 μMol/L); nobiletin (IC50=2 μMol/L); tangeretin (IC50=4 μMol/L); sinensitin (IC50=7 μMol/L) and heptamethoxyflavone (IC50=4 μMol/L). The effect of bergamottin on P-gp activity could not be estimated due its very low solubility. The direct inhibitory data suggests that naringin and 6',7'-dihydroxybergamottin seems to have a significant inhibitory effect on P-gp activity at the same concentration as found in commercially available grapefruit juices. 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.

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

• 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.
• 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, In Press
• 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. 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
• 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

Progress 10/01/04 to 04/30/05

Outputs
Grapefruit juice (GFJ) has been shown to interact with a variety of prescription medications leading to elevation of their serum concentrations from 1.5 to 15-fold after oral administration and raising 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, resulting in a significant reduction of the presystemic metabolism of drugs. An additional mechanism is the interaction with P-glycoprotein (P-gp), an energy-dependent membrane efflux-transporter that carries a wide range of substrates such as: immunosuppressive and antifungal agents, anti-HIV drugs, digoxin, talinolol, anti-cancer drugs, antibiotics and steroid hormones from the enterocyte back to the gut lumen. The modulation of P-glycoprotein activity by grapefruit juice, the duration of such interaction after intake of juice as well as its clinical relevance is still unclear and is a controversial issue. 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 interactions. Their contents in different commercially available and freshly squeezed GFJ as well as in different tissues of grapefruits were analyzed by HPLC. Naringenin was not detected in any sample tested. Wide brand-to-brand variability of naringin (174-1492 mcMol/L), bergamottin (1.0-36.6 mcMol/L) and 6,7-dihydroxybergamottin (0.2-52.5 mcMol/L) was observed. The white grapefruit showed the highest concentration of naringin and furanocoumarins located in albedo and peel when compared with ruby red. Findings from this study point out the importance to consider the concentration of each potential component responsible for grapefruit juice-drug interaction in order to correlate with the effect observed. The high variability in the polyphenolic profile of different brands also may explain the conflicting reports on grapefruit-induced effects on P-gp activity. In addition, we investigated the effects of naringin and 6,7-dihydroxybergamottin on P-gp transporter activity using the human colon carcinoma cell line Caco-2 as a model for the examination of intestinal absorption and talinolol, a P-gp substrate, as a drug model. The Caco-2 cell model was established and validated with standard reference substances. Initial results show that verapamil, as a positive control, promoted a significant inhibition of P-gp and that naringin and 6,7-dihydroxybergamottin only had modest inhibitory effect on P-gp activity when used in the same concentration range as found in commercially available grapefruit juices. Currently, different concentrations of talinolol, flavonoids and furanocoumarins are being tested. Results from this study will contribute to the a rational risk assessment of concomitant use of P-gp substrates with grapefruit juice and ultimately result in dosing recommendations for these drugs that will ensure better and safer therapeutic outcome of drug therapy.

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 contributing to 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 can be shown that a grapefruit-juice induced elevation of drug concentrations is of no 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 can be recommended which provide the same therapeutic benefit but do not interact significantly with grapefruit juice. Finally, the research in this project will help to develop and test alternative methods of juice production with the goal of designing products with lower drug interaction potential.

Publications

• W.V. de Castro, V. Butterweck, H. Derendorf. Variation of Flavonoids and Furanocoumarins in Grapefruit Juice: A Source of Variability in Grapefruit-Drug Interaction Studies. 18th Annual Research Showcase, University of Florida, April 14, 2005.
• S.U. Mertens-Talcott, I. Zdrojewki, W.V. De Castro, V. Butterweck, H. Derendorf. Drug-Interactions of Grapefruit- and Other Citrus- Polyphenolics: What have we learned? in F. Lam, S.M. Huang, S. Hall, "Drug-Herb Interactions", Marcel Dekker, 2005 (in press).
• Website www.druginteractioncenter.org (2005)