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Effect of Grapefruit Juice on P-Glycoprotein Activity

HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Variation of flavonoids and furanocoumarins...
 Grapefruit juice and its components...
 Modulation of P-glycoprotein mediated...
 Conclusions
 References
 Biographical sketch
 

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EFFECT OF GRAPEFRUIT JUICE ON P-GLYCOPROTEIN ACTIVITY By WHOCELY VICTOR DE CASTRO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Whocely Victor De Castro

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To my parents, my wife and my daughter.

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ACKNOWLEDGMENTS I would like to express my grateful thanks to Dr. Hartmut Derendorf for accepting me in his team and for his continuous encouragement during my Ph.D. program. My special gratitude goes to Dr. Veronika Butterweck for her invaluable assistance and technical support during in vitro and in vivo studies. Many thanks also go to the members of my supervisory committee Dr. Gnther Hochhaus, Dr. Jeffery Hughes and Dr. Susan Percival for their guidance and helpful advice throughout the years. I would like to thank friends and staff in the department of pharmaceutics for their friendship and support, especially to Sasiporn Sarawek and Immo Zdrojewski for their suggestions and contributions during the validation of the analytical methods. I would like to extend my thanks to the secretaries of the department of pharmaceutics Mr. James Ketcham, Mrs. Andrea Tucker, for their technical support. I also would like to thank all my assistants (Anke Rubner, Julia Hoevener) and the post-doc fellows, especially Dr. Vipul Kumar, for his friendship and technical support. My personal thanks go to my mother and my father for their support, guidance and encouragement throughout my life. Finally, I would like to express my sincerely gratefulness to my wife (Enda) and my daughter (Ana) for their support, friendship, kindness and love. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES .............................................................................................................ix LIST OF FIGURES ...........................................................................................................xi ABSTRACT .....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 The Grapefruit (Citrus paradisi)..................................................................................1 The Health Benefits of Polyphenolics..........................................................................2 Grapefruit Juice-Drug Interactions...............................................................................4 Phenolic Compounds in Grapefruit and Citrus with Potential for Drug Interactions...9 Mechanism of Action.................................................................................................13 Cytochrome P450 Family....................................................................................14 Permeability-glycoprotein: P-gp.........................................................................20 Organic Anion Transporting Polypeptide: OATP...............................................26 Oral Drug Absorption.................................................................................................28 Contribution of Small Intestine to Drug Absorption and Metabolism.......................29 Mechanisms of Transport of Molecules across Intestinal Epithelium........................32 Passive Transcellular Diffusion...........................................................................32 Paracellular Transport.........................................................................................34 Carrier-Mediated Transport.................................................................................34 Transcytosis.........................................................................................................35 In Vitro Methods for Studying Drug Absorption.......................................................35 Talinolol: Model Compound for Drugs Subjected to Intestinal Efflux......................37 Properties of Talinolol................................................................................................38 Hypothesis and Objectives.........................................................................................39 Specific Aim 1: Contents of Flavonoids and Furanocoumarins in Grapefruit and Grapefruit Juice.........................................................................................41 Specific Aim 2: In Vitro Studies.........................................................................41 Specific Aim 3: In Vivo Studies..........................................................................41 v

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2 VARIATION OF FLAVONOIDS AND FURANOCOUMARINS IN GRAPEFRUIT JUICES: A POTENCIAL SOURCE OF VARIABILITY IN GRAPEFRUIT JUICE-DRUG INTERACTION STUDIES......................................42 Background.................................................................................................................42 Specific Aim...............................................................................................................43 Material and Methods.................................................................................................43 Chemicals............................................................................................................43 Sample Preparation..............................................................................................44 Determination of Flavonoids (Naringin and Naringenin)...................................44 Extraction and Determination of Furanocoumarins (Bergamottin and 6,7-Dihydroxybergamottin)...........................................................................45 Stock and Work Solutions for the Preparation of Calibration Standards............46 Naringin stock solution................................................................................46 Naringenin stock solution.............................................................................46 Bergamottin stock solution...........................................................................46 6,7-Dihydroxybergamottin stock solution..................................................46 Naringin work solution.................................................................................47 Naringenin work solution.............................................................................47 Furanocoumarins work solution...................................................................47 Standard solutions of flavonoids..................................................................47 Standard solutions of furanocoumarins........................................................48 Validation............................................................................................................49 Statistical Analysis..............................................................................................50 Results and Discussion...............................................................................................51 Linearity..............................................................................................................51 Sensitivity............................................................................................................52 Specificity............................................................................................................52 Precision and Accuracy.......................................................................................52 Recovery..............................................................................................................57 Stability................................................................................................................57 Quantification of Naringin, Naringenin, Bergamottin, and 6,7-Dihydroxybergamottin in Grapefruit Juices....................................................58 Localization of Naringin, Naringenin, Bergamottin, and 6,7-Dihydroxybergamottin in Grapefruit...............................................................60 Conclusions.................................................................................................................64 3 GRAPEFRUIT JUICE AND ITS COMPONENTS INHIBIT P-GLYCOPROTEIN MEDIATED TRANSPORT OF TALINOLOL IN CACO-2 CELLS........................................................................................................................65 Background.................................................................................................................65 Specific Aim...............................................................................................................66 Material and Methods.................................................................................................66 Chemicals............................................................................................................66 Cell Culture.........................................................................................................67 Stock, Work Solutions, and Preparation of Calibration Standards.....................68 vi

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Transport Study...................................................................................................74 Sample Analysis..................................................................................................75 Data Analysis.......................................................................................................77 Statistical Analysis..............................................................................................78 Results.........................................................................................................................78 Linearity..............................................................................................................78 Specificity............................................................................................................79 Sensitivity............................................................................................................79 Precision and Accuracy.......................................................................................80 Stability................................................................................................................80 Concentration Dependence..................................................................................81 Inhibition Studies.................................................................................................82 Dose-response Studies.........................................................................................85 Effects of NAR and DHB on the Secretory Transport of Talinolol....................87 DISCUSSION.............................................................................................................88 Conclusions.................................................................................................................91 4 MODULATION OF P-GLYCOPROTEIN MEDIATED TRANSPORT OF TALINOLOL IN RATS BY GRAPEFRUIT JUICE, ORANGE JUICE, FLAVONOIDS AND FURANOCOUMARINS........................................................92 Background.................................................................................................................92 Specific Aim...............................................................................................................93 Materials and Methods...............................................................................................93 Chemicals............................................................................................................93 Stock, Work Solutions, and Preparation of Calibration Standards.....................94 Talinolol test solutions.................................................................................94 Talinolol stock solution I..............................................................................94 Talinolol stock solution II............................................................................95 Talinolol stock solution III...........................................................................95 Verapamil test solution I..............................................................................95 Naringin test solutions..................................................................................95 Naringenin stock solution.............................................................................96 Naringenin test solution...............................................................................96 Bergamottin test solutions............................................................................96 Talinolol standard solutions.........................................................................97 Animals and Experimental Protocol....................................................................97 Sample Preparation..............................................................................................99 Sample Analysis..................................................................................................99 Validation..........................................................................................................100 Pharmacokinetic Analysis.................................................................................101 Statistical Analysis............................................................................................102 Results.......................................................................................................................102 Validation of Analytical Method to Measure Talinolol in Rat Serum..............102 Linearity.....................................................................................................102 Specificity...................................................................................................103 Sensitivity...................................................................................................104 vii

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Precision, accuracy and recovery...............................................................104 Stability......................................................................................................105 Talinolol Pharmacokinetics...............................................................................105 Discussion.................................................................................................................108 Conclusions...............................................................................................................111 5 CONCLUSIONS......................................................................................................113 LIST OF REFERENCES.................................................................................................116 BIOGRAPHICAL SKETCH...........................................................................................135 viii

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LIST OF TABLES Table page 1-1 Pharmacokinetics parameters of talinolol after oral administration (50 mg) by health volunteers (192).............................................................................................39 2-1 Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of naringin (NAR) and naringenin (NAG)..........................48 2-2 Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of bergamottin (BG) and 6,7-dihydroxybergamottin (DHB).......................................................................................................................48 2-3 Intra-day (n = 3) and inter-day (n = 9) assay parameters of furanocoumarins (bergamottin and 6,7-dihydroxybergamottin) and flavonoids (naringin and naringenin)...............................................................................................................55 2-4 Recovery values (means SD) of bergamottin, 6,7-dihydroxybergamottin, naringin, and naringenin from the spiked orange juice............................................57 2-5 Naringin, bergamottin and 6,7-dihydroxybergamottin content in different brands of grapefruit juice sold in Florida.................................................................63 3-1 Concentrations of the work solutions of talinolol (TAL) used during the transport studies........................................................................................................71 3-2 Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of talinolol...........................................................................71 3-3 Concentrations of the inhibitory solutions of Verapamil hydrochloride (VER), naringin (NAR), naringenin, (NAG), bergamottin (BG), 6,7-dihydroxybergamottin (DHB), and 6,7-epoxybergamottin (EPBG)..............73 3-4 Concentrations of the inhibitory solution of naringin (NAR) plus 6,7-dihydroxybergamottin (DHB) used during the transport studies.....................74 3-5 Intra-day (n = 3) and inter-day (n = 9) assay parameters of talinolol. Precision expressed as CV% and accuracy as % of the theoretical concentration..................80 3-6 Concentration range that the grapefruit components are normally found in the juice..........................................................................................................................85 ix

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4-1 Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of talinolol...........................................................................97 4-2 Regression parameters obtained from calibration curves of talinolol in plasma in three different days.................................................................................................103 4-3 Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of talinolol in rat serum..............................................................................................104 4-4 Pharmacokinetics of talinolol in rats after a single dose oral dose (10 mg/kg)......108 x

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LIST OF FIGURES Figure page 1-1 Chemical structures of phenolic compounds in citrus.............................................12 1-2 Mucosa of small intestine.........................................................................................31 1-3 Possible transport routes a drug can follow to cross the intestinal epithelium........32 1-4 Molecular structure of talinolol. (*) chiral center....................................................38 2-1 Mean calibration curves (n = 9) of naringin (A) and naringenin (B) in methanol/water (1:1), and of bergamottin (C) and 6,7-dihydroxybergamottin (D) in DMSO/methanol (1:3)...................................................................................51 2-2 HPLC separation and absorbance-wavelength spectra of A furanocoumarins: 6,7-dihydroxybergamottin and bergamottin; B flavonoids: naringin and naringenin.................................................................................................................53 2-3 Chromatograms corresponding to the orange juice (OJ), standard solutions (Std) containing 6,7-dihydroxybergamottin (DHB) plus bergamottin (BG) (panel A), and naringin (NAR) plus naringenin (NAG) (panel B)...................................54 2-4 Absorbance-wavelength spectra of A bergamottin; B 6,7-dihydroxybergamottin; C naringin.........................................................................54 2-5 Results of the stability test after 36 hours at room temperature (A) and after 4 freeze/thaw cycles at -20 C (B)..............................................................................58 2-6 Distribution of naringin (A), bergamottin (B) and 6,7-dihydroxybergamottin (C) in different tissues of white (open bars) and ruby red (shaded bar) grapefruits.................................................................................................................62 3-1 Structures of selected flavonoids and furanocoumarins present in grapefruit juice and tested in this study.............................................................................................66 3-2 Mean calibration curve (n = 9) of talinolol in mobile phase. Vertical bars represent the standard deviations (SD) of the means...............................................78 3-3 Chromatograms corresponding to the talinolol (100 ng/mL) in HBSS-MES..........79 xi

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3-4 Stability of talinolol (low, medium, and high QCs).................................................81 3-5 Concentration dependent coefficient permeability (P app ) corresponding to the AP to BL and BL to AP transport of talinolol across Caco-2 cells monolayers............82 3-6 Coefficient of permeability (P app ) of talinolol (100M) across the Caco-2 cells monolayers...............................................................................................................84 3-7 Inhibitory dose-response effects...............................................................................86 3-8 Effect of the combination NAR:DHB (50:1.2; 125:3, 625:15, 1250:30, and 2000:48 M) on the secretory transport (BL-AP) of talinolol (100M) across the Caco-2 cells monolayers..........................................................................................87 4-1 Comparison of chromatograms corresponding to the talinolol (I) and the internal standard propranolol hydrochloride (II) after extraction from rat serum (A) with that obtained after extraction of a blank sample (B)..............................................103 4-2 Stability of talinolol (low, medium, and high QCs) in rat serum within 24 hours at room temperature (RT) (A) and during 3 weeks at -70 C (B). Data represents means of three replicates and vertical bars the standard deviation of the means...105 4-3 Mean serum concentration-time profiles (n = 8) of talinolol in rats after a single dose oral dose (10 mg/kg)......................................................................................107 xii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF GRAPEFRUIT JUICE ON P-GLYCOPROTEIN ACTIVITY By Whocely Victor de Castro December 2006 Chair: Hartmut Derendorf Cochair: Veronika Butterweck Major Department: Pharmaceutical Sciences Grapefruit juice (GJF) interacts with several medications increasing their oral bioavailability and the risk of toxicity. The predominant mechanism is the inhibition of intestinal CYP3A4 by flavonoids and furanocoumarins present in the juice. Additionally, it has been suggested that GFJ modulates the activity of the permeability-glycoprotein (P-gp). In the intestine, this efflux transporter acts as a biological barrier pumping a variety of drugs back from the enterocytes to the intestinal lumen. Inhibition of this back-secretion may increase the initial amount of drug that enters the systemic circulation and the possibility of side effects. The aim of this study was to compare the ability of GFJ and some of its specific flavonoids and furanocoumarins to interact with P-gp in vitro and in vivo in order to estimate the clinical relevance of this interaction. Additionally, the components of these interacting compounds in commercially available and fresh-squeezed GFJ as well as in xiii

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different tissues of grapefruit were quantified and compared in order to assess their potential influence on the data gained in drug interaction studies. Flavonoids and furanocoumarins were quantified by high performance liquid chromatography (HPLC) and considerable variation was observed among the juices tested. White grapefruit showed the highest concentration of naringin and furanocoumarins located in albedo (inner peel) and flavedo (yellow layer of the peel) compared with red varieties. Findings from my study suggest that the concentration of potentially contributing compounds may crucially influence the magnitude of the interaction and invalidate direct comparisons of studies where different juices have been used. The transport of talinolol, a non-metabolized P-gp substrate, across Caco-2 cell monolayers, was determined in the absence and presence of distinct concentrations of grapefruit juice and its constituents. A sigmoid dose-response model was used to fit the data and to estimate the potencies of the potential inhibitors. Results from this study show that isolated compounds of GFJ are able to inhibit the P-gp activity, but the overall effect seems not to be a simple additive effect. The effects of different types of GFJ and its components on the oral pharmacokinetics of talinolol in rats suggest that this interaction is unlikely to be of clinical relevance. xiv

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CHAPTER 1 INTRODUCTION The Grapefruit (Citrus paradisi) The word citrus is the Latin name of Kedros, a Greek word to designate trees like cedar, cypress and pine. Because the scent of citrus leaves and fruits was suggestive of cedar, the name citrus has been linked to the citron (Citrus medica), the first citrus with which the Europeans got acquainted. Later, Linnaeus grouped all other citrus fruits known to him in the genus Citrus (1). However, the taxonomy and phylogeny have not been completely elucidated yet and are subject of controversy, mainly, due to sexual compatibility between citrus and related genera, the high frequency of mutations, long history of cultivation, and wide dispersion (2). The grapefruit (Citrus paradisi), specifically, belongs to the Rutaceae family and received this denomination because the fruits grow in clusters similar to grapes (3). Studies using genetic markers, together with conventional and taxonomic approaches, strongly support that the grapefruit originated from an accidental hybrid between pummelo (Citrus maxima) and sweet orange (Citrus sinensis) (2-4). Almost all of Citrus species and its wild relatives are supposed to have been originated in the tropical and subtropical regions of Southeast Asia (5). Conversely, it is suggested that grapefruit emerged from the island of Barbados in the Caribbean, where Griffith Hughes, the rector of St. Lucys parish of Barbados, earliest described it as the forbidden fruit of Barbados in 1750 (3). However, the first scientific description of 1

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2 grapefruit is attributed to the botanist James Macfadyen in 1837, who named the forbidden fruit Citrus paradisi Macf. (3). Grapefruit seeds were probably brought to Florida in the beginning of the eighteenth century by Counte Odettte Phillippi and distributed to locate growers, where the settlers began to sow the seeds and acquired taste for the fruits (1, 6). Since that time, several cultivars have been created, especially in the Indian River citrus region stretching from Daytona Beach to central Palm Beach County. Today, in a typical season, Florida accounts for 2 million tons of grapefruit a year corresponding to about 80 percent of the U.S. total grapefruit production (7). The Health Benefits of Polyphenolics The health benefits associated with the consumption of vegetables and fresh fruits are largely accepted and recognized by many (8-11). They are related to the presence of polyphenolic compounds, particularly, flavonoids described to act as antioxidants in different biological systems (12). In fact, Sun et al. (13) demonstrated a linear relationship between the total phenolic content and the antioxidant activity in phytochemical extracts of different fruits, including grapefruit (r 2 = 0.98, p < 0.01), indicating that phenols seem to be the major contributor to the total antioxidant activities in fruits. The antioxidant activity of flavonoids is principally based on their ability to capture electrons providing great stability to the flavonoid radical and preventing the propagating chain reactions of the oxygen-free radicals formed during the natural metabolism of any aerobic cells (12). Flavonoids may also act by chelating iron, which is thought to catalyze the process leading to the appearance of free radicals (14). Included in the ordinary diet of many Americans, the grapefruit and the grapefruit juice are rich in flavonoids, such as narirutin, naringin, hesperidin, neohesperedin,

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3 didymin, quercetin, poncirin and kaempferol. Their total concentration in the grapefruit juice (GFJ) range from 200 to 840 mg/L (15). It has been demonstrated that flavonoids have a protective role against many degenerative disorders, such as cancer and cardiovascular diseases (11, 14, 16, 17). They can prevent or minimize the oxidation of lowand very low-density lipoproteins, disrupting a crucial step in the formation of the atherosclerosis lesion and then reducing thrombotic tendencies (14, 18). The low-density lipoprotein (LDL) can be normally oxidized or modified within sub-endothelial tissue by cells of the arterial wall including macrophages, vascular smooth cells, and endothelial cells (19). The modified LDL can trigger a receptor-mediated process, leading macrophages and smooth muscle cells to accumulate significant amounts of cholesteryl ester (CE). These CE-rich cells or foam cells are characteristic indicators of early atherosclerotic lesions. There is some evidence that after consumption of foods rich in flavonoids, such compounds were able to reach the sub-endothelial space of the arterial wall at concentrations enough to protect lipoproteins like LDL from oxidation (20). Le Marchand et al. (21) found decreased rates of lung cancer among the Hawaiian population whose diet included a large amount of onions and apples (rich in the flavone quercetin) and white grapefruit (rich in the flavanone naringin). Additionally, Kawaii et al. (16) assessed the chemopreventive properties of pure flavonoids from citrus fruits against four human cancer cell lines (lung, melanoma, leukemia and gastric cancer). The anticarcinogenic properties of flavonoids are related to the suppression of metabolic activity; antimutagenic, antioxidative, and antiproliferative effects; detoxification of

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4 carcinogens; inactivation of oncogenes; inhibition of DNA adduct formation and the carcinogenic cell invasion (11-14, 16, 18). Cerda and co-workers (22) also have shown that dietary supplementation with grapefruit pectin resulted in the decrease of plasma cholesterol (7.6%), low density lipoprotein cholesterol (10.8%) and the ratio of low-density to high-density cholesterol (9.8%) in healthy subjects. Furthermore, orange juice, grapefruit juice, and naringin, a major grapefruit flavonoid, have been shown to have a beneficial effect on the blood lipid profile and antioxidant capacity in rats (23, 24). A case-control study carried out in 299 women (154 had lung cancer and 145 were the control group) in Athens during a eighteen-month period did not find any correlation between intake of food with high content of flavonoids with the etiology of lung cancer (25). However, two distinct epidemiological studies involving around 11,000 volunteers conducted by different groups in Finland and Netherlands during 1966-1976 and 1985-1990, respectively, found that regular consumption of a diet rich in flavonoids was inversely related to coronary heart disease mortality; cerebrovascular disease, lung and prostate cancer, type 2 diabetes and asthma (26, 27). Therefore, in vitro, pre-clinical, clinical, and epidemiological studies have suggested that ingestion of flavonoid-rich foods such as grapefruit juice and grapefruit juice provide some benefits against heart disease and cancer. However, since the early nineties these potential health benefits have been over shadowed by possible risks of interactions between drugs and grapefruit and grapefruit juice. Grapefruit Juice-Drug Interactions The first report of grapefruit juice interacting with a drug, altering its bioavailability was published in 1991. This accidental discovery was made in a study on

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5 ethanol drug-interactions where GFJ was used to mask the taste of the alcohol: the bioavailability of felodipine, a calcium channel blocker, was increased when subjects were consuming GFJ concomitantly with the drug. Additionally, the subjects showed a decreased diastolic blood pressure and an increased heart rate (28). Subsequent research in the area of fruit-drug interactions focused on grapefruit and grapefruit compounds of which several were found to affect the absorption or metabolism of certain drugs. Grapefruit juice was shown to modify greatly the disposition of a variety of medications taken orally, including some 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (lovastatin, simvastatin) (29, 30), calcium channel blockers (felodipine, nicardipine, nisoldipine) (31-33), benzodiazepines (midazolam, diazepam) (34, 35), and HIV protease inhibitors (saquinavir) (36). Other fruits, vegetables and dietary supplements also have the potential to cause an adverse interaction with conventional drugs and are objects of concern (37). Additionally, consumers have become more aware of the health benefits of antioxidants, which increased the availability and consumption of dietary supplements, phytochemical extracts, and foods rich in antioxidant flavonoids, especially, together with prescription drugs. In fact, over 16% of all prescription drug users reported that they concurrently take at least one plant-based dietary supplement including grapefruit and citrus products (38). This may increase the likelihood of an adverse interaction between foods and certain drugs. For example, the absorption and/or metabolism of a drug may be harmfully affected, shifting the administered dose outside of the therapeutic range, which may lead to a lower effectiveness of the drug or to an overdose associated with undesired or even dangerous side effects (39).

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6 Byproducts from the citrus-processing industry such as grapefruit seed extract, flavonoids, essential oils from the peel, and pectins may be added to other food products in order to improve taste, consistency or overall quality. These byproducts also may be used in the production of dietary supplements (40). Consequently, consumption of food products containing such additives, which have a potential for an interaction with drugs, are not free of risk. Based on the current knowledge, grapefruit compounds interact with drugs, which are metabolized by cytochrome P450 3A4 (CYP3A4) and also have a low or variable oral bioavailability. In general, the drugs subjected to GFJ interaction have their area under the concentration-time curve (AUC) and maximum plasma concentration (C max ) increased from 1.2 to 15-fold after oral administration, and the magnitude of the effect maybe dependent on the brand and amount of GJF ingested, the timing of administration relative to the food intake and the intrinsic oral bioavailability of each drug (41-44). The enhancement of the oral bioavailability of some drugs such as felodipine, nicardipine, and halofantrine after consumption of GFJ has been associated with higher incidence of dose-dependent side effects among the subjects (32, 45, 46). Indeed, one case-report study depicted a 59-year old patient receiving 60 mg of atorvastatin (Lipitor) daily who developed rhabdomyolysis after drinking GFJ. Such condition was reverted and his cholesterol levels normalized after replacing the current drug by pravastatin, another HMG-CoA inhibitor but not but not metabolized by CYP3A4 (47). Grapefruit and GFJ have a potential to interact with several oral medications when consumed in moderate amounts such as 1-2 servings (48). The concern, that the concomitant administration of grapefruit products with certain drugs may result in

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7 toxicity has lead to the recommendation to avoid consuming grapefruit products in combination with these medicines. The Food and Drug Administration (FDA) now requires drugs such as cyclosporine, sirolimus, simvastatin, lovastatin, and felodipine, to carry a warning label regarding the possibility of an interaction. For example, Neoral, an immuno-suppressant drug, carries a label stating Grapefruit and GFJ affect metabolism, increasing blood concentrations of cyclosporine, thus should be avoided (49). Procardia is labeled Co-administration of nifedipine with GFJ resulted in approximately a 2-fold increase in nifedipine AUC and C max with no change in half-life. The increased plasma concentrations are most likely due to inhibition of CYP3A4 related first-pass metabolism. Co-administration of nifedipine with GFJ is to be avoided (50). Interactions with a number of medications also have been shown for Seville orange juice, although Seville oranges are usually not processed to juice (51, 52). For most drugs in question, definitive recommendations regarding their concomitant administration with GFJ are not available, since conclusions concerning the clinical significance of the observed or predicted grapefruit-drug interactions are still limited and many data available are derived from in vitro experiments and not directly applied to human responses. Furthermore, the determination of the clinical relevance of observed interactions in human intervention trials is complicated by individual variability. Additionally, predicting the clinical significance of pharmacokinetic drug interactions is sometimes difficult especially for drugs where there are no robust methods to quantify effects or side effects. There has been recent effort in the United States by the FDA and the Pharmaceutical Research and Manufacturers of America (PhRMA) to establish some general guidelines to help drug companies, prescribers, and patients

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8 interpret the clinical significance of drug interactions(53). These are the result of clinical experience gained from some well-known drug interactions, such as the inhibition of CYP3A4. For this interaction, specifically, it could be shown that the benzodiazepine midazolam is a reproducible probe that allows quantitative determination of a potential interaction with an inhibitor of the CYP3A4 enzyme. The degree of interaction can be measured based on the augmentation in the AUC of the midazolam serum concentrations. It was recently proposed to classify changes of midazolam AUC being less than 2-fold as weak interaction, which is the case observed on midazolam co-administration with ranitidine, relatively small volumes of GFJ, roxithromycin, fentanyl, or azithromycin(53). AUC changes that range from 2to 4.9-fold, which occur on midazolam co-administration with erythromycin, diltiazem, fluconazole, verapamil, relatively large volumes of GFJ, and cimetidine are classified as moderate interaction. Changes that are 5-fold or higher in midazolam AUC are labeled as strong interaction. Examples of drugs demonstrating strong midazolam interactions include: ketoconazole, itraconazole, mibefradil, clarithromycin, and nefazodone. Strong drug interactions are considered clinically significant and result in contraindications or warnings on the product label. The clinical significance of moderate inhibitors may include decisions about dose adjustments that should be based on the concentration-effect relationship. It should be noted, however, that almost all drugs, which show an interaction with grapefruit, can be replaced by alternative drugs belonging to the same therapeutic class but without a recognized potential for interaction. The major drug classes for which grapefruit or other citrus interactions include antiallergics, antibiotics, anticoagulants, antimalaria drugs, antiparasitics, anxiolytics,

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9 calcium channel blockers, HMG-CoA reductase inhibitors, anti-HIV drugs, hormones, immunossupressants, anti-tumors, and beta blockers (40, 42, 43, 48, 54-57). Phenolic Compounds in Grapefruit and Citrus with Potential for Drug Interactions Phenolic compounds represent the major group of citrus compounds. Included are hydroxycinnamic acids, flavonoids, such as flavanones, flavones, and flavonols, anthocyanins, and furanocoumarins (Figure 1-1) (58). As previously described, many of these components have antioxidant properties and may play an important role in preventing chronic diseases such as cancer, coronary heart disease, diabetes, among others (26, 27). GFJ components such as the flavonoids naringin (NAR), naringenin (NAG), quercetin, kaempferol and the furanocoumarins bergamottin (BG), 6,7-dihydroxybergamottin (DHB) and its dimers, bergapten, bergaptol, and 6`,7`-epoxybergamottin (EPBG) (Figure 1-1) have been suggested to contribute to GFJ-drug interaction (54, 59-63). In vitro and in vivo studies have demonstrated interactions between many drugs and the furanocoumarins, BG and DHB. These GFJ compounds decreased the hepatic metabolism of saquinavir in vitro and the concentrations necessary to reach half of the maximal effect (IC 50 ) was shown to be 0.33 M and 0.74 M, respectively (64). Additionally, BG was able to augment the C max and AUC of diazepam in dogs (65), of nifedipine in rats (66), and of felodipine in humans (67). Several human intervention studies also confirm the contribution of BG and DHB to drug-interactions with GFJ (41, 68-70).

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10 GFJ components have been demonstrated to interact with susceptible drugs by different mechanisms besides the reduction of metabolism. For example, BG, DHB, bergaptol and bergapten were shown to elevate the steady state uptake of [ 3 H]-vinblastine sulfate in Caco-2 cells (71), whereas BG and DHB caused a decrease in the organic anion transport peptide (OATP)-B-mediated uptake of estrone-3-sulfate into human embryonic kidney cells (72). Therefore, the effects of grapefruit and GFJ are not restricted on the activity of the enzyme CYP3A4 but also involve modulation of membrane influx and efflux transporters such as the permeability glycoprotein (P-gp) and the OATP, respectively. Interactions with drugs also have been demonstrated for flavonoids from citrus. Naringenin and naringin have been shown to inhibit the OATPB-mediated uptake of estrone-3-sulfate in human embryonic kidney cells (72) and to interact with saquinavir (64) and simvastatin (73) in vitro. Compared to the control group, NAR increased by 2-fold the C max and the AUC of diltiazem in rats, while there was no significant change in the time to reach the C max (T max ) and terminal plasma half-life (t 1/2 ). NAR also increased the oral bioavailability of quinine in rats by 42% (74). However, this flavoinoid did not show any effect on the pharmacokinetics and pharmacodynamics of nisoldipine in healthy volunteers (75). Quercetin inhibited P-gp-mediated efflux of ritonavir in Caco-2 cells (76), increased the C max and AUC of verapamil in rabbits by 2-fold (77), and inhibited the metabolism of midazolam and quinidine in human liver microsomes (78). It did not have an effect on CYP3A4-mediated metabolism and P-gp mediated transport of saquinavir

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11 (64). Nobelitin and tangeretin inhibited OATP-B-mediated uptake of estrone-3-sulfate into human embryonic kidney cells (72). For several phenolics from citrus such as eriocitrin, poncirin, and sinapic acid and anthocyanins, which occur in red grapefruit varieties and blood oranges, no interactions with CYP3A4 and P-gp have been reported yet. In vitro studies of single compounds can help to understand the mechanism of action of each component as well as their relative contribution to the overall effect observed after ingestion of a food with potential for interacting with drugs. However, the clinical relevance of data obtained from in vitro studies with single compounds is questionable, because they have a limited predictability of the effects of the examined compounds in vivo. Moreover, some phenolics were shown to modify the absorption or metabolism of drugs only at very high concentrations, which are likely to exceed the expected in vivo concentration after the consumption of a moderate amount of a grapefruit/citrus product. Additionally, the overall effect of GFJ in drug interactions studies is probably the result of the mixture of several compounds present in the juice. Therefore, the existence of synergism, antagonism or additive effects needs to be investigated by testing, simultaneously, different combinations of phenolic compounds normally found in GFJ. Nevertheless, studies with single compounds have demonstrated that furanocoumarins and their dimers are primarily responsible for the interactions of GFJ and drugs. In conclusion, grapefruit, sour orange (Seville), and limes, which contain BG (69) seem to have the highest potential among the citrus species for interacting with drugs, whereas other citrus varieties such as sweet orange seem to have an overall low potential

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12 for interfering with medications. However, in studies using juices rather than single compounds, orange juice (C. sinensis), which does not contain NAR and only small amount of BG and DHB, has also been shown to interact with some drugs. A more recent study in rats demonstrated that orange juice (and apple juice) decreased the oral exposure of fexofenadine, possibly through an inhibition of the influx transporter OATP (79). The interaction of orange juice and fexofenadine has also been demonstrated in HeLa cells, where orange juice at 5% of the normal strength potentially reduced the uptake of fexofenadine, mediated through the human OATPA (80). In general, the effect of orange juice on CYP3A4 is negligible and in some GFJ-drug interaction studies orange juice was even used as control (81, 82). Overall, it can be concluded that orange juice has a minor potential for drug interactions. O O OMe OMe OMe MeO MeO O O OMe OMe OMe MeO MeO OMe O O OMe OMe MeO MeO OMe O O OMe OMe OMe MeO MeO OMe OMeOOHOHOHO OOHOHO OH O O OH OH OH O O OH OH O O O O O O O O OH OH O O O O O O O O OH O OH O O O MeO OMe OH O O OH OH OH OH O O OH OH OMe O O O Tangeretin Nobeletin Sinensetin Heptamethoxyflavone Naringin Naringenin Bergamottin 6',7'-Dihydroxybergamottin 6',7'-Epoxybergamottin Bergaptol 7-Hydroxycoumarin Limetin Eriodictiol Hesperetin 7-Geranosyloxylcoumarin Figure 1-1. Chemical structures of phenolic compounds in citrus.

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13 Mechanism of Action A food-drug interaction can be defined as the alteration of drug exposure and drug effects by food. The underlying mechanisms can be classified into two broad categories. The first category is pharmacokinetics, which includes alterations in absorption, distribution, metabolism, and excretion. The second category is pharmacodynamics, which describes alterations in the drug concentration-effect relationship (83). Changes in the pharmacokinetics of drugs are the more common consequences of citrus-drug interactions, which may shift the effect of the drug outside of its therapeutic window, possibly leading either to loss of effect or undesired side effects, or even toxicity (48). Because the majority drugs exhibiting an interaction with GFJ are metabolized mostly by CYP3A4, which is present not only in the hepatocytes, but also in the epithelial cells of the intestine, it has been suggested that the effect of GFJ may be due, primarily, to the inhibition of the activity of this drug metabolizing enzyme (48, 57, 83). However, differently from the most potent CYP3A4 inhibitors such as ketoconazole, where the main site of action is the liver, GFJ when consumed in usual volumes, is an example of inhibitor that appears to act preferentially in the enteric CYP3A4, resulting in a significant reduction of the presystemic metabolism of drugs without, therefore, modify their elimination half-life (54, 56). For several drugs such as felodipine, cyclosporine, midazolam, and nifedipine, the gastrointestinal mucosa has been demonstrated to be a major metabolic organ, where an inhibition of CYP3A4 caused an increase in oral bioavailability (84). Additionally, GFJ intake did not change the pharmacokinetics and pharmacodynamics parameters of midazolam (85), and felodipine (45) after intravenous administration, but increased their systemic bioavailability from oral dosage forms,

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14 supporting that inhibition of intestinal CYP3A4 by maybe particularly important when the drug is given orally together with the juice. Additionally, it has been suggested that GFJ can delay the gastric emptying rate contributing, in part, to the double peak phenomenon observed after co-administration of nifedipine with the juice (86). In fact, the lower the pH of the gastric chyme that reaches the small intestine, the slower will be rate of gastric emptying (87). It is also suggested that GFJ influences not only the first-pass metabolism of drugs like nifedipine, but also its secondary oxidative steps (86). In summary, the major mechanism leading to a grapefruit-drug interaction appears to be the reduction of the "first-pass" metabolism through the inhibition of intestinal CYP3A4 (57), although inhibition of secondary metabolic pathways and decrease of gastrointestinal motility cannot be excluded. In addition, GFJ also can modify the disposition of several drugs substrates of P-gp and recent reviews compile the effects and the clinical relevance of such interaction (43, 88). Moreover, GFJ and other fruit juices have been shown to inhibit in vitro and in vivo a number of organic anion-transporting polypeptides (OATPs) (72, 79, 80, 89, 90). Cytochrome P450 Family The cytochrome P450 (CYP) enzyme family is the major catalyst of phase I drug biotransformation reactions and accounts for the metabolism, at least in part, of about 60% of all drugs subjected to oxidation in the body (91). CYP enzymes are bound to membranes of the endoplasmatic reticulum and are predominantly expressed in the liver, although they are also present in extra-hepatic tissues such as the gut mucosa (84). They can catalyze a notable number of metabolic processes including aliphatic oxidation, aromatic hydroxylation, N-dealkylation, O-demethylation, S-demethylation, oxidative

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15 deamination, sulfoxide formation, N-oxidation, N-hydroxylation (83). Such biotransformation reactions usually lead to inactivation and elimination of most pharmaceuticals, therefore, ingestion of xenobiotics able to inhibit or activate the CYP enzymes can affect the disposition of many of CYP-drug substrates. More than 50 different human CYPs have been recognized to date and their nomenclature is determined by resemblance of amino acid constitution (83). For instance, the isoform CYP3A4 belongs to family 3 (matching 40% of amino acid sequencing) and subfamily 3A (matching 55% of amino acid sequencing) (48). Among the CYP3A family, the CYP3A4 isoform is the most abundantly expressed and represents approximately 30% to 40% of the total CYP protein in human adult liver (92). It also can be found in the enterocytes of the small intestine at levels of 10 to 50% lower than those found in the liver (84). However, CYP3A4 concentrations equivalent or above of those found in liver have already been verified in some subjects (93-95), which can explain much of the inter-subject variability observed during clinical trials studying GFJ-drug interactions. The relatively high expression levels in the intestinal mucosa and the broad substrate specificity may contribute to the high susceptibility of CYP3A4 for citrus-drug interactions (96). Many drugs for which interactions with citrus have been demonstrated are metabolized by CYP3A4 (48, 57, 83). It is currently recognized that the major mechanism for grapefruit-drug interaction is the inhibition of the drug-metabolizing enzyme CYP3A4 in the small intestine resulting in a significant reduction of metabolism of drugs during their first passage from the intestinal lumen into the systemic circulation (54). Types of intestinal CYP3A4

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16 inhibition by GFJ and its components comprise both competitive and mechanism-based, the latter resulting in accelerated degradation of the enzyme through suicide inhibition (97, 98). In fact, the amount of CYP3A4 in the small intestine of healthy volunteers has been shown to decrease by 50-60% after 4-6 h administration of 8 oz (~237 mL) GFJ, without any change in corresponding messenger RNA levels (60, 97). Several drug classes such as dihydropyridine calcium antagonists and HMG-CoA-reductase inhibitors are affected by grapefruit-induced inhibition of CYP3A4. GFJ increased the AUC and maximal plasma concentration (C max ) for these calcium antagonists within an approximated range of 1.5to 4-fold on average of the values obtained with water (48). For the HMG-CoA reductase inhibitor atorvastatin, double-strength GFJ increased the AUC 2.5-fold but not the C max when the GFJ was administered over three days and the drug was given on day three (99). In a very similar study design performed by the same group with simvastatin, double-strength GFJ increased the AUC 16-fold and C max 9-fold (100). Additionally, repetitive ingestion of high volumes of grapefruit juice has augmented the AUC values of lovastatin over 10fold compared with water (29). Furthermore, a daily volume of 250 mL of grapefruit juice had a modest effect on lovastatin, however, in this study grapefruit juice had been taken about 12 h apart from statin (101). All drugs mentioned above are, to a certain extent, subject to CYP3A4 metabolism. Lundhal et al. (102) compared the acute effect of GFJ intake (200 mL) on the pharmacokinetics and haemodynamics parameters of the CYP3A4 substrate felodipine with the interaction following14-days intake of drug with juice. The AUC and C max of

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17 felodipine after acute treatment with GFJ and on day 14 were similar and around 57% to 73% and 114% to 138% higher than those from the control group, respectively. Additionally, the authors reported a decrease of heart rate and increase of vascular adverse events during the GFJ period compared with the control. These results suggest that one glass of GFJ is enough to produce the maximum inhibitory effect on the intestinal CYP3A4 activity. A moderate consumption does not appear to lead to an inhibition of hepatic CYP3A4 activity (103), however, the repeated consumption (200 mL three times a day) of double-strength GFJ induced a higher increase in triazolam concentrations and prolonged its half-life. The repeated consumption of GFJ may have caused an inhibition of hepatic CYP3A4 (104). In a three day study, GFJ increased the AUC of simvastatin 3.6-fold and that of its active metabolite, simvastatin acid, 3.3-fold. C max of simvastatin and simvastatin acid were increased 3.9-fold and 4.3-fold, respectively, when the GFJ was administered for three days and simvastatin on day three (30). In an in vitro study performed with several grapefruit compounds, it was shown that BG, DHB, and the furanocoumarin dimers GF-I-1 and GF-I-4 inhibited CYP3A4-catalyzed nifedipine oxidation in a concentration-and time-dependent manner, which is consistent with the mechanism-based inhibition. DHB was more potent than BG, while the dimers were more potent than the monomers. Not only CYP3A4 but also CYP2C9, CYP2C19, and CYP2D6 seem to be affected by citrus compounds. In the same study, the inhibitory effect of BG was stronger on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on CYP3A4 (105). In an intervention trial with healthy volunteers, GFJ (twice daily) decreased the activity of CYP1A2, as determined with caffeine as a probe (106). In

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18 another human study, GFJ and naringenin caused a minor reduction of the activity of CYP1A2 (107). In addition, ingestion of 8 oz of regular-strength GFJ once or three times a day did not affect liver CYP3A4 activity, colon levels of CYP3A5, or small bowel concentrations of P-glycoprotein, villin, CYP1A1, and CYP2D6 (60, 97). Overall, the inhibition of CYP enzymes other than CYP3A4 does not appear to be of great magnitude and may clinically be relevant only for drugs with a narrow therapeutic range (96). There is a lack of information available regarding the reversible and mechanism-based inhibition kinetics for grapefruit compounds. In a study conducted with human intestinal microsomes by Paine and coworkers (103), DHB induced a substrate-independent reversible and mechanism-based inhibition on CYP3A4. In contrast, BG, a more lipophilic compound, was a substrate-dependent reversible inhibitor and a substrate-independent mechanism-based inhibitor. For BG, the inhibition for testosterone was more potent than for midazolam, possibly due to the higher affinity of BG for the testosterone-binding site than for the midazolam-binding site. As mechanism-based inhibitors, BG and DHB are definitely substrates for CYP3A4, but the binding sites are not known. The authors suggested that both furanocoumarins inactivate CYP3A4 by the binding of the furanoepoxide to the apoprotein, presumably at or near their respective substrate domains. The same group determined the onset time of inhibition by both compounds (63). It was found that DHB inhibited 85% of CYP3A4 activity independent of substrate within 30 minutes, whereas the onset for BG-induced inhibition was much later (70% inhibition was reached after three hours). The substrate-dependent inhibition caused by BG was more than 50% after 0.5 to 3 hours for testosterone 6-hydroxylation, while midazolam 1-hydroxylation was unaffected, or activated, within one hour. Both

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19 furanocoumarins caused 40% to 50% reduction of CYP3A4 protein, probably due to intracellular degradation of the enzyme caused by mechanism-based inactivation. These data imply that, after the consumption of GFJ, DHB causes the enzyme inhibition earlier than BG. Lilja and coworkers (108) conducted a crossover study in which 10 healthy volunteers ingested 40 mg simvastatin with water (control), or with "high-dose" grapefruit juice (200 mL double-strength grapefruit juice three times a day for 3 days), or with water on days 1, 3, and 7 after ingestion of GFJ. When simvastatin was taken with GFJ, the mean C max and AUC of simvastatin were increased 12.0-fold and 13.5-fold, respectively, compared with control. The C max values when simvastatin was administered with water 24 hours, and 3 days after ingestion of the last dose of GFJ, were increased 2.4and 1.5-fold, respectively, compared with the control. Similarly, the AUC values were 2.1and 1.4-fold higher than those of the control group after 24 hours and 3 days of GFJ intake, correspondingly. Seven days after ingestion of GFJ, no difference in the C max or AUC of simvastatin were observed. Furthermore, Greenblatt et al. (109) determined, in a human intervention trial, the time of recovery of intestinal CYP3A4 after the consumption of 300 mL of regular-strength GFJ and a single dose of midazolam at 2, 26, 50, or 74 hours after administering the juice. After two hours, the AUC was 1.65-fold increased and after 26, 50, and 74 hours, the AUC was 1.29-, 1.29-, and 1.06-fold higher, respectively, in comparison to the control. The recovery half-life was estimated at 23 hours. These results indicate that the interaction of grapefruit juice with CYP3A4 substrates dissipates within 3 to 7 days after ingestion of the last dose of GFJ, consistent with a mechanism-based inhibition.

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20 In summary, the presented in vitro studies confirm the inhibitory effects of grapefruit compounds on CYP-enzymes, with major effects on CYP3A4, involving both mechanism-based and reversible inhibition. DHB appears to be more potent than BG; however, furanocoumarin dimers seem to be more effective than monomers in the inhibition of CYP3A4. The human intervention trials examining the pharmacokinetic interaction of GFJ revealed a great inter-individual variability, where subjects with the highest content of CYP3A4 showed the largest reduction of this enzyme after the consumption of grapefruit. Permeability-glycoprotein: P-gp The interest in transporters as mediators of interactions between grapefruit and drugs is increasing and one of the most studied drug transporters is the permeability-glycoprotein or P-gp. This efflux transporter consists of a 170KDa glycosylated plasma membrane protein that belongs to the ATP-binding cassette (ABC) transporters superfamily (110, 111). In humans, the multidrug resistance genes MDR1 and MDR3 encode the P-gp whereas, three members of this family (mdr1a, mdr1b, and mdr2) are found in mice (88, 112). The P-glycoprotein encoded by human MDR1 and mouse mdr1a/1b genes are drug transporters, while human MDR3 and mouse mdr2 P-glycoprotein are thought to be implicated mostly with the transport of phospholipids (113, 114). Structure-function analyses revealed that P-gp consists of two homologous halves, each containing six transmembrane domains where are located the drug-binding sites and a cytoplasmatic nucleotide-binding domain where the ATP hydrolysis takes place generating the driving force for the drug efflux (115, 116).

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21 P-glycoprotein was first characterized in tumor cells contributing to multidrug resistance (MDR) (117), but it is also expressed constitutively at high levels on the apical membrane of luminal epithelial cells of different tissues, which are involved in drug absorption and disposition such as hepatocytes cannalicular membrane, renal proximal tubules, intestinal mucosa and capillaries in the brain (110, 118-120). One of the most intriguing aspects of P-glycoprotein is its ability to recognize and transport many drugs with a wide array of chemical structures. So far, a clear structure activity relationship for predicting P-glycoprotein substrates has not been established and the only common feature is that most of P-gp substrates are hydrophobic in nature, suggesting that partitioning within the lipid membrane of cells is the first step for the interaction of substrate with the active sites of P-glycoprotein (110). Several models have been proposed to describe the mechanism for drug transport activity. The more favorable model proposes that P-glycoprotein intercepts lipophilic drugs either from outer membrane of lipid bilayer, before they enter the cell cytosol, or from the inner membrane of the bilayer, and extrudes them into the extracellular medium (115, 116, 121). The development of mdr1a, mdr1b, and mdr1a/b knockout mice was a landmark on P-glycoprotein research (122). In mdr-knockout mice one or both genes responsible for P-gp expression are deleted, allowing investigation of the function of P-gp in a very clear experimental design. For example, the AUC of paclitaxel was reported to be 2and 6-fold higher in mdr1a (-/-) knockout mice than in the wild type animals after intravenous bolus (i.v.) and oral administration, respectively. The increased AUC of paclitaxel after i.v. administration in mdr1a (-/-) mice was attributed to the decrease in elimination clearance,

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22 whereas the higher AUC after oral administration in mdr1a (-/-) mice resulted from combination of a decrease in the elimination clearance and enhancement of drug absorption. Based on the AUC values after i.v. and oral administration, the bioavailability of paclitaxel was calculated to be 11 and 35% for wild type and mdr1a (-/-) knockout mice, respectively (123). Wetsphal et al. (124) demonstrated that rifampin (a P-gp inducer) was able to reduce the AUC of intravenously (30 mg) and orally administrated (100 mg) P-gp substrate talinolol in healthy volunteers by 21% and 35%, respectively. Experiments in mice with cannulated gallbladder revealed that within 90 minutes after intravenous bolus administration of [ 3 H]digoxin approximately 16% of the dose was secreted into intestinal contents of wild-type mice, whereas in mdr1a (-/-) knockout animals this value was reduced to only 2% (125). In summary, the intestinal P-glycoprotein hinder the absorption of distinct classes of drugs like anti-cancer, anti-HIV, cardiac glycosides, steroids, -adrenergic agents, immuno-suppressants, and antibiotics by carrying them from the enterocytes back to the gut lumen (126, 127). Therefore, inhibition of the intestinal P-gp function may be expected to be followed by enhanced oral bioavailability of its substrates, increasing the risk of dose related side effects particularly for drugs with very low therapeutic index. Due the remarkable overlaps in tissue distribution, substrates and inhibitors specificity between CYP3A4 and P-gp (128) it is not surprising that GFJ and its components can also interact with this membrane efflux transporter. Among the many components of grapefruit juice (GFJ), the flavonoids (naringin, naringenin, kaempferol, quercetin), the furanocoumarin (bergamottin,

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23 6,7-dihydroxybergamottin, and 6,7-epoxybergamottin), and some polymethoxylated flavones (tangeretin, nobelitin) derivatives (Figure 1-1) are able to modulate the activity of P-glycoprotein and have been suggested to contribute to grapefruit juice-drug interaction (56, 62, 71, 129-131). In contrast to CYP3A4, the levels of P-gp in the small intestine are not affected by GFJ or its components. Ingestion of 8 oz of GFJ (three times per day for six days) did not alter the expression of intestinal P-gp in healthy volunteers (97). Takanaga et al. (132) suggested that GFJ and its components were able to inhibit the P-gp activity in Caco-2 cells using vinblastine as probe. Since vinblastine also is a substrate of CYP3A4 (133), the GFJ effects regarding this inhibition were not conclusive. However, GFJ and phenolic compounds from orange, such as tangeretin, nobiletin, and heptamethoxyflavone, which have been demonstrated not to alter CYP3A4 activity, increased the net influx of vincristine into adriamycin-resistant human myelogenous leukemia cells conclusively, through the inhibition of P-gp (131). Several in vitro studies with different probes, such as vinblastine (71), talinolol (134), and digoxin (135) also confirm the findings that GFJ inhibits the efflux of P-gp substrates. In addition to GFJ, orange juice and pomelo juice also have been shown to inhibit the activity of P-gp in vitro (135). Flavones from orange juice have been described to be more potent than compounds from grapefruit in the inhibition of P-gp (132). In contrast to these findings, Soldner et al. (136) suggested that GFJ significantly activates the P-gp-mediated efflux of drugs in Madin-Darby canine kidney (MDCK)-MDR1 cells model.

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24 Five GFJ components (quercetin, naringin, naringenin, bergamottin and 6,7-dihydroxybergamottin) were screened as modulators of P-gp activity by assessing the directional transport of saquinavir (a P-gp substrate) across Caco-2 cell monolayers (64). Only naringin and 6,7-dihydroxybergamottin had any appreciable effect, reducing the net secretion of saquinavir transport from 25 to 7.6 and 7.1, respectively. In order to evaluate the effect of grapefruit juice on P-glycoprotein-related transport processes, measurements of permeability through Caco-2 cells and in vivo drug absorption studies in Sprague-Dawley rats were performed using talinolol, which is a P-gp substrate but not metabolized by CYP3A4, as a model compound (134). Apical to basolateral talinolol transport across Caco-2 monolayers was increased 3-fold when GFJ was present. During in vivo study in rats, doubled maximum plasma concentrations, enhanced AUC values, and decreased apparent oral clearance were found for both talinolol enantiomers at the same order of magnitude, suggesting an inhibition of P-gp by GFJ. A recent clinical trial, however, reported that a single glass of GFJ (300 mL) reduced the talinolol AUC, and C max to 56% and 57%, respectively, of those with water. Similar effect was observed after repeated ingestion of GFJ (300 mL three times a day for 6 days) (137). These results suggest that constituents in grapefruit juice preferentially inhibited an intestinal uptake process rather than P-glycoprotein, although it is not clear yet if talinolol is a substrate of any influx transporter, such as OATP. Therefore, activation of P-gp cannot be discharged as a grapefruit effect in this study. Edwards et al. (130) evaluate the effect of GFJ and Seville orange juice on the disposition of cyclosporine, a P-gp substrate, in man. Compared with the control

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25 experiment, the AUC and peak concentrations of cyclosporine were increased to 55% and 35%, respectively, by GFJ, whereas Seville orange juice did not have an influence on cyclosporine, while both juices reduced enterocyte concentrations of CYP3A4. The authors additionally demonstrated that DHB did not inhibit P-gp in vitro. In parallel, Malhotra et al. (138) through a randomized three-way crossover intervention trial studied the effect of Seville orange juice, diluted GFJ (normalized to contain equivalent total concentration of BG and DHB as in Seville orange juice), and sweet orange juice on the disposition of felodipine in healthy volunteers. Seville orange juice and GFJ increased the AUC of felodipine. Considering both studies, while Seville orange juice and GFJ probably interact with felodipine through inactivation of intestinal CYP3A4, the lack of interaction between Seville orange juice and cyclosporine indicates that grapefruit may cause interactions also through the inhibition of intestinal glycoprotein. In addition, these data imply that the inhibition of P-gp activity by other compounds in GFJ (different from those present in Seville orange) may be responsible for the increased bioavailability of cyclosporine. When a tablet containing 0.5 mg of digoxin was co-administrated with GJF in healthy subjects the pharmacokinetics parameters were slightly increased (1.2and 1.1-fold increase in AUC and C max respectively) (139), or not affected at all (140). However, in both studies it should not be concluded that there is no interaction between GFJ and P-gp since digoxin has a high oral bioavailability (70-80%) and even a total inhibition would be expected to enhance its oral bioavailability by only 1.2-1.3-fold (141). Similarly, the pharmacokinetics of indinavir, another known P-gp substrate was not altered by GFJ (52), suggesting that possible other unknown mechanisms and factors

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26 such as strength of the administered juices and length of consumption are relevant for the interactions of citrus with drugs (142). Overall, it can be stated that grapefruit and other citrus may interact with several drugs through the combined inhibition of CYP3A4 and P-gp. Additionally, there are significant differences among the published papers depending on the animal (rats, mice, dogs) or cell model (Caco-2 or MDCK cell lines) used as well as culture conditions, study design, and clinical relevance. Furthermore, the magnitude of interaction may strongly depend on the variations in the polyphenolic profile of grapefruit juice, which is related to the origin, quality of raw material, the manufacturing procedure, and storage conditions (40). In conclusion, the modulation of P-gp activity by grapefruit juice or its components and the clinical relevance of such interaction is still unclear and is a controversial issue, since some authors have reported activation (136, 143), others inhibition of P-gp (61, 71, 144), or absence of effect (52, 139, 140). Therefore, additional studies are required in order to clarify the P-gp-related interactions between drugs and GFJ. Organic Anion Transporting Polypeptide: OATP The organic anion-transporting polypeptides (OATPs) are sodium-independent influx membrane transporters belonging to the superfamily of solute carriers (SCL) (145). So far, 39 members of the OATP superfamily have been identified in mammalian species such as human, rat and mouse (146, 147) all of them with broad substrate specificity (148). OATP transporters are composed by 12 transmembrane domains and a large extracellular region between the helices 9 and 10, which is believed to have a important role on solute transport (149). Most of OATP members are expressed in the basolateral

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27 membrane of polarized cells of multiple tissues, exceptions are the OATP1B1 and OATP1B3 present predominantly on human hepatocytes (145, 150, 151) and the OATP2B1 that has been detected in the apical membrane of enterocytes (152). The whole superfamily is classified into families and each family into subfamilies. The individual families contain proteins with not less than 40% in amino acid sequence identity and are indicated by numbers (e.g., OATP1, OATP2, OATP3, etc.). Subfamilies contain proteins with amino acid sequence identities equal or higher than 60% and are designated by letters (e.g., OATP1A, OATP1B, OATP1C, OATP2A, etc.). Individual paralogues within a subfamily are indicated by different final numbers (e.g., OATP1B1, OATP1B3) (152). Considering the co-localization of these absorptive transporters and the secretory P-gp at the apical plasma membrane of the enterocytes, their potential significance in the oral absorption cannot be neglected and might be related with a number of drug-drug and food-drug interactions (120, 127). GFJ and orange juice (OJ) have been reported to decrease the AUC and C max values of fexofenadine to 30 and 40%, respectively, of those when the drug was taken with water (80, 90). Similar results were also find when celiprolol was co-administrated with GFJ or OJ (153, 154). Both drugs are substrates for P-gp and OATP, but not CYP3A4 (155, 156). If P-gp had played a major role in the observed interactions, the bioavailability would have been increased instead of decreased. This led to the conclusion GFJ and orange juice preferentially decreased the OATP-mediated transport of fexofenadine and celiprolol, than the transport carried-out by P-gp.

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28 In a study with human embryonic kidney cells expressing OATP1B, different citrus juices were tested in their effect on the uptake of estrone-3-sulfate. GFJ, orange juice, BG, DHBG, quercetin, naringin, and naringenin significantly inhibited OATP1Bmediated uptake of estrone-3-sulfate. The citrus compounds DHB and tangeretin significantly inhibited OATP1B-mediated influx of the probe glibenclamide (72). The effects of fruit juices on the oral availability of fexofenadine also have been tested in rats (79). In this study, orange juice decreased the oral bioavailability of the drug to a lesser extent than that observed in humans. Overall, it has to be considered that genetic differences in the OATPs between humans and other species may contribute to differences in the susceptibility to grapefruit-induced inhibition, which also is true for other transporters and enzymes. In summary, OATPs appear to play an important role in the influx of a number of drugs into enterocytes and hepatocytes. The inhibition of OATP activity has been demonstrated for orange and GFJ in in vitro and in vivo experiments. The clinical relevance of this mechanism remains to be investigated in further human intervention trials. Oral Drug Absorption Most of prescription drugs are administered orally due to convenience of administration, patient compliance, safety, ease of production and distribution. However, this administration route is frequently associated with issues of low bioavailability, defined as the rate and extent that a drug is absorbed from its dosage form and becomes available at the site of action (157, 158). Among the factors with a potential to influence the bioavailability are the physical-chemical characteristics of the drug molecules (partition coefficient, ionization constant, solubility, polymorphism, particle size and particle shape distribution, stability at various

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29 pH values and against gastrointestinal enzymes), the pH and physiological conditions of the absorptions site, gastric emptying, intestinal motility, absorption windows, transporters, biotransformation and the manufacturing factors as excipients and pharmaceutical technology (87, 157). Additionally, the active ingredient of the dosage form should be appropriately released, solubilized in the GI fluids, and should cross the epithelial barriers in order to be absorbed throughout the gastrointestinal (GI) tract and reach the systemic circulation. In such scenario, the term release may include several processes like disintegration, deaggregation, diffusion, and dissolution of the drug in the GI fluids (87, 157, 159). Contribution of Small Intestine to Drug Absorption and Metabolism In a very simplistic point of view, the human gastrointestinal tract can be functionally divided into a preparative and primary storage region (mouth and stomach), a secretory and absorptive region (small intestine), a water reclamation system (ascending colon), and finally, a waste-product storage system (the descending and sigmoid colon regions and the rectum) (160). The small intestine, comprising of duodenum, jejunum and ileum, has two major functions: it can efficiently absorb nutrients, fluids, electrolytes, and many of the drugs already developed, and simultaneously act as a physical and metabolic barrier against of potentially toxic xenobiotics and pathogens (161). Such peculiarity can be explained by its morphology and physiology, which provide an enormous surface area with ability to absorb liquids and solutes selectively. The mucosa of small intestine is consisted of three groups of projections: circular folds, villi and microvilli (Figure 1-2). The absorptive cells or enterocytes are elongated, polarized and intimately connected by tight junctions, forming a physical barrier between the contents of GI tract and the intercellular space

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30 (87). Another physical barrier located just above the enterocytes is the mucus, a viscous and elastic gel layer consisted of water, electrolytes, and high-molecular-weight (2 x 10 6 Da) glycoproteins, produced by the goblet cells (162, 163). The basolateral and apical membrane of the enterocytes are markedly distinct conferring a polarized feature due the presence of a striated end (brush border) at the top of the cells (apical side) towards to the interior of the tract (Figure 1-2). These microvilli multiply by a factor of 20 the surface area of the small intestine and are consisted, especially, of protein fibers of actin and myosin (87, 162). Additionally, most of functionally active transporters protein systems (sugar transporters, P-gp, OATPs) and metabolizing enzymes (peptidases, esterases, CYP3A isoforms, N-acetyl transferases and glutathione transferases) are expressed in the mature enterocytes (163, 164) affecting, therefore, the fraction of the drug initially administrated available for absorption. The cellular membrane contains fundamentally a phospholipid bilayer, in which glycoprotein molecules are located. The phospholipids are arranged such that the hydrophilic ends face the two membranes surfaces, inner and outer, and the hydrophobic tails are directed towards the membrane center (87, 162). Such characteristic makes lipophilic compounds be rapidly and completely absorbed, while slowly and incompletely passively absorbed drugs are, in general, hydrophilic and with poor distribution into the cell membranes (165). Although the intrinsic selectiveness of the small intestinal may impair the bioavailability of many drugs, it is still the principal target for drug absorption following an oral administration due to its vast surface area. Physicochemical properties of drug

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31 molecules and formulations parameters can be modified in order to obtain a better absorption profile. However, co-administration of two or more drugs subjects to similar metabolic pathways or sharing the same transporter systems increase the likelihood of drug-drug interaction. Similarly, the ingestion of dietary supplements and foods, like grapefruit juice, able to affect the normal physiology of the small intestine, the activity of the metabolizing enzymes, and carriers have a potential to modify markedly the disposition of drugs taken orally. Figure 1-2. Schematic representation of the mucosa of small intestine consisted of circular folds, villi finger-like structures covered by a monolayer of epithelial cells separating the lumen from the blood capillary network, and microvilli the brush board projections located at the apical side of the enterocytes Source: http://instruct1.cit.cornell.edu/courses/biog105/pages/demos/105/unit6/ media/villus. structure.jpg.

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32 Mechanisms of Transport of Molecules across Intestinal Epithelium Orally administrated drugs can enter the systemic circulation by crossing the intestinal epithelium via two major routes, the paracellular or the transcellular pathways (164). Paracellular can be further divided in passive transcellular diffusion, carrier-mediated transport or transytosis (Figure 1-3). The importance of each route for drug transport depends on the physicochemical properties of the compound being transported such as lipophilicity, size, pKa, hydrogen bond potential, affinity for transport proteins (166). Figure 1-3. Possible transport routes a drug can follow to cross the intestinal epithelium: (1) passive transcellular transport, (2) passive paracellular transport, (3) active and/or carrier mediated transport, (4) transcytosis. In addition, drug transport can be modulated by active efflux carrier mechanisms such as P-gp (5) (167) Passive Transcellular Diffusion The majority of drugs given orally reaches the systemic circulation by passive transcellular diffusion (164). This process consists on permeation of the apical membrane of the enterocytes, movement across the cytoplasma, and permeation of the lateral or basolateral membranes (87). Since lipid barriers need to be crossed, passive diffusion of drugs through the small intestine depends to a large degree on their lipophilicity. The diffusion process can be mathematically described by the Ficks first law (equation 1-1):

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33 )11.eq()(..21 CCAPJ where J is the flux or mass of transport over time, P is the permeability coefficient, A is the surface area of the absorptive membrane, and (C 1 C 2 ) is the concentration gradient of the uncharged from of the solute over the membrane. Assuming that the drug does not accumulate in any region at the basolateral membrane of the enterocytes because the blood and lymph flow provide a sink condition, the C 2 becomes insignificant and the flux can be expressed just in term of the apical concentration, C 1 Therefore, the driving force of diffusion is mainly controlled by the concentration of drug remaining to be transported from one to the other side of the enterocytes (87). The permeability coefficient across the membrane, defined as the velocity of the drug transport (cm/sec) into the body (127), can be defined by the equation 2. )21.eq(.mDKP where K is the partition coefficient between the water and the membrane, D m is the membrane diffusion coefficient and is the thickness of the of the membrane (162). Transport of ionized compounds rely on some the physical-chemistry properties such as the pKa of the drug molecule and pH of the absorption site, since these parameters will define the equilibrium of the charge-state of the compound (162). In general, these principles are followed by many drugs with the exception of highly branched molecules, which are less permeable than expected due stereochemistry issue since branched molecules results in greater disturbance in the arrangement of the lipid layer (87).

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34 Paracellular Transport This transport route takes place via the tight-junctions between the enterocytes and works as a semipermeable barrier, which protects against xenobiotics and controls the passage of ions, water and small hydrophilic molecules (168). The apically located tight-junctons restrict the paracellular transport of molecules that are larger than a certain threshold size (molecular weight 350 Da) (164). Additionally, the surface area for the paracellular transport has been estimate to be about 1000-fold smaller than the total intestinal surface, suggesting a low efficiency of this pathway on drug transport (165). Such characteristic has stimulated investigations to enhance the permeability of this route and, therefore, increase the bioavailability of very hydrophilic drugs (165, 166, 169). The tight-junctions are also a fence separating lipids and proteins of the apical and basolateral domains of the enterocytes (168). The tight-junctions become progressively tighter and the surface area decreases from the small intestine to the colon, which can explain why the permeability of hydrophilic compounds decrease in the same direction (170, 171). Experiments using Caco-2 cells demonstrated that paracellular route can contribute significantly to the absorption of many drugs including: atenolol, pindolol, cimetidine, metformin, didanosine, and acyclovir (172). These molecules have a moderate molecular weight and are relative hydrophilic. Carrier-Mediated Transport Generally, carrier-mediated transport processes consist of a reversible interaction between the solute and a membrane protein, which acts as a transporter. Such transport has unique characteristics such as specificity, competition, saturation kinetics, and energy requirement (87). Transporters that use the energy of ATP hydrolysis to maintain a

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35 gradient across the membrane are named primary active transporters, whereas, secondary active transporters utilize the ion gradient across the membrane generated by the primary active carriers to transport their substrates. These carriers can be responsible for the uptake (influx transporters) or secretion (efflux transporter) of many drug molecules across the intestinal epithelium and in both cases it may result in non-linear pharmacokinetics and dose-dependent absorption. The permeability-glycoprotein is a primary active efflux transporter, whereas the organic anion transport peptide (OATP) is an example of secondary active influx carrier. The relevance of a carrier-mediated transport for drug absorption depends on the selectivity, capacity, direction, distribution, and expression of the transporters along the intestine (127). Transcytosis Transcytosis is a specialized pathway where particles and macromolecules are entrapped in vesicles due to invagination of the apical membrane before to be released into the cytoplasma (87). Characteristics of this route include a very low capacity of transport and the large amount of proteolytic enzymes inside the vesicles, which can hydrolyze many of the solutes. The transport of vitamin B12 is the best example of transport mediated by transcytosis (165). In Vitro Methods for Studying Drug Absorption Intestinal drug permeability is considered to be one of the two major barriers to intestinal drug absorption, solubility being the other. For assessment of the mechanistic aspects of intestinal drug permeability, several in vitro systems have been developed. Advantages of using in vitro models include small sample volumes, low variability between replicates, the fact that many external factors that influence transport can be

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36 manipulated easily, and, in general, the outputs obtained by the in vitro assessment can be extrapolated to the in vivo scenario (165, 173, 174). Epithelial cell culture models such as Caco-2 are commonly used (165). This human colon adenocarcinoma cell line undergoes enterocytic differentiation in culture allowing studies not only of passive diffusion processes but also, active drug uptake, efflux carrier systems and presystemic drug metabolism since they are able to naturally express transporters like P-gp, sugar uptake transporters, multi drug resistance protein, and metabolizing enzymes such as CYP3A4, N-acetyl transferases, and glutathione transferase (89, 163, 167, 173, 175-177). The ability of the cells to form and maintain a confluent monolayer, with characteristics similar to the in vivo barrier being modeled, on some type of semipermeable support is a prerequisite for transport study. Despite of the good versatility some potential weakness of the Caco-2 cell culture model should be considered. For instance, the Caco-2 form very tight monolayers compared to the human small intestine, which has been explained by the colonic origin of the cell line (165, 173). Also, a unexpectedly large percentage of the compounds are found to be substrates for P-gp, whereas the in vivo relevance of these findings remains unclear, probably because the higher P-gp expression observed in this cell line compared to the small intestine (163, 165). The Madin Darby canine (MDCK) and porcine (LLC-PK1) kidney epithelial cell lines consist of fast and simple additional in vitro models mostly used for screening tests and measurement of passive diffusion, since they do not express transporters under the normal conditions. They also can be used to study the effect of one single transport system or the interplay of efflux and influx carriers, having a untransfected wild-type cell

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37 line as control (62, 71, 76, 178). In contrast to Caco-2 cells, they do not need to be kept 3 weeks in culture before use. Limitations include the fact that MDCK and LLC-PK1 cells are non-intestinal and non-human cell models (163, 179). Excised segments of human or rat intestines properly positioned in an Ussing chamber and the everted gut sac of rat small intestine placed in a oxygenated tissue culture media are sensitive detection methods to study mechanisms of drug absorption at different sites in the intestine, to evaluate intestinal metabolism of xenobiotics, to quantify the paracellular transport of hydrophilic molecules, and to estimate the effects of potent enhancers on their absorption (171, 180-183). Potential disadvantages of these approaches are the presence of muscularis mucosa which may lead to underestimation of compounds with propensity to bind to muscle cells, short duration of the experiments due to limited cell viability, and the availability of human tissue is scarce (163). Therefore, distinct in vitro techniques are accessible for the prediction of the relevant parameters of drug absorption and their advantages, applicability, and limitations should be carefully considered. Talinolol: Model Compound for Drugs Subjected to Intestinal Efflux Talinolol, a highly selective 1-adrenoceptor antagonist, was introduced into clinical practice in 1975 in the former German Democratic Republic by AWD-Pharma GmbH & Co. KG (Dresden, Germany) under the trade mark Cordanum. It is used in the therapy of arterial hypertension, angina pectoris, ischemic heart disease, and rhythmic disturbances of the heart (184, 185). Studies in vitro (186, 187) and in vivo (188-190) have demonstrated that talinolol is a substrate for the P-glycoprotein efflux system. Additionally, this drug is subjected to

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38 minor metabolism in humans, dogs, and rats with less than 1% of the administered dose being excreted into urine in form of hydroxylated metabolites (124, 191-193). Due the remarkable overlaps in tissue distribution, substrates, inhibitors, and inducers specificity between CYP3A4 and P-gp (128), talinolol is considered a good probe for mechanistic studies involving interactions of drugs or food constituents, like grapefruit juice, with P-glycoprotein. The intestinal efflux of talinolol can be estimated without an interference of any biotransformation processes, and the variability of its oral pharmacokinetics should reflect the variability of the P-gp function (193). Additionally, talinolol has a moderate passive membrane permeability, which prevents rapid passive absorption of the drug (187). Properties of Talinolol The chemical structure of talinolol ((1-(4-cyclohexylureidophenoxy)-2-hydroxy-3-tertbutylaminopropane) (molecular weight = 363.5 Da) and its pharmacokinetics parameters after oral administration are presented on Figure 1-4 and Table 1-1, respectively. N N O O N OH HHH Figure 1-4. Molecular structure of talinolol. (*) chiral center. The basicity of the weak base talinolol (pKa = 9.4) refers to a secondary amino functional group located close to the chiral center. Since the nitrogen of this amino group can be protonated, talinolol features a pH-dependent solubility, which decreases with increasing pH values (162). The log P of 3.2 indicates a moderate lipophilicity, compared to other common -adrenoceptor antagonists. It is less lipophilic than propranolol (log P: 4.6) and more lipophilic than atenolol (log P: 0.2) (194). Solubility in water (pH = 7.0)

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39 have been reported to be 0.2 mg/mL and recommended daily doses range from 50 to 300 mg, in healthy volunteers safety of the drug was proven up to single doses of 400 mg (186, 192). Table 1-1. Pharmacokinetics parameters of talinolol after oral administration (50 mg) by health volunteers (192). Parameters Values SD AUC 0-36 1180 337 ng.h/mL Vd ss 3.3 0.5 L/kg Cl tot 11.9 2.4 mL/min.kg C max 168 47 ng/mL T max 3.2 0.8 h Elimination half life (t 1/2 ) 11.9 2.4 h Biotransformation < 1% Renal Excretion 60 % of the bioavailable dose Extrarenal excretion 40% of the bioavailable dose Bioavailability 55 22%; dose dependent Vd ss was calculated after a constant rate intravenous infusion of 30 mg of talinolol over 30 min Hypothesis and Objectives Grapefruit juice has been demonstrated to inhibit the metabolizing enzyme CYP3A4 in the small intestine via suicide inhibition mechanism. As the result of such inhibition, the oral bioavailability of many drugs of different therapeutic classes is increased and in some cases dose dependent side effects are observed.

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40 Because CYP3A4 and P-glycoprotein share many substrates, inhibitors, and inducers, ingestion of GFJ is likely to modify the disposition of drugs transported by this efflux system. However, the influence of different GFJ compounds on P-gp is still unclear and is a controversial issue particularly, regarding the in vivo relevance of this potential interaction. Therefore, we will test the hypothesis that grapefruit juice and its constituents change significantly the disposition of talinolol, a substrate of P-glycoprotein. Concentrations of potential compounds able to interact with P-gp will be determined in different brands and lots of grapefruit juice in order to assess the potential influence of the variability of these juice components on the variability of data gained in drug interaction studies. Additionally, experiments using Caco-2 cells and talinolol as a probe compound will be performed with the purpose of study the mechanistic aspects of the interaction of grapefruit juice and its components with P-gp. Furthermore, the effect of GFJ and some of its constituents on the pharmacokinetics of talinolol in male Sprague-Dawley will be evaluated and the data obtained from both in vitro and in vivo experiments will be correlated. We expected that this study will aid to clarify the predominant mechanisms and the major compounds contributing to the overall effect of grapefruit juice on drug disposition. Therefore, to test the hypothesis of this study the following specific aims were proposed:

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41 Specific Aim 1: Contents of Flavonoids and Furanocoumarins in Grapefruit and Grapefruit Juice 1. To measure selectively, using validated and sensitive HPLC methods, the contents of the specific flavonoids (naringin and naringenin) and furanocoumarins (bergamottin and 6,7-dihydroxybergamottin) in commercially available and fresh-squeezed GFJ as well as in different tissues of grapefruit. Specific Aim 2: In Vitro Studies 1. To determine the permeability of talinolol across Caco-2 cell monolayer cultures in the absence and in the presence of different concentrations of grapefruit juice and several of its components. 2. To estimate the kinetic parameters Km and P max of P-glycoprotein regarding the transport of talinolol. 3. To calculate concentrations of grapefruit juice and its components able to produce a half maximum inhibition (IC 50 ) of the P-gp-mediated transport of talinolol across the Caco-2 cell monolayers. Specific Aim 3: In Vivo Studies 1. To evaluate the effect of grapefruit juice and its components on the pharmacokinetics of talinolol in male Sprague-Dawley rats.

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CHAPTER 2 VARIATION OF FLAVONOIDS AND FURANOCOUMARINS IN GRAPEFRUIT JUICES: A POTENCIAL SOURCE OF VARIABILITY IN GRAPEFRUIT JUICE-DRUG INTERACTION STUDIES Background The amount of active ingredients of grapefruit juice ingested may be an important factor influencing the mechanism, magnitude, and reproducibility of the grapefruit-drug interaction (48), compromising the comparison of data gained in different clinical studies. A clinical trial with healthy subjects has revealed that the AUC and the C max of felodipine were increased by about 73 and 138% compared with water, respectively, when a single oral dose (10 mg) of the drug was administrated with 200 mL of GFJ (prepared by diluting 50 mL of frozen concentrate with 150 mL of tap water) (102). However, in a similar study co-administration of the same oral dose of felodipine and 250 mL of a commercial GFJ increased the AUC and C max values by 305 and 228%, respectively (68). Although the variability in magnitude of the grapefruit juice-drug interaction might be at least partially explained by inherent differences in enterocyte CYP3A4 content (68, 97), it is also possible that more concentrated or larger volumes of grapefruit juice caused a more pronounced interaction. Furthermore, differences in the concentrations of drug-interacting compounds in the juices used in both studies may have contributed to the discrepancy of results. This points out the potential role of the concentration of components causing drug interactions in juices used in clinical trials. 42

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43 Specific Aim The objective of this study was to compare the contents of the specific flavonoids (naringin and naringenin) and furanocoumarins (bergamottin and 6,7-dihydroxybergamottin) (Figure 1-1) in commercially available and fresh-squeezed GFJ as well as in different tissues of grapefruit in order to assess the potential influence of the variability of these juice components on the variability of data gained in drug interaction studies. These compounds have been already described as the most abundant present in GFJ and play a role in the GFJ-drug interaction (98, 195, 196). Material and Methods Chemicals Naringin (NAR) and naringenin (NAG), both > 95% pure, were from Roth GmbH & Co. (Karlsruhe, Germany), bergamottin (BG) (> 98% purity) was bought from Indofine Chemical Company, Inc. (Somerville, NJ, USA), 6,7-dihydroxybergamottin (DHB), was kindly supplied by Dr. John Manthey at the U.S. Department of Agriculture (USDA), Citrus and Subtropical Products Laboratory, Agricultural Research Service, Winter Haven, FL. Upon isolation, the compound identification and purity (> 98%) was measured by analytical thin layer chromatography, HPLC-MS and melting point comparisons with authentic standard at the USDA laboratories. Ethyl acetate was bought from Sigma Chemical Company (St. Louis, MO, USA), methanol HPLC grade, dimethylsulfoxide, and orthophosphoric acid were acquired from Fisher Scientific (Fair Lawn, NJ, USA) and purified water was obtained using a NANOPure system from Barnstead (Dubuque, IA, USA).

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44 Sample Preparation Twenty-nine commercially available GFJ samples and two types of fresh grapefruit (white and red) were purchased at local supermarkets. Concentrations of flavonoids (naringin and naringenin) and furanocoumarins (bergamottin and 6,7-dihydroxybergamottin) in fresh grapefruit were determined in the flavedo layer (0.1 g), pulp (0.1 g), albedo (1.0 g), seeds (1.0 g), and fresh-squeezed juice. Fruit tissues were first homogenized with 6 mL (flavedo and pulp) or 12 mL (albedo and seeds) of purified water using a PowerGen homogenizer (Fisher Scientific, Pittsburgh, PA) prior to the extraction step. The freshly squeezed juices were obtained by using an HJ 29 handy juicer (Black & Decker, Towson, MD). Samples were analyzed by a Shimadzu VP series HPLC system (Kyoto, Japan) equipped with an SPD-M10Avp diode array detector, an LC-10ATvp solvent delivery unit, an SIL-10AF autosampler, a CTO-10Avp column oven, an SCL-10Avp system controller, a DGU-14A on-line degasser, an FCV-10ALvp low-pressure gradient unit, and Class VP 7.2 SP1 chromatographic software. Additionally, the peak purity software (Class VP 7.2 SP1 chromatographic software, Shimadzu) was applied to the diode array data to test for impurities in all of the chromatographic peaks of interest. The same software was used to determine the similarity of the ultraviolet (UV) spectra of the target compounds present in the grapefruit with those from the respective standard compounds. Determination of Flavonoids (Naringin and Naringenin) The GFJ and the homogenate of tissues (200 L) were mixed with cold methanol (400 L), vortexed for 1 min, and centrifuged at 2500 g for 15 min, as previously described (197). After filtration through a 0.45 m PVDF membrane filter (Millipore

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45 Corp., Bedford, MA), the supernatant (25 L) was injected and analyzed at 285 nm. The flow rate and the temperature were set to 0.5 mL/min and 35 C, respectively. Mobile phases A and B consisted of water (pH 2.4) (adjusted with orthophosphoric acid) and water (pH 2.4) (adjusted with orthophosphoric acid)/ methanol (40:60), respectively. The 250 x 4.6 mm i.d., 5 m, Lichrospher RP-18 column and Lichrospher 100 RP-18 guard column (Merck KGaA, Darmstadt, Germany) were initially equilibrated during 30 min with solvent A. After sample injection, an initial isocratic run for 5 min was followed by a linear gradient from 100% of A at 5 min to 100% of B at 55 min. This condition was maintained until 70 min and then returned to 100% of A, which was kept constant during 5 min before proceeding to the next injection. Extraction and Determination of Furanocoumarins (Bergamottin and 6,7-Dihydroxybergamottin) GFJ and the homogenate of tissues (3 mL) were mixed with ethyl acetate (2 mL). The extraction was performed by shaking the mixtures four times over 30 min. The mixture was centrifuged at 3200 g for 20 min; the organic phase was collected and evaporated under vacuum. The residue was reconstituted with 600 L of a DMSO/methanol solution (1:3 v/v). The reconstituted residues were filtered through a 0.45 m PVDF membrane filter (Millipore Corp.). Volumes of 25 L of each sample were injected and analyzed at 310 nm. The flow rate and the temperature were set to 1 mL/min and 35 C, respectively. Solvents A and B consisted of water and methanol, respectively. The column and guard column (as used for flavonoids) were initially equilibrated with mobile phase consisting of a mixture of solvents A and B (45:55), respectively. Twenty minutes after injection, solvent B was increased linearly from 55 to 100% in 20 min. This condition was maintained for 5 min, after which the system

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46 returned to the original mobile phase and was equilibrated for a further 5 min before the next injection. Stock and Work Solutions for the Preparation of Calibration Standards All stock solutions were prepared in dimethylsulfoxide (DMSO) in order to obtain a final concentration of 50 mmol/L. The stock solutions were kept at -20 C and used within 4 weeks. Naringin stock solution The amount of 58.5 mg of naringin (MW = 580.53 g/mol) was accurately weighed, and transferred quantitatively to a 2.0 mL volumetric flask. The standard was then dissolved in DMSO, the volume was completed with the same solvent, and the final solution mixed thoroughly. Naringenin stock solution The amount of 27.2 mg of naringenin (MW = 272.26 g/mol) was accurately weighed and transferred quantitatively to a 2.0 mL volumetric flask. The standard was then dissolved in DMSO, the volume was completed with the same solvent, and the final solution mixed thoroughly. Bergamottin stock solution The amount of 33.8 mg of bergamottin (MW = 338.42 g/mol) was accurately weighed and transferred quantitatively to a 2.0 mL volumetric flask. The standard was then dissolved in DMSO, the volume was completed with the same solvent, and the final solution mixed thoroughly. 6,7-Dihydroxybergamottin stock solution The amount of 37.2 mg of 6,7-dihydroxybergamottin (MW = 372.42 g/mol) was accurately weighed and transferred quantitatively to a 2.0 mL volumetric flask. The

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47 standard was then dissolved in DMSO, the volume was completed with the same solvent, and the final solution mixed thoroughly. Naringin work solution Volume of 1 mL from the naringin (NAR) stock solution was accurately transferred to a 5 mL volumetric flask, the volume was completed with a solution of methanol/water (1:1) and mixed thoroughly. The final concentration of NAR was 10 mmol/L. Naringenin work solution Volume of 100 L from the naringenin (NAG) stock solution was accurately transferred to a 5 mL volumetric flask, the volume was completed with a solution of methanol/water (1:1) and mixed thoroughly. The final concentration of NAG was 1 mmol/L. Furanocoumarins work solution Volumes of 250 L and 100 L from the bergamottin (BG) and 6,7-dihydroxybergamottin (DHB) stock solutions, respectively, were accurately transferred to a 5 mL volumetric flask, the volume was completed with a solution of DMSO/methanol (1:3) and mixed thoroughly. The final concentrations of BG and DHB were 2.5 mmol/L and 1 mmol/L, respectively. Standard solutions of flavonoids From the NAR and NAG work solutions, five different concentrations of standard solutions of naringin and naringenin and three quality controls (QC) were prepared in methanol/water (1:1) according Table 2-1. All solutions were filtered through a 0.45 m PVDF membrane filter (Millipore Corp.) before analysis.

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48 Table 2-1. Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of naringin (NAR) and naringenin (NAG). Concentration (mol/L) Standard NAR work solution (L) NAG work solution (L) methanol/water (1:1) q.s.p. (mL) NAR NAG 1 50 100 10 50 10 2 150 200 10 150 20 3 250 300 10 250 30 4 350 400 10 350 40 5 450 500 10 450 50 QC1 50 150 10 50 15 QC2 250 250 10 250 25 QC3 450 450 10 450 45 Standard solutions of furanocoumarins From the furanocoumarins work solution, six different concentrations of bergamottin and 6,7-dihydroxybergamottin and three QCs were prepared in DMSO/methanol (1:3) according Table 2-2. All solutions were filtered through a 0.45 m PVDF membrane filter (Millipore Corp.) before analysis. Table 2-2. Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of bergamottin (BG) and 6,7-dihydroxybergamottin (DHB). Concentration (mol/L) Standard Furanocoumarins work solution(L) DMSO/methanol (1:3) q.s.p. (mL) BG DHB 1 10 10 2.5 1 2 25 10 6.25 2.5 3 100 10 25 10 4 250 10 62.5 25 5 500 10 125 50 6 1000 10 250 100 QC1 50 10 12.5 5 QC2 125 10 31.25 12.5 QC3 750 10 187.5 75

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49 Validation An external standardization method was validated over the range of concentration of the target compounds present in the grapefruit juice and tissues. The validation parameters of linearity, sensitivity, specificity, precision, accuracy, recovery and stability were determined. For each level of concentration, standards solutions and QCs were injected in the HPLC in triplicate in three different days. GraphPad Prism version 4.0 (GraphPad Software Inc, San Diego, CA, USA) was used to construct the calibration curves obtained by plotting the mean area versus the corresponding concentration of the each standard solution. The linearity of the standard curves was determined by least-squares linear regression method and expressed in terms of coefficient of determination (r 2 ). The intraand inter-day precision and accuracy of the quantification were measured by replicate analyses of three different concentration levels (low, medium and high QCs) on the same day and on alternate days. The precision was based on the calculation coefficient of variation (CV %), and the accuracy was expressed as percent of the found amount compared to the theoretical one. The calibration was considered suitable if not more than 1/3 of the quality controls showed a deviation from the theoretical values equal or greater than 20% at the lower limit of quantification (LLOQ) and within 15% at all other calibration levels. The limit of detection (LOD) and LLOQ of NAR, NAG, BG, and DHB were expressed on the basis of the mean values of the intercept (Y bl ), the standard deviation (S bl ) of the blank responses, and the slope (b) of the original calibration curve (equations 2-1 and 2-2) (198). )22.eq(3 bSYLOQblbl )12.eq(10bSYLODblbl

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50 Briefly, a second standard curve for each grapefruit juice component was obtained, injecting each point in triplicate, but at lower concentrations than those of the used for quantification. The Y bl values represent the intercepts of these new curves, corresponding to the output as the concentrations of the analytes are equal to zero. Similarly, the S bl values were determined after plotting the standard deviation versus the correspondent concentration level. The recovery of each compound was evaluated by adding known amounts of pure compounds to orange juice, from which they are naturally absent (199), and extracting them under the same conditions used in the grapefruit samples. The analytical results for extracted samples at three concentration levels were compared with unextracted standards that represent 100% recovery. To assess the flavonoids and furanocoumarins stability at room temperature, three QCs (low, medium, and high) were prepared using fresh stock solutions and analyzed at 0 and 36 hours, as previously described. The stock solutions were then frozen at -20 C and freeze/thaw cycles were carried out once week up to 4 cycles. After each cycle, solutions of flavonoids and furanocoumarins corresponding to the medium QC level were prepared and analyzed by the validate HPLC method mentioned formerly. The areas were compared to those obtained from fresh solutions for each concentration level and results expressed as % of the control. Three replicates of each concentration level were prepared for each test performed. Statistical Analysis Validation parameters were expressed as mean of three to nine determinations SD. The concentration of each compound in the juice and tissues was expressed as the

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51 mean of the three determinations SEM. Asterisks indicate significant differences for white grapefruit compared to red grapefruit (p < 0.05). Data were analyzed by one-way analysis of variance (ANOVA) with JMP5 software (SAS Institute Inc., Cary, NC,1996). Mean separation was conducted using the Tukey-Cramer HSD comparison for all pairs (p < 0.05). Values with different letters are significantly different (p < 0.05). Results and Discussion Linearity Calibration curves (n = 9) for all four grapefruit juice constituents were linear within the tested range of concentration with r 2 > 0.999 (Figure 2-1), indicating that both methods showed good linearity. 0 100 200 300 400 500 0 5.0106 1.0107 1.5107 2.0107 2.5107Ar2 = 0.9998Naringin (mol/L)Area 0 10 20 30 40 50 60 0 2.5105 5.0105 7.5105 1.0106 1.3106Br2 = 0.9997Naringenin (mol/L)Area 0 60 120 180 240 300 0 1.0106 2.0106 3.0106 4.0106 5.0106 6.0106Cr2 = 0.9991Bergamottin (mol/L)Area 0 25 50 75 100 125 0 5.0105 1.0106 1.5106 2.0106 2.5106Dr2 = 0.99946',7'-Dihydroxybergamottin (mol/L)Area Figure 2-1. Mean calibration curves (n = 9) of naringin (A) and naringenin (B) in methanol/water (1:1), and of bergamottin (C) and 6,7-dihydroxybergamottin (D) in DMSO/methanol (1:3). Vertical bars represent the standard deviations (SD) of the means.

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52 Sensitivity The Y bl values for the blank responses of NAR, NAG, BG, and DHB were 6170.9, 7462.9, 271.1, and 671.6, respectively. In the same way, the S bl values were 5026, 1790, 6.2, 30.7. Therefore, the LODs values for naringin, naringenin, bergamottin, and 6,7-dihydroxybergamottin were 0.47, 0.56, 0.01, and 0.04 mol/L, respectively, whereas the LLOQs were 1.25, 1.11, 0.02, and 0.05 mol/L. Specificity The methods provided good resolutions between naringin and naringenin as well as between bergamottin and 6,7-dihydroxybergamottin. Peaks of the flavonoids and furanocoumarins had similar retention times and the UV spectra (200-400 nm) in all samples when compared to the standards. The wavelengths 285 and 310 nm used to quantify flavonoids and furanocoumarins at their maximum absorption, respectively, were confirmed by their UV spectra (Figure 2-2). There was no endogenous interference from GFJ or orange juice (Figure 2-3) in either assay, indicating specificity of both methods to the tested compounds. The results of the analysis of the peak purity software suggested the absence of impurities for all compounds (purity > 95% for each individual peak). Additionally, the UV spectra of all tested compounds showed more than 99% of similarity with those obtained using the respective standard compounds (Figure 2-4). Precision and Accuracy The precisions intraand inter-day for both flavonoids and furanocoumarins were satisfactory with CV values between 0.55 and 6%. Similarly, the accuracy of the assay was between 93 and 114.8% for all compounds tested at three different concentrations. The results are summarized in Table 2-3.

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53 Figure 2-2. HPLC separation and absorbance-wavelength spectra of A furanocoumarins: 6,7-dihydroxybergamottin and bergamottin; B flavonoids: naringin and naringenin.

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54 Figure 2-3. Chromatograms corresponding to the orange juice (OJ), standard solutions (Std) containing 6,7-dihydroxybergamottin (DHB) plus bergamottin (BG) (panel A), and naringin (NAR) plus naringenin (NAG) (panel B). Figure 2-4. Absorbance-wavelength spectra of A bergamottin; B 6,7-dihydroxybergamottin; C naringin. (1) represents the spectra of the standard compound and (2) represents the spectra of the peak with same retention time of the corresponding standard but obtained after injection of the grapefruit sample. 2 1 2 1 2

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Table 2-3. Intra-day (n = 3) and inter-day (n = 9) assay parameters of furanocoumarins (bergamottin and 6,7-dihydroxybergamottin) and flavonoids (naringin and naringenin). Precision expressed as CV %and accuracy as % of the theoretical concentration. Bergamottin QC1-12.5 mol/L QC231.25 mol/L QC3187.5 mol/L Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 0.7 0.6 3.8 1.0 1.8 0.9 1.1 2.3 0.7 Accuracy 107.4 109.4 108.4 105.0 108.4 106.2 104.4 108.0 103.4 Inter-day QC1-12.5 mol/L QC231.25 mol/L QC3 187.5 mol/L Precision 2.4 1.3 1.5 Accuracy 108.6 106.5 105.3 6,7-Dihydroxybergamottin QC1-5 mol/L QC212.5 mol/L QC375 mol/L Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 1.5 1.2 1.9 1.2 4.9 1.7 5.2 0.8 0.9 Accuracy 110.3 114.8 114.2 106.0 107.0 99.0 94.6 107.4 105.0 Inter-day QC1-5 mol/L QC212.5 mol/L QC375 mol/L Precision 2.9 4.1 4.03 Accuracy 113.1 104.0 102.3 Naringin QC1-50 mol/L QC2250 mol/L QC3450 mol/L Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 4.3 0.6 0.8 0.6 1.2 1.5 3.2 2.1 1.6 Accuracy 98.2 95.1 93.0 104.7 106.0 100.9 98.3 98.3 100.8 Inter-day QC1-50 mol/L QC2250 mol/L QC3450 mol/L Precision 6.1 2.0 5.0 Accuracy 95.4 103.9 99.1 55

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Table 2-3 Continued Naringenin QC1-15 mol/L QC225 mol/L QC345 mol/L Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 2.4 0.8 3.6 2.3 1.2 0.7 1.3 0.6 5.9 Accuracy 100.9 96.6 96.6 106.1 103.1 96.2 100.8 99.5 99.8 Inter-day QC1-45 mol/L QC225 mol/L QC315 mol/L Precision 3.0 5.8 4.5 Accuracy 98.1 101.8 102.7 56

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57 Recovery The recoveries from orange juice spiked samples were between 97.7 and 106% (Table 2-4). Concentrations were back calculated using the equation obtained from linear regression of the standard curves. Table 2-4. Recovery values (means SD) of bergamottin, 6,7-dihydroxybergamottin, naringin, and naringenin from the spiked orange juice. Values in the parenthesis represent the mean % recovered from each added concentration. GFJ components added to the orange juice Theoretical Concentration (mol/L) Mean measured Concentration (n = 3) 6.25 6.1 0.3 (97.7) Bergamottin 25 24.8 1.7 (99.2) 150 156.6 12.4 (102.0) 5 5.2 0.1 (103.7) 6,7-Dihydroxybergamottin 25 24.9 0.8 (99.8) 100 101.3 2.8 (101.3) 50 52.2 3.3 (103.4) Naringin 250 265.9 25.6 (106.2) 450 475.5 32.3 (105.9) 15 15.4 0.4 (102.7) Naringenin 25 25.6 1.5 (102.5) 45 44.2 1.2 (98.4) Stability The standard solutions of flavonoid and furanocoumarins were found stable at room temperature within 36 hours, whereas the stock solutions showed adequate stability during at least 1 month after freeze/thaw cycles at -20 C. In both cases, the shifting of the areas of each sample tested was less than 5% of those obtained from a fresh solution at the same level of concentration (Figure 2-5).

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58 A B 1 2 3 4 0 25 50 75 100 125BG (31.25mol/L) DHB(12.5mol/L) NAR(25mol/L) NAG(25mol/L) Cycles% Control BGDHBNARNAG 0 25 50 75 100 125Low QC Medium QC High QC % Control Figure 2-5. Results of the stability test after 36 hours at room temperature (A) and after 4 freeze/thaw cycles at -20 C (B). Data represents means of three replicates and vertical bars the standard deviation of the means. Quantification of Naringin, Naringenin, Bergamottin, and 6,7-Dihydroxybergamottin in Grapefruit Juices. The values found for all compounds (Table 2-5) are in the same general range as those reported in previous publications except for naringenin, which was absent in all samples tested. In contrast to a previous study in which concentrations of naringenin were found from 19.5 to 595 mol/L in 8 different brands of GFJ (196), we did not detect this flavonoid in any of the 14 brands of juices analyzed, which was also in agreement with

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59 other authors (41). However, a great variability of naringin (from 174 to 1492 mol/L), bergamottin (from 1.0 to 36.6 mol/L), and 6,7-dihydroxybergamottin (from 0.22 to 52.5 mol/L) contents was observed among all tested brands of GFJ. Although Bronner and Beecher (195) found similar concentrations of naringin (850 mol/L) in two different brands of GFJ (Giant and Minute Maid), a large variability of naringin, bergamottin, and 6,7-dihydroxybergamottin in GFJ has also been confirmed by different authors with ranges of 218-2062 mol/L (196), 2.0-28.3 mol/L (71), and 9.1-42 mol/L (71), respectively. In the fresh-squeezed GFJ the highest concentration of naringin (312.6 mol/L) was measured in the white variety, whereas the red fruit provided the highest contents of bergamottin (3.1 mol/L) and 6,7-dihydroxybergamottin (10.3 mol/L). In the pink GFJ (A-C) the concentrations of 6,7-dihydroxybergamottin were not statistically different intraand inter-lot (p > 0.05) and brand B showed a higher content of naringin when compared with the other two brands, although the variability of this flavonoid between the lots was not significant. Additionally, the content of bergamottin in lot 2 of brand C was 2.4-fold higher than in lot 1. Intra-lot comparisons revealed that the content of naringin was significantly different (p < 0.05) in all brands of white GFJ used in this study. The white brands D and E showed similar concentrations of bergamottin within lots. In red juices only brand H showed significant intra-lot variability of naringin and 6,7-dihydroxybergamottin. The concentrations of bergamottin and 6,7-dihydroxybergamottin in brand E (frozen from concentrate) were significantly higher when compared to all other juices. Although there is a lack of detailed information regarding the stability of furanocoumarins (bergamottin and 6,7-dihydroxybergamottin) present in GFJ, brand E is sold as a frozen product, which may possibly have prevented

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60 extensive degradation of furanocoumarins compared to juices stored at room temperature. In general, the white GFJ showed the highest levels of naringin, bergamottin, and 6,7-dihydroxybergamottin with mean concentrations of 1010 87, 24.5 2.3, and 14.5 6.7 mol/L, respectively. Additionally, the red variety showed higher amounts of bergamottin (9.5 1.8 mol/L) and 6,7-dihydroxybergamottin (5.6 1.9 mol/L) compared to the pink variety, although the mean concentration of naringin was 1.7fold lower. Localization of Naringin, Naringenin, Bergamottin, and 6,7-Dihydroxybergamottin in Grapefruit. A comparison of concentrations and distribution of the target compounds between fresh white and red grapefruit varieties (Figure 2-6) revealed that extracts from white grapefruit showed higher concentrations of naringin (3152 g/g), bergamottin (19.8 g/g), and 6,7dihydroxybergamottin (106 g/g) located in the albedo and flavedo. The lowest concentrations were found in the seeds and pulp of red grapefruit. These results are in agreement with the values found in the commercial juices where, in general, the white grapefruit juices showed the highest contents of naringin, bergamottin, and 6,7-dihydroxybergamottin compared to the pink or red varieties. A similar study was conducted to determine the content of the dimers of bergamottin and 6,7-dihydroxybergamottin in the white and red grapefruits (200). Bergamottin and 6,7-dihydroxybergamottin were able to inhibit the human microsomal CYP3A-mediated testosterone 6-hydroxylation by half at 22 and 2 mol/L, respectively (98). With regard to their effects on P-gp activity, bergamottin (10 mol/L) and 6,7-dihydroxybergamottin (33 mol/L) have been reported to reduce the activity of P-gp in vitro by 58 and 50%,

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61 respectively (41, 80). In addition, 6,7-dihydroxybergamottin has been shown to be a potent inhibitor of rat OATP3 (IC 50 = 0.28 mol/L) (80). Although the flavonoid naringin has been excluded as the principal CYP3A4 inhibitor in GFJ (41, 75, 201), it promoted a significant inhibition on P-gp and OATP activity in vitro at concentrations of 1000 and 5 mol/L, respectively (80, 202). Naringin has also been shown to be hydrolyzed into the more potent CYP3A4 inhibitor naringenin by the gastrointestinal microflora located preferentially in the distal part of the small intestine and in the colon (203). The clinical relevance of this interaction is uncertain because most of the drug absorption takes place in the small intestine. Overall, the concentrations of naringin, bergamottin, and 6,7-dihydroxybergamottin in the samples analyzed in this study appear to be high enough to considerably decrease CYP3A4, P-gp, and OATP activities. Other GFJ constituents (not tested in this study) have been shown to inhibit the CYP3A4 activity, such as the dimeric compounds (IC 50 < 1 mol/L) (98) and epoxybergamottin (IC 50 = 4.2 mol/L) (94). Additionally, the flavonoids quercetin and kaempferol have been shown to inhibit the organic cation transporters (OCT) by half at 32 and 38 mol/L, respectively (202). In general, the GFJ-drug interactions studies are characterized by a wide variability in the pharmacokinetics and pharmacodynamics data among patients from the same study, as well as between different studies. Variations in the intestinal concentrations of CYP3A4 among individuals seem to contribute to the differences in the intensity of this interaction (94). In addition, it was speculated that the high variability of components in GFJ may also increase the risk of an interaction in patients who drink GFJ habitually and appear to be equilibrated during a drug therapy, if the brand or even the lot is switched during drug therapy with a susceptible drug (43). However, further studies comparing the

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62 effect of different brands on the magnitude of the GFJ-drug interaction are needed to clarify this issue. Additionally, the correlation between the content of the interacting compounds in a GFJ preparation and their inhibitory effects would help to estimate the role of each component on the overall GFJ-mediated inhibition and also to predict and circumvent such interactions (109). Flavedo Albedo Pulp Seeds 0 1500 3000A*Grapefruit tissuesNaringing/g Flavedo Albedo Pulp Seeds 0 10 20B****Grapefruit tissuesBergamotting/g Flavedo Albedo Pulp Seeds 0 50 100C**Grapefruit tissues6'7'-dihydroxybergamotting/g Figure 2-6. Distribution of naringin (A), bergamottin (B) and 6,7-dihydroxybergamottin (C) in different tissues of white (open bars) and ruby red (shaded bar) grapefruits. Values are means (SEM) (n = 3). Asterisks indicate significant differences between values of white compared to red grapefruit.

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63 Table 2-5. Naringin, bergamottin and 6,7-dihydroxybergamottin content in different brands of grapefruit juice sold in Florida. Concentration (mol/L) Grapefruit Juices Variety Lots Naringin Bergamottin 6,7-DihydroxyBergamottin Brand A L1 777.6 (4.3) a h-k b 9.76 (0.5) klm 0.44 (0.01) hij L2 772.3 (10.1) h-k 9.03 (0.2) lmn 0.44 (< 0.01) hij Brand B* L1 878.9 (32.4) gh 8.79 (1.0) lmn 0.65 (0.04) hij L2 939.3 (34.1) fgh 12.13 (0.6) jkl 1.07 (0.02) hij Brand C L1 668.5 (28.1) i-l 5.98 (0.02) mno 0.35 (< 0.01) hij Pink L2 654.6 (6.3) i-l 14.24 (0.1) h-k 0.76 (0.02) hij Brand B* L1 1,262 (42.1) bc 18.81 (1.3) fgh 1.14 (0.1) hij L2 1,492 (44) a 25.86 (1.0) de 2.21 (0.1) hij Brand C L1 820.4 (43.9) ghi 21.95 (0.5) ef 1.03 (0.03) hij L2 589.9 (14) lm 28.04 (0.3) cd 2.29 (0.3) hij Brand D* L1 915.6 (8.8) fgh 15.54 (0.9) g-j 3.37 (0.2) g-j L2 651.8 (15.4) i-l 14.71 (0.7) g-j 1.34(0.2) hij Brand E ** L1 810.9 (58.5) hij 31.77 (1.1) bc 45.04 (1.8) b L2 1,056 (22.6) def 34.37 (1.6) ab 49.01 (2.5) ab L3 1,318 (36.3) b 36.34 (1.6) a 52.51 (2.8) a Brand F L1 1,216 (57) bcd 17.14 (0.4) ghi 0.73 (0.05) hij White L2 980.6 (7.8) efg 25.42 (0.7) de 1.15 (0.03) hij Brand C L1 174.1 (2.8) o 2.95 (0.03) op 0.28 (< 0.01) ij L2 238.9 (3.8) o 5.06 (0.1) nop 0.28 (0.01) ij Brand F L1 234.0 (3.1) o 3.59 (0.3) op 0.22 (< 0.01) j L2 303.6 (12.6) no 4.02 (0.1) op 0.25 (< 0.01) ij Brand D L1 667.5 (15.7) i-l 13.56 (0.2) g-j 14.57 (0.4) c L2 648.4 (14.8) jkl 14.16 (0.20) g-j 17.92 (0.7) c Brand G L1 303.9 (5.1) no 5.17 (0.3) m-p 0.25 (< 0.01) ij L2 291.5 (2.8) o 2.58 (0.2) op 0.24 (0.02) ij Brand H L1 469.4 (18) mn 12.19 (0.5) i-l 4.55 (0.05) j-i L2 1,147 (97) cde 12.73 (0.2) jkl 7.38 (0.02) efg Brand I L1 584.4 (10) lm 19.18 (1.3) fg 8.74 (0.4) def Ruby Red L2 613.4 (18.8) klm 18.92 (0.9) fg 12.10 (0.8) cd White 312.6 (9.5) no 0.97 (< 0.01) qr 4.71 (0.1) fgh Fresh squeezed Ruby Red 245.7 (28.3) o 3.10 (0.1) p 10.28 (0.6) cde a Values are means (n=3) with SEM in parenthesis. b Values with same letter(s) are not significantly different (Tukey-Cramer multiple comparison, p0.05). = from concentrate; ** = frozen from concentrate. Brands: A Blue Bird; B Thirft Maid; C Ocean Spray; D Publix; E Minute Maid; F Win Dixie; G Sams Choice; H Floridas Natural; I Indian River.

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64 Conclusions The analytical methods applied were suitable to quantify the predominant flavonoids and furanocoumarins in the juice and in different tissues of the grapefruit. On the basis of the demonstrated variability and lack of information regarding the profile flavonoids and furanocoumarins of juices used for human trials, a direct comparison between different studies is difficult. Therefore, to minimize prevalent variability in GFJ-drug interaction trials, it is important to correlate measured endpoints to the concentrations of those grapefruit components that are considered to be relevant for the interaction with drugs. In future studies investigating grapefruit-drug interactions, concentrations of compounds with a suspected interaction should be considered. When possible, juices of very similar composition should be used for studies designed for direct comparison. In addition, for animal studies the concentrations of critical compounds may be adjusted by supplementation to normalize the administered juices.

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CHAPTER 3 GRAPEFRUIT JUICE AND ITS COMPONENTS INHIBIT P-GLYCOPROTEIN MEDIATED TRANSPORT OF TALINOLOL IN CACO-2 CELLS Background Due to the striking overlaps in tissue distribution, substrates and inhibitors specificity of CYP3A4 and permeability-glycoprotein (P-gp) (128) it is not surprising that grapefruit juice (GFJ) and its components can also interact with this membrane efflux transporter, which inhibition would be expected to be followed by augment of the oral bioavailability of its substrates. However, modulation of P-gp activity by GFJ and the clinical relevance of such interaction is still unclear and is discussed controversially, since some authors have reported activation (136, 143) and others inhibition of P-gp (61, 71, 144). Recently, the -blocker talinolol, which is a non-CYP substrate, yet a P-gp substrate, showed an increase in bioavailability when associated with GFJ in both in vitro and in vivo models (134). However, a clinical trial had shown an opposite outcome, i.e. reduction of talinolol bioavailability (137) following its co-administration with GFJ in healthy subjects. Many GFJ compounds have been proposed to interact with CYP enzymes and P-gp. These include both flavonoids (eg. naringenin, naringin, quercetin and kaempferol) and nonflavonoids (eg. bergamottin, 6,7-dihydroxybergamottin). However, the influence of different GFJ constituents on P-gp is uncertain, particularly regarding the in vivo relevance of this potential interaction mechanism. 65

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66 Specific Aim It was the aim of this study to systematically investigate in vitro, using a Caco-2 cell model and talinolol as a marker compound, the potential drug interaction between selected ingredients of GFJ (Figure 3-1) alone and in combinationand the P-glycoprotein transporter system, which controls many barriers in the body. Additionally, the concentration dependent transport of talinolol was demonstrated and its kinetics parameters estimated. OOHOHOHO OOHOHO OH O O OH OH OH O O OH OH O O O O O O O O OH OH O O O O O Naringin Naringenin Bergamottin 6',7'-Dihydroxybergamottin 6',7'-Epoxybergamottin Figure 3-1. Structures of selected flavonoids and furanocoumarins present in grapefruit juice and tested in this study. Material and Methods Chemicals The following materials were used: (rac)-verapamil hydrochloride, Hanks balanced salt solution (HBSS), 2-(N-morpholino)ethanesulfonic acid solution (MES); sodium pyruvate solution (1 M), Lucifer yellow dipotassium salt, all of which were from

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67 Sigma Chemical Company (St. Louis, MO, USA); Dulbeccos modified Eagles medium (1X) high glucose without sodium pyruvate (DMEM), nonessential aminoacids, trypsin (0.05%)-EDTA (0.02%) solution, Fungizone antimycotic solution containing 250 g of amphotericin B and 205 g of sodium deoxycholate per mL as solubilizer, penicillin G (10,000 units/mL) and streptomycin sulfate (10,000 g/mL) solution, (N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] buffer solution (1 M) (HEPES) were obtained from Gibco BRL Life Technology (Grand Island, NY, USA); fetal bovine serum heat inactivated and Dulbeccos phosphate buffered saline (DPBS) (pH 7.4) were purchased from Mediatech Inc. (Herdon, VA, USA); talinolol (99.9% purity) was gently provided by AWD-Pharma GmbH & Co. KG (Dresden, Germany), naringin (NAR) and naringenin (NAG), both > 95% pure, were from Roth GmbH & Co. (Karlsruhe, Germany), bergamottin (BG) (98% purity) was bought from Indofine Chemical Company, Inc. (Somerville, NJ, USA), 6,7-dihydroxybergamottin (DHB), and 6,7-epoxybergamottin (EPBG) were kindly supplied by Dr. John Manthey at the U.S. Department of Agriculture (USDA), Citrus and Subtropical Products Laboratory, Agricultural Research Service, Winter Haven, Florida. Upon isolation, the compounds identification and purity (> 98%) were measured by analytical thin layer chromatography, HPLC-MS and melting point comparisons with authentic standards at the USDA laboratories. Triethylammonium phosphate buffer (1 M) was obtained from Fluka (Buchs, Switzerland). All others reagent were analytical grade. Cell Culture Caco-2 cells were obtained from ATCC (Rockville, MD, USA) and maintained as stock cells in T flasks of 150 cm 2 (Corning Costar Corp., Cambridge, MA, USA). The

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68 growing medium consisted of DMEM containing 10% of fetal bovine serum, 1% of nonessential amino acids, 100U/mL penicillin G, 100 g/ml streptomycin, 1.25 g/mL amphotericin B, and 10 mM sodium pyruvate. The atmosphere was kept with 90-95% of relative humidity, 37 C, 5% CO 2 and 95% air. The cells were passaged upon reaching 80-90% confluence using trypsin-EDTA (5mL) and plated at densities of 1:5 1:7 in new T flasks. After reaching passages 20-40 the Caco-2 cells were seeded at density of 60,000 cells/cm 2 onto each 12-mm transparent polyester cell culture insert 12 well plates with area of 1.13 cm 2 and pore size of 0.4 m (Transwell, Corning Costar Corporation, Cambridge, MA, USA). The DMEM medium was added in both apical (AP) and basolateral (BL) compartments and changed 4 days after seeding, and every 2 days thereafter. The cells were used on days 18 to 25 post-seeding to obtain differentiated monolayers and higher expression of P-glycoprotein (173, 174). Transepithelial electrical resistance values (TEER) were monitored with EndOhm Voltohmmeter equipped with a STX-2 chopstick electrode (World Precision Instruments Inc, Sarasota, FL, USA) in order to check cell confluence and integrity. Confluent Caco-2 monolayers with TEER values >350 cm 2 after correction for the resistance obtained in control blanks, were used in the transport experiment. All volumes amounted to 0.5 mL at the apical side and 1.5 mL at the basolateral side. Stock, Work Solutions, and Preparation of Calibration Standards The stock solutions at 50 mmol/L of NAG, BG, and DHB were prepared in dimethylsulfoxide (DMSO) as previously described in Chapter 2. Talinolol stock solution I was prepared in ethanol, verapamil hydrochloride (HCl) in buffer pH 6.0, NAR and EPBG in DMSO.

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69 Talinolol stock solution I The amount of 36.3 mg of talinolol (MW = 363.5 g/mol) was accurately weighed, and transferred quantitatively to a 2.0 mL volumetric flask. The standard was then dissolved in ethanol, the volume was completed with the same solvent, and the final solution mixed thoroughly in order to obtain a final concentration of 50 mmol/L Talinolol stock solution II The amount of 25 mg of talinolol (TAL) was accurately weighed and transferred quantitatively to a 25 mL volumetric flask. The standard was then dissolved in ethanol, the volume was completed with the same solvent, and the final solution mixed thoroughly in order to obtain a final concentration of 1 mg/mL. Talinolol stock solution III Volume of 1 mL from the talinolol stock solution II was accurately transferred to a 100 mL volumetric flask, the volume was completed with a solution of 0.025 mol/L triethylammonium phosphate buffer (pH 3.0 adjusted with 1 M NaOH) and acetonitrile (77:23) and mixed thoroughly. The final concentration of talinolol was 10 g/mL. Talinolol stock solution IV Volume of 1 mL from the talinolol stock solution III was accurately transferred to a 10 mL volumetric flask, the volume was completed with a solution of 0.025 mol/L triethylammonium phosphate buffer (pH 3.0 adjusted with 1 M NaOH) and acetonitrile (77:23) and mixed thoroughly. The final concentration of talinolol was 1000 ng/mL. Verapamil hydrochloride stock solution The amount of 49.1 mg verapamil HCl (MW = 491.05 g/mol) was accurately weighed, and transferred quantitatively to a 2.0 mL volumetric flask. The standard was then dissolved in buffer pH 6.0, the volume was completed with the same solvent, and the

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70 final solution mixed thoroughly. The final concentration of verapamil HCl was 50 mmol/L. Naringin stock solution I The amount of 145.1 mg of naringin (MW = 580.53 g/mol) was accurately weighed, and transferred quantitatively to a 5.0 mL volumetric flask. The standard was then dissolved in 1 mL of DMSO, the volume was completed with HBSS-MES (pH 6.0) and mixed thoroughly in order to obtain a final concentration of 50 mmol/L. 6,7-Epoxybergamottin stock solution The amount of 35.4 mg of 6,7-epoxybergamottin (MW = 354.41 g/mol) was accurately weighed, and transferred quantitatively to a 2.0 mL volumetric flask. The standard was then dissolved in DMSO, the volume was completed with the same solvent and solution mixed thoroughly in order to obtain a final concentration of 50 mmol/L Talinolol work solutions For the inhibitory study, a dosing solution of talinolol (100 M) was prepared in HBSS-MES transport medium (HBSS buffered with 10mM MES and adjusted to pH 6 with 1 M NaOH) or in HBSS-HEPES transport medium (HBSS buffered with 10 mM HEPES and adjusted to pH 7.4 with 1 M NaOH). The concentration-dependent transport of talinolol was studied in the concentration range of 25 to 1000 M (25, 50, 200, 400, 600, 800 and 1000 M). Solutions were prepared according Table 3-1 using the talinolol stock solution I. In all cases the ethanol concentrations of the final solutions did not exceed 2 %. Yamashita et al. (204) already reported that ethanol (< 5 v/v %) did not affect the permeability of dexamethasone and the integrity of Caco-2 cells monolayers, suggesting its usefulness as a solubilizing agent of drugs during in vitro experiments.

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71 Table 3-1. Concentrations of the work solutions of talinolol (TAL) used during the transport studies. Solution TAL stock solution I (L) Buffer pH 6.0 or pH 7.4 q.s.p. (mL) Talinolol (mol/L) 1 5 10 25 2 10 10 50 3 20 10 100 4 50 10 250 5 80 10 400 6 120 10 600 7 160 10 800 8 200 10 1000 Talinolol standard solutions From the talinolol stock solutions III and IV nine different concentrations of standard solutions and three quality controls (QC) were prepared in 0.025 mol/L triethylammonium phosphate buffer (pH 3.0 adjusted with 1 M NaOH) and acetonitrile (77:23) according Table 3-2. All solutions were filtered through a 0.45 m PVDF membrane filter (Millipore Corp.) before analysis. Table 3-2. Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of talinolol. Standard TAL stock solution III (L) TAL stock solution IV (L) Buffer/ACN (77:23) q.s.p. (mL) Talinolol (ng/mL) 1 5 10 5 2 10 10 10 3 25 10 25 4 50 10 50 5 100 10 100 6 250 10 250 7 500 10 500 8 750 10 750 9 1000 10 1000 QC1 5 10 5 QC2 100 10 100 QC3 1000 10 1000

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72 Inhibitory solutions For the inhibitory experiments solutions of verapamil HCl (50, 100, 250, 500, and 1000 M) (positive control) or NAR (100, 250, 500, 625, 1250, 2500 M), NAG (10, 50, 100, 250, 500 M), DHB (0.5, 10, 25, 50, 500 M) and EPBG (0.5, 1, 5, 10, 50 M) were prepared in HBSS-MES in the presence or absence of the marker compound talinolol (100 M), according shown in Table 3-3. BG was tested only in two concentrations (1 and 10 M) due to solubility issues. Similarly, solutions containing a fixed ratio of the flavonoid NAR to the furanocoumarin DHB (50:1.2, 125:3, 625:15, 1250:30, and 2000:48 M) were prepared considering the range these compounds are found in the juice (Table 3-4). The effect of the combination of these major compounds present in the GFJ on the P-gp mediated transport of talinolol was evaluated. The white GFJ frozen concentrated (Minute Maid) was bought in the local market and diluted with HBSS-MES to 0.5, 1, 5, 10, 25 and 50% in the presence or absence of talinolol (100 M). The GFJ solutions were previously passed to a 45 m filter PVDF membrane filter (Millipore Corp., Bedford, MA) before addition of talinolol. Concentrations of NAR, NAG, BG, and DHB in the juice were 811 M, < 0.56 M, 31.8 M, 45 M, respectively, as previously reported (205). The concentration of DMSO in these inhibitory solutions was < 1%.

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73 Table 3-3. Concentrations of the inhibitory solutions of Verapamil hydrochloride (VER), naringin (NAR), naringenin, (NAG), bergamottin (BG), 6,7-dihydroxybergamottin (DHB), and 6,7-epoxybergamottin (EPBG) used during the transport studies. VER VER stock solution (L) Buffer pH 6.0 or pH 7.4 q.s.p. (mL) VER (mol/L) 1 25 25 50 2 50 25 100 3 125 25 250 4 250 25 500 5 500 25 1000 NAR NAR stock solution (L) Buffer pH 6.0 or pH 7.4 q.s.p. (mL) NAR (mol/L) 1 50 25 100 2 125 25 250 3 250 25 500 4 312.5 25 625 5 625 25 1250 6 1250 25 2500 NAG NAG stock solution (L) Buffer pH 6.0 or pH 7.4 q.s.p. (mL) NAG (mol/L) 1 5 25 10 2 25 25 50 3 50 25 100 4 125 25 250 5 250 25 500 BG BG stock solution (L) Buffer pH 6.0 or pH 7.4 q.s.p. (mL) BG (mol/L) 1 1 50 1 2 10 50 10 DHB DHB stock solution (L) Buffer pH 6.0 or pH 7.4 q.s.p. (mL) DHB (mol/L) 1 0.5 50 0.5 2 5 25 10 3 12.5 25 25 4 25 25 50 5 250 25 500 EPBG EPBG stock solution (L) Buffer pH 6.0 or pH 7.4 q.s.p. (mL) EPBG (mol/L) 1 0.5 50 0.5 2 0.5 25 1 3 2.5 25 5 4 5 25 10 5 25 25 50

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74 Table 3-4. Concentrations of the inhibitory solution of naringin (NAR) plus 6,7-dihydroxybergamottin (DHB) used during the transport studies. Concentration (mol/L) NAR:DHB inhibitory solution NAR stock solution (L) DHB stock solution (L) Buffer pH 6.0 or pH 7.4 q.s.p. (mL) NAR DHB 1 25 0.6 25 50 1.2 2 62.5 1.5 25 125 3 3 312.5 7.5 25 625 15 4 625 15 25 1250 30 5 1000 24 25 2000 48 Transport Study On the study day, the DMEM was removed, the Caco-2 monolayers were rinsed with DPBS, and incubated for 30 min with the transport medium (TM) under the same atmospheric conditions described previously. The TM on the AP and BL side consisted of HBSS-MES and HBSS-HEPES, respectively. After 30 min incubation, the drug-free transport medium on the AP side was replaced by the HBSS-MES medium (pH 6.0) containing talinolol or talinolol plus inhibitor in order to investigate the AP to BL transport. Alternatively, for the BL to AP transport assessment, the buffer of the donor chamber was replaced by a solution of talinolol in HBSS-HEPES medium (pH 7.4), additionally the volume of the acceptor chamber was replaced by HBSS-MES medium (pH 6.0) containing the inhibitor. For the control experiments only solutions of talinolol were used. Thereafter, sample aliquots (200 L) were taken at 20, 40, 60, 90 and 120 min from the AP or BL side and the amount of talinolol transported at each time point was determined by HPLC. Following the transport experiment, the TEER values were measured again and an additional control of monolayer integrity was performed using Lucifer yellow (LY) (173)

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75 Sample Analysis A reversed phase-HPLC method previously described (124) was optimized to enable talinolol to be quantified. Succinctly, samples from Caco-2 (25 L) were directed injected into a Shimadzu VP series HPLC system (Kyoto, Japan) equipped with a RF-10AXL fluorescence detector, a LC-10ATvp solvent delivery unit, an SIL-10AF autosampler, a CTO-10Avp column oven, a SCL-10Avp system controller, a DGU-14A on-line degasser, a FCV-10ALvp low-pressure gradient unit, and Class VP 7.2 SP1 chromatographic software. The excitation and emission wavelengths were set at 252 and 332 nm, respectively, the flow rate at 1 mL/min and the temperature at 40 C. The analytical column was a Lichrospher 60RP-Select B column, 250 x 4.6 mm i.d., 5 m, preceded by a Lichrospher 60RP-Select B guard column (Merck KGaA, Darmstadt, Germany). Talinolol was eluted isocratically using a mobile phase consisted of 0.025 mol/L triethylammonium phosphate buffer (pH 3.0) and acetonitrile (77:23). The validation parameters of linearity, specificity, sensitivity, precision, accuracy, and stability of this external standardization method for talinolol were determined. For each level of concentration, standards solutions and QCs were injected in the HPLC in triplicate in three different days. GraphPad Prism version 4.0 (GraphPad Software Inc, San Diego, CA, USA) was used to construct the calibration curves obtained by plotting the mean area versus the corresponding concentration of the each standard solution. The linearity of the standard curves was determined by least-squares linear regression method and expressed in terms of coefficient of determination (r 2 ). Specificity was determined by comparing the chromatogram of talinolol with that obtained from a blank solution containing only the inhibitory compounds. The intraand inter-day precision and

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76 accuracy of the quantification were measured by replicate analyses (n = 3) of three different concentration levels (low, medium and high QCs) on the same day and on alternate days. The precision was based on the calculation coefficient of variation (CV%), and the accuracy was expressed as percent of the found amount compared to the theoretical one. The calibration model was accepted if the residuals (% difference of the back-calculated concentration from the nominal concentration) were within 20% at the lower limit of quantification (LLOQ) and within 15% at all calibration levels. Calibration curves were rejected if more than 1/3 of the QCs showed a deviation from the theoretical concentration equal or greater than 20%. The lower limit of quantification (LLOQ) was established as the lowest concentration used in the calibration curve. The stability at room temperature and at 37 C of a solution of talinolol in HBSS-MES (pH 6.0) and HBSS-HEPES (pH 7.4) transport medium was assessed within 48 hours and 4 hours, respectively. The same samples were frozen at -20 C during one week in order to check the freeze/thaw stability. Similarly, the stability of talinolol (low, medium, high QCs) in mobile phase was determined within 24 hours at room temperature. The samples were analyzed by the validate HPLC method mentioned formerly, the areas were compared to those obtained from fresh solutions for each concentration level, and results expressed as % of the control. Three replicates of each solution were prepared for each test performed. An additional stability test was conducted during 3 hours in HBSS-MES at 37 C for all grapefruit juice constituents tested in this study. Samples were analyzed as

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77 described in Chapter 2. The 6,7-epoxybergamottin was analyzed using the same method as described for furanocoumarins in Chapter 2. Data Analysis The coefficients of permeability (P app ) talinolol were calculated by using equation (1): )1(CoATQPapp where Q/T is the amount of drug (ng/min) appearing in the acceptor compartment as a function of time obtained from the slope of the linear portion of the amount transported vs time plot., Co is the initial concentration of talinolol in the donor compartment (ng/mL), and A the surface area of the semi-permeable membrane in cm 2 (62, 135). All solutions were kept under 37 C before use. The passive and the Michaelis-Menten parameters of talinolol were estimated using Scientist v. 2.0 (MicroMath Inc., St. Louis, MO, USA). To apply the model the equation (2) was used: where the Papp (BL-AP) is the observed permeability (cm/sec) of talinolol from basolateral to )2(00max)()(CKCPpassivePPMAPBLappAPBLapp apical side of Caco-2 monolayers. The P app (BL-AP) passive is the permeability due only to diffusion, P max (cm/sec) is the maximal permeability due to the active transport and the K M (M) is the Michaelis-Menten constant.

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78 The IC 50 values (concentration to reach half of the maximum inhibition) of verapamil, GFJ and its components were then determined using nonlinear regression according to the Hill equation with GraphPad Prism v.4.0 (GraphPad Software Inc, San Diego, CA, USA). For NAR and verapamil, the best fit was obtained according to the sigmoid dose-response model without the variable slope. Statistical Analysis The mean of at least three experiments and their standard error (SEM) were used to express the values of P app and the kinetics parameters. The confidence interval of each IC 50 value was determined. Statistical analyses were performed with one-way ANOVA followed by Dunnets multiple comparison tests as post-hoc analysis. A probability of less than 0.05 (p < 0.05) was considered to be statistically significant. Results Linearity The range of validation (5 to 1000 ng/mL) showed good linearity with r 2 > 0.998 (Figure 3-2). 0 250 500 750 1000 1250 0 1.0106 2.0106 3.0106 4.0106 5.0106 6.0106r2= 0.9996Talinolol (ng/mL)Area Figure 3-2. Mean calibration curve (n = 9) of talinolol in mobile phase. Vertical bars represent the standard deviations (SD) of the means.

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79 Specificity The method provided good specificity since the chromatograms of the blank solution did not show any peak with similar retention time as expected for talinolol (Figure 3-3) Figure 3-3. Chromatograms corresponding to the talinolol (100 ng/mL) in HBSS-MES with retention time about 9.3 min (A) and a blank solution (B) containing the inhibitory compounds NAR, NAG, BG, DHB, EPBG and VER all at 50 mol/L in HBSS-MES. Sensitivity The LLOQ of 5 ng/mL was accepted as the lowest concentration of the calibration curve since their concentrations could be determined with acceptable precision (%CV < 20), and accuracy (%error < 20).

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80 Precision and Accuracy The precisions intraand inter-day for both flavonoids and furanocoumarins were satisfactory with CV values between 0.7 and 8%. Similarly, the accuracy of the assay was between 93 and 113% for all compounds tested at three different concentrations. The results are summarized in Table 3-5. Table 3-5. Intra-day (n = 3) and inter-day (n = 9) assay parameters of talinolol. Precision expressed as CV% and accuracy as % of the theoretical concentration. Intra-day CQ 5 ng/mL CQ 100 ng/mL CQ 1000 ng/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 0.8 0.7 7.9 4.7 4.2 4.8 4.2 0.8 2.9 Accuracy 109.9 105.6 113.1 93.0 93.3 98.3 100.4 100.1 98.9 Inter-day CQ 5 ng/mL CQ 100 ng/mL CQ 1000 ng/mL Precision 5.3 4.8 2.7 Accuracy 109.9 94.9 99.8 Stability The standard solutions of talinolol were found stable at room temperature within 48 hours and at 37 C in both transport medium for 4 hours. Additionally, in general, all grapefruit components tested showed adequate stability under the test conditions for 3 hours. The 6,7-epoxybergamottin showed a 20% of degradation and a peak at the same retention time as the DHB was observed concomitantly, indicating that EPBG was partially converted into DHB. For the others compounds, the shifting of the areas of each sample tested was less than 5% of those obtained from a fresh solution at the same level of concentration (Figure 3-4).

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81 12 h (RT) 24 h (RT) 0 25 50 75 100 125Low QC Medium QC High QC % Control C)2 h (37 C)(37 4 h 24 (RT) 48 (RT) C)1 week (20 0 25 50 75 100 125Talinolol in MES (5.5mol/L) Talinolol in HEPES (5.5mol/L) % ControlABC ml/L)NAR (450 ml/L)NAG (450 ml/L)BG (5 ml/L)DHB (50 ml/L)EPBG (50 0 25 50 75 100 125% Control Figure 3-4. Stability of talinolol (low, medium, and high QCs) in mobile phase within 24 hours at room temperature (RT) (A). Stability of talinolol in transport medium within 4h at 37 C, during 48 h at RT, and after one week at -20 C (B). Stability of GFJ components in after 3h in HBSS-MES (pH 6) at 37 C (C). Data represents means of three replicates and vertical bars the standard deviation of the means. Concentration Dependence The absorptive permeability (P app (AP-BL) ) of talinolol was concentration independent and significantly lower than the permeability in the secretory direction (P app (BL-AP) ) at all concentrations (Figure 3-5). On the other hand, the P app (BL-AP) decreased considerably as

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82 the concentration of talinolol on the basolateral side of the Transwell system was increased above 200 M. The apparent P max P app (BL-AP) passive, and K M values and their standard deviations associated with the transport of talinolol in the secretory direction are (2.2 0.33) x 10 -6 cm/sec, (2.0 0.05) x 10 -6 cm/sec, and 737 258 M, respectively. 0 200 400 600 800 1000 0 5 10 15 20 25BL to AP AP to BL *********Talinolol (M)Papp x 107 (cm/sec) Figure 3-5. Concentration dependent coefficient permeability (P app ) corresponding to the AP to BL and BL to AP transport of talinolol across Caco-2 cells monolayers. Data are expressed as mean SEM (n = 3-6). Fitting was performed using Scientist 2.0 as described in Material and Methods. All points of P app (BL-AP) were compared with the talinolol 25 M and all data of P app (AP-BL) were compared with the respective pair representing the secretory transport of talinolol. p < 0.05; ** p < 0.01; p < 0.001; p < 0.01. Inhibition Studies The Figure 3-6 shows the permeability of talinolol across the Caco-2 cells monolayers in the absence and presence of different concentrations of verapamil, GFJ, and its components. Verapamil, a known inhibitor of P-gp, decreased the ratio P app (BL-AP) to P app (AP-BL) of talinolol from 7.4 to 3. This effect was due to the attenuation of secretory (14 x 10 -7 to 9.4 x 10 -7 cm/sec) and to the enhancement of absorptive (1.9 x 10 -7 to 3.1 x

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83 10 -7 cm/sec) transport of talinolol. However, compared with the respective controls reduction of P app (BL-AP) was more significant than the enlargement of P app (AP-BL) Similarly, grapefruit juice (GFJ) reduced the P app (BL-AP) of talinolol from 22 x10 -7 to 4.5 x 10 -7 cm/sec but did not affect significantly its absorptive transport. Although NAR, the main constituent of GFJ, was found in the juice at relative high concentration (811 M), it showed a minor effect on the secretory transport of talinolol. Concentrations higher than 625 M decreased the P app (BL-AP) to around 1.3-1.5 fold compared to the control. Likewise GFJ, the effect of NAR on the absorptive transport of talinolol was negligible. On the other hand, the aglycone NAG, which is not normally found in GFJ (205), decreased the BL to AP transport more than 2-fold compared to the control as its concentration was increased beyond 250 M. Analogous to 6,7-dihydroxybergamottin, bergamottin at 1 M and 10 M did not change the permeability of talinolol however, in vitro studies of further concentrations were prejudiced because solubility issues. The furanocoumarins 6,7-dihydroxybergamottin (DHB) at concentrations higher than 10 M and 6,7-epoxybergamottin (EPBG) exerted a considerably reduction on the BL to AP transport of talinolol. In fact, the P app (BL-AP) of talinolol in the presence of DHB (500 M) and EPBG (50 M) decreased 5and 2.5fold, respectively, when compared with the controls. The effective concentrations of these inhibitory compounds are in the same range as they are found in the juice (Table 3-2).

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84 0501002505001000 0 5 10 15 20 25********AP/BL BL/AP Verapamil (M)Papp x 107 (cm/sec) 00.515102550 0 5 10 15 20 25***********GFJ (%)Papp x 107 (cm/sec) 010025050062512502500 0 5 10 15 20 25Naringin (M)Pappx 107 (cm/sec) 0102550100250500 0 5 10 15 20 25*Naringenin (M)Pappx 107 (cm/sec) 00.5102550100500 0 5 10 15 20 25*******6',7'-Dihydroxybergamottin (M)Papp x 107 (cm/sec) 00.5151050 0 5 10 15 20 25*****6',7' Epoxybergamottin (M)Pappx 107 (cm/sec)ABCDEF Figure 3-6. Coefficient of permeability (P app ) of talinolol (100M) across the Caco-2 cells monolayers in the absence and presence of distinct concentrations of A: verapamil HCl (25, 50, 100, 250, and 500 M); B: NAR (100, 250, 500, 625, 1250, 2500 M); C: DHB (0.5, 10, 25, 50, 500 M); D: GFJ (0.5, 1, 5, 10, 25, and 50 %); E: NAG (10, 50, 100, 250, 500 M); F: EPB (0.5, 1, 5, 10, 50 M). Data are expressed as mean SEM (n = 3-6). p < 0.05; ** p < 0.01.

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85 Table 3-6. Concentration range that the grapefruit components are normally found in the juice. Inhibitor Conc. in GFJ (M) Naringin 174 1492 (205)a Naringenin < 0.56 (205) Bergamottin 1 36.6 (205) 6,7-Dihydroxybergamottin 0.22 52.5 (205) 6,7-Epoxybergamottin 0.1 7.4 (60) a references are in parenthesis Dose-response Studies The dose-response curves for the inhibitory effects and the IC 50 values of verapamil, GFJ, and its components are presented in Figure 3-7, respectively. Except for NAR, the range of concentration tested was enough to obtain a sufficient estimation (narrow confidence interval range) of the potency and efficacy of all tested compounds and GFJ. The lower potency of NAR affected the accuracy of its IC 50 value (2409 M), since further concentrations (> 2500 M) could not be tested because of the negative impact on the integrity of the Caco-2 monolayers (decreased the TEER values and increased LY transport) and due to solubility problems. Verapamil, however, showed a relatively high potency (28 M) but did not present a good efficacy reducing the activity of P-gp by only 35% compared to the control. GFJ (IC 50 = 0.6%) diluted in HBSS-MES buffer solution (pH 6.0) decreased the secretory transport of talinolol to 27% of that without any inhibitor. Correspondingly, NAG was 10-fold more potent than its glycoside NAR reducing the P-gp mediated secretory transport of talinolol by 60% when compared to the control. Interestingly, the furanocoumarin EPBG was about 50-fold more potent than DHB with IC 50 values of 0.7 M and 34 M, respectively. Additionally, compared

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86 at the same concentration (50 M), EPBG and DHB showed similar efficacy reducing the P app(BL-AP) of talinolol by 55% and 60%, correspondingly. 0 0IC50 = 28MC.I. = 10 77 60 70 80 90 100 100 101 102 103Verapamil (M)% Control 0 0 25 50 75 100IC50 = 0.6 %C.I. = 0.5 0.8 100 101 102GFJ (%)% Control 0 0Naringin (M) 60 70 80 90 100IC50 = 2409MC.I. = 221 26306 102.0 102.5 103.0 103.5% Control 0 0 40 50 60 70 80 90 100IC50 = 236MC.I. = 69 809 101 102 103Naringenin (M)% Control 0 0 25 50 75 100IC50 = 34MC.I. = 24 47 100 101 102 1036',7'Dihydroxybergamottin (M)% Control 0 0 40 60 80 100IC50 = 0.7MC.I. = 0.5 0.9 10-2 10-1 100 101 1026',7'-Epoxybergamottin (M)% ControlABCDEF Figure 3-7. Inhibitory dose-response effects of A: verapamil HCl (50, 100, 250, 500, and 1000 M); B: GFJ (0.5, 1, 5, 10, 25, and 50 %); C: NAR (100, 250, 500, 625, 1250, 2500 M); D: NAG (10, 50, 100, 250, 500 M); E: DHB (0.5, 10, 25, 50, 500 M; F: EPBG (0.5, 1, 5, 10, 50 M) on the transport of talinolol in the secretory (BL-AP) direction. Data are expressed as mean SEM (n = 3-6). The IC50 values of each compound and their respective 95% of confidence interval (C.I.) were estimated by nonlinear regression using GraphPad Prism 4.0 as described in Material and Methods.

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87 Effects of NAR and DHB on the Secretory Transport of Talinolol Although screening isolated compounds is an important tool to understand the mechanism of action and the relevance of each one on the interaction between GJF and P-gp, combinations of two or more compounds is a more realistic approach since the compounds are present all together in the juice and the overall outcome might be a result of an additive, or a synergistic or an antagonistic effect. Therefore, several combinations using a fixed NAR to DHB ratio of 41.7 were prepared and tested for their effect on the activity of P-gp. NAR is the main component present in the juice whereas, DHB has been shown to be a potent inhibitor of P-gp. Interestingly, the P app (BL-AP) of talinolol went up considerably as the concentration of each compound was increased (Figure 3-8). In fact, the combinations NAR:DHB (1250:30 M and 2000:48 M) increased the secretion of talinolol about 2and 2.7-fold, respectively, of that without inhibitor. ControlM)M):DHB (1.2NAR (50M)M):DHB (3NAR (125M)M):DHB (15NAR (625M)M):DHB (30NAR (1250M)M):DHB (48NAR (2000 0 10 20 30 40 50 60****Papp x 107(cm/sec) Figure 3-8. Effect of the combination NAR:DHB (50:1.2; 125:3, 625:15, 1250:30, and 2000:48 M) on the secretory transport (BL-AP) of talinolol (100M) across the Caco-2 cells monolayers. Data are expressed as mean SEM (n = 3). p < 0.05; ** p < 0.01.

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88 DISCUSSION In the present study we systematically investigated in vitro the potential drug interaction between selected ingredients of GFJ alone and in combinationand the P-glycoprotein transporter system, which controls many barriers in the body using talinolol as a probe substrate. The stability studies demonstrated that any modification on talinolol concentration over the time on both apical and basolateral sides was regulated only by the transport phenomenon, which includes passive and active transport. Additionally, the GFJ components showed sufficient stability under the study conditions. However, since several metabolizing enzymes are present in Caco-2 the effect of GFJ metabolites can not be neglected and further studies are necessary to answer this issue. The polarized transport of talinolol across the Caco-2 cell monolayers was noticeable with the BL-AP permeability exceeding significantly the AP-BL transport and exhibiting ratios higher than 5 (Figure 3-5 and Figure 3-6). Additionally, the decrease on the P app (BL to AP) of talinolol was concentration-dependent demonstrating saturable efflux transport. Such behavior is consistent with the presence of the P-gp highly expressed in Caco-2 cells (173, 174). Similarly, it was demonstrated that the apparent oral clearance (CL/F) of talinolol in healthy volunteers decreased, approximately, from1000 mL/min to 500 mL/min as the dose of talinolol increased from 25 mg to 400 mg. This phenomenon can be attributed to the saturation of the intestinal secretion of talinolol mediated by P-gp with consequent augment of fraction of the drug absorbed (186). The P app (AP-BL) of the marker compound remained unchanged at all concentrations (Figure 3-5). This asymmetry between the absorptive and secretory P-gp mediated transport was already described for talinolol (206) and other P-gp substrates like digoxin,

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89 rhodamine 123 (207), fexofenadine (89), and taxol (175). It was suggested that P-gp was more rapidly saturated by substrates approaching from the BL than from AP side (207). Reasons for these observations were attributed to the differences in the lipid and protein composition between the AP and BL membranes (89). The apical membrane has about 2-fold more glycosphingolipids than the basolateral membrane. Such lipids have high capacity to form interand intra-molecular hydrogen bonds, which increase the microviscosity of the apical membrane (208). Kinetic analyses revealed that the passive permeability of talinolol during the secretory transport was 2.0 x 10 -6 cm/sec and similar of that obtained in previous studies 3.6 x 10 -6 cm/sec (206). The P max value of 2.2 x 10 -6 cm/sec, similar to the passive diffusion, supports the observation that P-gp-mediated transport exhibits a strong influence on the total permeability of talinolol over a wide range of concentrations (206) In general, the P app (AP-BL) of talinolol was not affected by the presence of any of the inhibitors tested (Figure 3-6). Verapamil up to 1000 M promoted a slight increase in the absorptive transport from (1.95 0.23) x 10 -7 cm/sec to (3.1 0.34) x 10 -7 cm/sec and the maximum effect on the secretory transport was reached at concentration of 500 M (Figure 3-6). However, verapamil (IC 50 = 28 M) diminished the secretory activity of P-gp by only 35% (Figure 3-7). A complete inhibition would be characterized by total suppression of the polarized transport of talinolol. In contrast to others studies where verapamil was added on both the apical and basolateral side and sometimes pre-incubated for 15 to 30 min before addition of the substrate (186, 207, 209, 210), in the present study verapamil was present only in the apical side without previous incubation. Such approach

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90 tried to simulate the effects of the several inhibitors tested when co-administrated with an oral dose of talinolol. Although GFJ had a remarkable inhibitory effect on P-gp-mediated secretory transport of talinolol, and the half maximum effect was achieved at concentration of around 0.6%, its main constituent NAR had only a minor direct effect (IC 50 = 2409 M). However, NAR may contribute indirectly to the overall inhibitory effect of GFJ, since this flavonoid can be converted by intestinal microflora to its aglycone NAG (211) which promotes a significant effect on P-gp activity (IC 50 = 236 M) as presented in Figure 3-6 and Figure 3-7. Such observation supports that lipophilicity is an important parameter for P-gp substrates. Among all inhibitors tested, the furanocoumarin EPBG (IC 50 = 0.7 M) seems to be the most potent compound against the P-gp mediated transport (Figure 3-7). Similarly, Dresser and co-workers (80) using vinblastine and digoxin as marker compounds, found that 6,7-dihydroxybergamottin was the most potent inhibitor (IC 50 = 33 M), bergamottin did not alter P-gp activity at concentrations up to 50 M, and naringin at 3000 M reduced P-glycoprotein to 51% 9% of control. Additionally, the components of grapefruit are present in the juice at enough concentration to promote a significant effect on P-gp activity (Table 3-2). Interestingly, when combinations of NAR and DHB were tested a completely different profile was observed (Figure 3-8). The P app (BL-AP) of talinolol increased from (21.5 1.85) x 10 -7 cm/sec (control) to (57.1 2.33) x 10 -7 cm/sec. So far, there is a lack of information regarding the simultaneous effect of multiple GFJ components on the activity of P-gp. The NAR to DHB ratio of 41.7 employed in this study differed from that found in the juice ( 18DHBNAR ), suggesting that the relative concentrations of these

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91 components may also play an important role on the mechanism and on the grapefruit juice-drug interaction outcomes. Since expression of others transporters such as OATP and organic cation transporters (OCT) in Caco-2 is uncertain (89), and talinolol is not substrate of multiple drug resistant protein (MRP) or breast cancer resistant protein (BCRP) which can be expressed in this cell line (89, 212, 213), we speculate that the effect observed was due to activation of P-gp activity or modification of membrane fluidity. Further in vitro and animal studies using different ratios of NAR and DHB, additional compounds, such as the dimers of furanocoumarins, and new combinations of GFJ components are necessary to clarify this issue. Conclusions The results of this study support the importance of P-gp mediated transport on drug disposition. Modulation of P-gp activity by GFJ and its components may have some clinical relevance. Isolated compounds present in GFJ are able to inhibit directly or indirectly the P-gp activity. This study demonstrated that the modulation of P-gp by GFJ seems not to be a simple additive effect, instead a more complex association is likely, especially considering the variability of these components present in the grapefruit and in the juice. This may help to explain the controversial results previously reported for GFJ-Pgp interaction studies. Additionally, taking into account the lack of information about the effect on P-gp activity when two or more grapefruit juice constituents are tested simultaneously, further investigations are necessary to elucidate the mechanisms behind the GFJ-drug interactions and their clinical relevance.

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CHAPTER 4 MODULATION OF P-GLYCOPROTEIN MEDIATED TRANSPORT OF TALINOLOL IN RATS BY GRAPEFRUIT JUICE, ORANGE JUICE, FLAVONOIDS AND FURANOCOUMARINS Background There is a lack of information regarding the effect of different brands and strengths of grapefruit juices (GFJ) and its constituents on the disposition of drugs substrates for the permeability-glycoprotein (P-gp) intestinal efflux transporter. Most of the reported studies focus on evaluating the interaction between GFJ and its components with the drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) or the effect of only one juice on the activity of P-gp (40, 134, 137, 214). Reports of interaction between GFJ and talinolol, a well described non-metabolized P-gp substrate (215), in rats and humans have shown opposite results. Co-administration of white GFJ (Tropicana) 50% diluted in saline pH 7.0 with talinolol (10mg/kg) was described to increase the maximum talinolol serum concentration (C max ) and the area under the concentration-time profile curve (AUC) in rats by 2and 1.4fold, respectively (134). In contrast, the C max and AUC of talinolol when taken orally with GFJ (Paradiso-Succo Germany) by healthy volunteers were reduced by half (137). The magnitude of the GFJ-drug interaction maybe dependent on the brand, the amount of GJF ingested, the timing of drug administration relative to the food intake, the intrinsic oral bioavailability of each drug, and the relative concentration of each grapefruit component in the juice (40, 128). Thus, the extent of which GFJ modifies P-gp activity remains unclear and more studies are desirable to clarify this issue. 92

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93 Specific Aim It was the aim of this study to investigate the effect of different strengths and brands of GFJ and its components naringin (NAR), naringenin (NAG), bergamottin (BG) alone and in combination on the disposition of talinolol, in male Sprague-Dawley rats. These compounds together with 6,7-dihydroxybergamottin (DHB), 6,7-epoxybergamottin, and their dimers have been suggested to contribute to GFJ-drug interaction (68, 105). Additionally, the effects of both acute and one-week long of juice intake before drug administration were evaluated. The effect of orange juice on talinolol disposition was also estimated as preliminary data for futures studies. We expect that this study will aid to clarify the predominant mechanism, the major compounds contributing to the overall effect of grapefruit juice on drug disposition and the in vivo relevance of this potential interaction mechanism. Materials and Methods Chemicals The following materials were used: (rac)-verapamil hydrochloride and halothane were obtained from Sigma Chemical Company (St. Louis, MO, USA); talinolol (99.9% purity) was a gently provided by AWD-Pharma GmbH & Co. KG (Dresden, Germany), naringin (NAR) and naringenin (NAG), both > 95% pure, were from Roth GmbH & Co. (Karlsruhe, Germany), bergamottin (BG) (98% purity) was bought from Indofine Chemical Company, Inc. (Somerville, NJ, USA); triethylammonium phosphate buffer (1M) was obtained from Fluka (Buchs, Switzerland); methanol HPLC grade was purchased from Fisher Scientific (Fair Lawn, NJ, USA). All others reagent were analytical grade.

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94 Stock, Work Solutions, and Preparation of Calibration Standards The stock solution of BG 50 mM was prepared in dimethylsulfoxide (DMSO) as previously described in Chapter 2 and NAG 2.5 mM in propylene glycol. Talinolol test solution I and II were prepared in propylene glycol:saline (20:80) and saline, respectively, whereas the talinolol stock solution I was obtained using ethanol. The final concentration of propylene glycol (PPG) was not higher than 20%. Talinolol test solutions The amount of 50 mg of talinolol (TAL) was accurately weighed and transferred quantitatively to a 25 mL volumetric flask. The standard was dissolved in 5 mL of propylene glycol and the volume completed with saline solution which pH has been previously adjusted to 3.0 in order to simulate the pH of the juice and guarantee a complete dissolution of the drug. The final dose solution containing 20% of PPG was mixed thoroughly to obtain a talinolol concentration of 2 mg/mL and was used as a control when evaluating the effects of GFJ constituents and verapamil on the disposition of talinolol in rats. Same solution was prepared in absence of propylene glycol and was used as a control when evaluating the effects of grapefruit juices and OJ on the disposition of talinolol in rats Talinolol stock solution I The amount of 25 mg of talinolol (TAL) was accurately weighed and transferred quantitatively to a 25 mL volumetric flask. The standard was then dissolved in ethanol, the volume was completed with the same solvent, and the final solution mixed thoroughly in order to obtain a talinolol concentration of 1 mg/mL.

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95 Talinolol stock solution II Volume of 100 L from the talinolol stock solution I was accurately transferred to a 10 mL volumetric flask, the volume was completed with a solution of 0.025 mol/L triethylammonium phosphate buffer (pH 3.0 adjusted with 1 M NaOH) and acetonitrile (77:23) and mixed thoroughly. The final concentration of talinolol was 10 g/mL. Talinolol stock solution III Volume of 1 mL from the talinolol stock solution II was accurately transferred to a 10 mL volumetric flask, the volume was completed with a solution of 0.025 mol/L triethylammonium phosphate buffer (pH 3.0 adjusted with 1 M NaOH) and acetonitrile (77:23) and mixed thoroughly. The final concentration of talinolol was 1000 ng/mL. Verapamil test solution I The amounts of 20 mg of verapamil hydrochloride and 50 mg of TAL were transferred quantitatively to a 25.0 mL volumetric flask. The compounds were dissolved in 5 mL of propylene glycol and the volume completed with saline solution which pH has been previously adjusted to 3.0. The solution was mixed thoroughly and the final concentrations of verapamil.HCl and talinolol were 0.8 mg/mL and 2 mg/mL, respectively. Naringin test solutions The amounts of 11.8 mg or 47 mg of naringin (MW = 580.53 g/mol) and 50 mg of TAL were transferred quantitatively to a 25.0 mL volumetric flask. The compounds were dissolved in 5 mL of propylene glycol and the volume completed with saline solution which pH has been previously adjusted to 3.0. The solutions were mixed thoroughly and the final concentrations of NAR and talinolol were 813 M or 3238 M and 2 mg/mL, respectively. The concentrations of NAR in these solutions approximately correspond to

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96 that found in the white grapefruit juice normal strength (811 M) or frozen concentrate (3240 M) used in this study. Naringenin stock solution The amount of 17 mg of naringin (MW = 272.26 g/mol) was transferred quantitatively to a 25.0 mL volumetric flask. The compound was dissolved in 15 mL of propylene glycol, the volume was completed with the same solvent, and the final solution mixed thoroughly. The final concentration of NAG was 2.5 mM. Naringenin test solution The amount of 50 mg of talinolol was transferred quantitatively to a 25.0 mL volumetric flask containing 5 mL of the naringenin stock solution. Talinolol was then dissolved and the volume was completed by adding saline solution which pH has been previously adjusted to 3.0. The solution mixed thoroughly and the final concentrations of NAG and talinolol were 500 M and 2 mg/mL, respectively. Bergamottin test solutions From the bergamottin stock solution (50 mM), 16 L or 64 L were accurately transferred to a 25 mL volumetric flask containing 5 mL of propylene glycol. The amount of 50 mg of talinolol was transferred quantitatively to the same flask and dissolved. The volume was completed with saline solution which pH has been previously adjusted to 3.0 and mixed thoroughly. The final concentrations of BG and talinolol were 32 M or 128 M and 2 mg/mL, respectively. The concentrations of BG in these solutions correspond to that found in the white grapefruit juice normal strength or frozen concentrate used in this study.

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97 Talinolol standard solutions From the talinolol stock solutions II (10 g/mL) and III (1000 ng/mL), nine different concentrations of standard solutions and three quality controls (QC) were prepared in rat plasma (Biomeda, Foster City, CA) according Table 4-1. Table 4-1. Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of talinolol. Standard TAL stock solution II (L) TAL stock solution III (L) Rat plasma q.s.p. (mL) Talinolol (ng/mL) 1 5 1 5 2 10 1 10 3 25 1 25 4 50 1 50 5 10 1 100 6 25 1 250 7 50 1 500 8 75 1 750 9 100 1 1000 QC1 25 1 25 QC2 45 1 450 QC3 85 1 850 Animals and Experimental Protocol All animals were housed and all experiments performed according to the policies and guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Florida, Gainesville, USA. Male Sprague-Dawley rats weighing 350 to 400 g were used in this study. The animals were not fasted and had free access to food and water during the experiment except for the first 2 hours after drug administration. An oral dose of talinolol (10mg/kg; 5ml/kg) was prepared in the absence or presence of verapamil HCl (4mg/kg) (positive control), or NAG (500 M), NAR (813 and 3238 M) and BG (32 and 128 M) in propylene glycol:saline solution (20:80). The pH of saline solution was previously adjusted to 3.0 with 1mM HCl in order to simulate

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98 the juice pH and guarantee the talinolol dissolution. Furthermore, a solution containing the same dose of talinolol and a fixed ratio of the flavonoid NAR to the furanocoumarin BG (3238:128 M) was prepared based on the concentration of these compounds in frozen concentrate GFJ (Minute Maid). Similarly, the effects orange juice (OJ) (Tropicana), Ruby Red GFJ (Florida Natural) normal strength, White GFJ (Minute Maid) concentrated (GFJC) and normal strength (GFJNS) (prepared by diluting one part of the concentrated juice into 3 parts of saline) on the P-gp mediated transport of talinolol were evaluated. In this scenario, the doses of talinolol were prepared directly in the juice or in pure saline pH 3.0 and administered by gavage. Ruby Red GFJ was also given twice daily (5ml/kg) during one week prior talinolol administration. Concentrations of NAR, NAG, BG and DHBG in GFJNS and Ruby Red GFJ were 811 and 469.4M, < 0.56M, 31.8 and 12.2M, 45 and 0.28M, respectively, as previously reported (205). Concentrations of components present in the orange juice with potential to interact with P-gp were not estimated in this study. After drug administration, blood samples (500 L) were collected from the sublingual vein of each rat at 0, 45 min, 1, 2, 3, 4, 5, and 6 hours which were separated into two different days apart by one week of washout period (4 blood collections per day per animal were performed). The variability of the weight of each animal on both periods was not higher than 20%. Prior to blood collection, the rats were anaesthetized with halothane and after each sampling, approximately 1000 L of isotonic saline were replaced by i.p. injection in order to maintain the blood fluid. The samples were centrifuged at 2800 g for 15 min at room temperature and the serum separated. The collected samples were stored at -70 C until analysis.

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99 Sample Preparation Talinolol was extracted from the samples by using 96-well solid-phase extraction (SPE) disks (Polaris, Varian, Lake Forest, CA, USA). Briefly, the disks were equilibrated with 400 L of methanol and pre-conditioned by adding the same volume of 1mM HCl. Samples were diluted (1:2) with the conditioning solvent but containing the internal standard (propranolol hydrochloride at 500 ng/mL) and vortexed for 30 sec before they were applied to the SPE disks at a final volume of 750 L. The interferents were eluted with 400 L of methanol:water (20:80). Finally, the analytes were recovered rising the SPE disks twice with 200 L of methanol:water (90:10). The solvent was evaporated to dryness and the residue redissolved in 125 L of mobile phase before analysis. Sample Analysis A reversed phase high-performance liquid chromatograph (HPLC) method with ultraviolet detection was applied for the quantification of talinolol in plasma. Samples (75L) were injected into a Shimadzu LC2010C HPLC system (Kyoto, Japan) equipped with a 100L loop and Class VP 7.2 SP1 chromatographic software. The wavelength was set at 241 nm, the flow rate at 1.00 mL/min and the temperature at 40 C. The analytical column was a Lichrospher 60RP-Select B column, 250 x 4.6 mm i.d., 5 m, preceded by a Lichrospher 60RP-Select B guard column (Merck GaA, Darmstadt, Germany). Talinolol was eluted isocratically using a mobile phase consisted of 0.025 mol/L triethylammonium phosphate buffer (pH 3.0) and acetonitrile (77:23).

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100 Validation The validation parameters of linearity, specificity, sensitivity, precision, accuracy, recovery, and stability were determined. For each concentration, standard solutions and QCs were injected into the HPLC in triplicate on three different days. Calibration curves were obtained by plotting the mean standard/internal standard peak-area ratios versus the standard concentrations in Microsoft Excel spreadsheets. The linearity of the standard curves was determined by least-squares linear regression method and expressed in terms of coefficient of determination (r 2 ) using the Linest function. A weighing factor of 1/Conc 2 was chosen to achieve the homogeneity of variance. Specificity was determined by comparing the chromatogram of talinolol with that obtained from blank plasma. The intraand inter-day precision and accuracy of the quantification were measured by replicate analyses of three different concentrations (low, medium and high QC) on the same day and on alternate days. The precision was based on the calculation coefficient of variation (CV%), and the accuracy was expressed as percent of the measured concentration compared to the theoretical one. The calibration model was accepted if the residuals (% difference of the back-calculated concentration from the nominal concentration) of the weighted curve were within 20% at the lower limit of quantification (LLOQ) and within 15% at all calibration levels. Calibration curves were rejected if more than 1/3 of the QCs showed a deviation from the theoretical concentration equal or greater than 20%. The LLOQ was established as the lowest concentration used in the calibration curve. The recovery was evaluated by adding known amounts of three standard solutions of talinolol to blank rat serum to get final concentrations of 10, 250 and 500 ng/mL. Samples were extracted under the same conditions mentioned previously. A standard

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101 curve was prepared in mobile phase considering the same range of concentration of that used for the rat samples. The analytical results for extracted samples at three concentration levels were then back-calculated using the equation obtained from the weighted linear regression of the standard curve and compared with their nominal values that represent 100%. Stability of talinolol in rat serum was tested at room temperature and at -70 C. Three QCs (low, medium, and high) were prepared by spiking the blank serum with fresh talinolol stock solution II (10 g/mL) or III (1000 ng/mL). Analyses of room temperature samples started immediately following the preparation of the test solutions and after 6, 12 and 24 hours. Another set of QCs in serum was prepared, stored at -70 C and analyzed once a week for 3 weeks. Samples were prepared following the steps mentioned previously and analyzed by the validated HPLC method already described. Results were expressed in terms of % of drug remaining compared to the original concentration. On-instrument stability was verified by injecting another set of QCs (low, medium, and high) at the end of each run. The concentrations in these samples were compared to the initial injections of QCs and expressed in terms of % error. Three replicates of each concentration level were prepared for each test performed. Pharmacokinetic Analysis The pharmacokinetics of talinolol was evaluated by noncompartmental method with WinNonlin Professional Edition (version 3.1; Pharsight Corp, Mountain View, CA, USA). The maximum talinolol serum concentration (C max ) and the time to reach C max (t max ) were obtained directly from the concentration-time profile curves. The AUC (0-6), corresponding to the area under the concentration-time profile curve from time 0 to the

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102 time of the last measurable concentration (C 6h ), was determined by the linear trapezoidal method. The terminal elimination rate constant (z) was determined by log-linear regression of at least the last 3 points. The terminal half-life (t 12 ) was calculated as ln2/z. The AUC from time 0 to infinity (AUC (0-) ) was the sum of AUC (0-6) and C 6h /z. Statistical Analysis Descriptive and comparative statistics were calculated with the use of GraphPad Prism v.4.0 (GraphPad Software Inc, San Diego, CA, USA). Pharmacokinetic data are presented as mean values (n = 8) standard error of the mean (SEM). Comparison among the groups initially used one-way ANOVA followed by Tukeys multiple comparison tests as post-hoc analysis. A probability of less than 0.05 (p<0.05) was considered to be statistically significant. Grubbs test was applied to detect possible outliers ( = 0.05) and Kolmogorov-Smirnov test was used to determine if the obtained data were normally distributed. Results Validation of Analytical Method to Measure Talinolol in Rat Serum The validation parameters of the analytical method used to assess the concentration of talinolol in rat serum are now presented. Linearity The range of validation (5 to 1000 ng/mL) was linear with coefficients of correlation more than 0.995 (Table 4-2).

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103 Table 4-2. Regression parameters obtained from calibration curves of talinolol in plasma in three different days. Regression parameters Day 1 Slope (1/Conc 2 ) Intercept (1/Conc 2 ) r 2 Rep1 0.003708 0.011549 0.9981 Rep2 0.002551 0.002643 0.9976 Rep3 0.003665 0.006256 0.9977 Day 2 Slope (1/Conc 2 ) Intercept (1/Conc 2 ) r 2 Rep1 0.002939 0.016177 0.9982 Rep2 0.002805 0.017454 0.9977 Rep3 0.002749 0.023680 0.995 Day 3 Slope (1/Conc 2 ) Intercept (1/Conc 2 ) r 2 Rep1 0.003111 0.011471 0.9958 Rep2 0.003152 0.011726 0.9998 Rep3 0.003206 0.010758 0.9982 Specificity The method provided good specificity since the chromatograms of the blank solution did not show any peak with similar retention time as expected for talinolol (Figure 4.1) Figure 4-1. Comparison of chromatograms corresponding to the talinolol (I) and the internal standard propranolol hydrochloride (II) after extraction from rat serum (A) with that obtained after extraction of a blank sample (B).

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104 Sensitivity The LLOQ of 5 ng/mL was accepted as the lowest concentration of the calibration curve since their concentrations could be determined with acceptable precision (%CV < 20), and accuracy (%error < 20). Precision, accuracy and recovery The precisions intraand inter-day for talinolol were satisfactory with CV values between 1.2 and 7.6%. Similarly, the accuracy of the assay obtained with quality control samples containing 25, 450, and 850 ng/mL talinolol was between 96.4 and 106.8% of the nominal values. The mean recovery assessed at three distinct levels of concentration (10, 250 and 500 ng/mL) ranged from 89.8 to 103.2% of the expected values. The results are summarized in Table 4-3. Table 4-3. Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of talinolol in rat serum. Precision expressed as CV%, accuracy and recovery as % of the theoretical concentration. Intra-day CQ 25 ng/mL CQ 400 ng/mL CQ 850 ng/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 1.2 1.7 4.8 3.2 5.1 7.6 7.3 5.8 1.7 Accuracy 96.4 97.1 102.5 106.7 104.9 100.4 103.2 104.1 105.2 Inter-day CQ 25 ng/mL CQ 400 ng/mL CQ 850 ng/mL Precision 3.9 5.0 5.7 Accuracy 98.4 104.2 104.7 Recovery CQ 10 ng/mL CQ 250 ng/mL CQ 500 ng/mL % 103.2 89.8 91.3 CV% 3.5 7.9 4.6

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105 Stability Results of stability tests in rat serum are presented in Figure 4-2. Talinolol was stable under the tested conditions. The mean % remaining in rat serum after 24 hours at room temperature was 99.1 3.3, 97.8 1.4, and 99.1 1.0 for the low, medium and high concentrations, respectively. After 3 weeks at -70 C the mean % remaining was 99.6 2.9, 101.3 3.7, and 98.1 2.9 for the low, medium and high concentrations, respectively. No degradation of talinolol was observed in the on-instrument assay. The % error of all samples was < 5. 0 61224 50 60 70 80 90 100 11025 ng/mL 400 ng/mL 850 ng/mL ATime (hours)% Remaining 0 123 50 60 70 80 90 100 11025 ng/mL 400 ng/mL 850 ng/mL BWeeks% Remaining Figure 4-2. Stability of talinolol (low, medium, and high QCs) in rat serum within 24 hours at room temperature (RT) (A) and during 3 weeks at -70 C (B). Data represents means of three replicates and vertical bars the standard deviation of the means. Talinolol Pharmacokinetics The concentration-time profiles and the pharmacokinetic parameters of talinolol are presented on Figure 4-3 and Table 4-3, respectively. According the Kolmogorov-Smirnov test the outcomes followed a normal distribution and any outlier was identified by Grubbs test. No difference was observed in the pharmacokinetic profiles when talinolol was given with or without propylene glycol (PPG). Verapamil (VER) (4 mg/kg), a known

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106 inhibitor of P-gp, produced a slightly increase in C max and AUC (0-) of talinolol by 1.4and 1.7-fold, correspondingly. Different from the other treatments, where no effect was observed, VER delayed the absorption (t max ) of talinolol from 2 to 2.8 hours. The elimination half-life (t 1/2 ) remained practically unchanged among all treatments. Orange juice, white GFJNS and GJFC had minor but distinct effects on talinolol pharmacokinetics. The GFNS provided a slight increase (1.3-fold) on C max and AUC (0-) while GFJC or OJ reduced or had no effect on talinolol absorption, respectively. Among the GFJ components, the furanocoumarin BG (128 M) modified significantly the talinolol disposition, augmenting the C max and AUC (0-) by 2.4and 1.8-fold, respectively. In general, the flavonoids NAG (500 M) and NAR (813 M and 3238 M) had a similar effect increasing the talinolol C max and AUC (0-) by 1.5to 1.8-fold, respectively, compared to the control group. When the combination of BG (128 M) and NAR (3238 M) was tested, the C max and AUC (0-) of talinolol were reduced to around 63% of those obtained in the control group. This result was opposite of that when both compounds were given separately. Similarly, Ruby Red GFJ (acute) decreased the pharmacokinetic parameters of talinolol by half when contrasted with saline and by 2.7-fold when compared with the white GFJNS. However, a distinct result was observed after repeated ingestion of the same juice before talinolol administration. In this scenario, the C max of talinolol was increased by 1.6-and 3.6-fold and the AUC (0-) by 1.5and 3-fold,when compared with the control or acute administration of Ruby Red GFJ, respectively.

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107 0 1 2 3 4 5 6 0 100 200 300 400White GFJNS White GFJC Control without PPG Time (hours)Talinolol (ng/mL) 0 1 2 3 4 5 6 0 100 200 300 400 500Ruby Red GFJ Acute Ruby Red GFJ Chronic Control without PPG OJ Time (hours)Talinolol (ng/mL) 0 1 2 3 4 5 6 0 250 500 750Control PPG BG 32M BG 128M BG 128M + NAR 3238M Verapamil (4m/kg) Time (hours)Talinolol (ng/mL)ABCD 0 1 2 3 4 5 6 0 200 400 600Control PPG NAR 813M NAR 3238M NAG 500M Time (hours)Talinolol (ng/mL) Figure 4-3. Mean serum concentration-time profiles (n = 8) of talinolol in rats after a single dose oral dose (10 mg/kg) with: A white GFJ normal strength (NS), or white GFJ concentrate (GFJC); B Ruby Red GFJ acute or chronic treatment, or orange juice (OJ); C bergamottin (BG) (32 M or 128 M), BG (128 M) plus naringin (NAR) (3238 M), or verapamil; D naringenin (NAG) 500 M, NAR (813 M or 3238 M). Vertical bars represent the standard error of the means (SEM).

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108 Table 4-4. Pharmacokinetics of talinolol in rats after a single dose oral dose (10 mg/kg) in saline (control) or with verapamil, white grapefruit juice concentrate (GFJC), white GFJ normal strength (NS), Ruby Red GFJ, naringenin (NAG) 500 M, naringin (NAR) 813 and 3238 M, bergamottin (BG) 32 and 128 M, and a solution containing NAR (3238 M) plus BG (128 M). Data are expressed as mean (n=8) SEM. Treatment AUC(0-6) (ng.h/mL) AUC(0-) (ng.h/mL) C max (ng/mL) t max (h) t 1/2 (h) Control with PPG 501 (57) 512 (55) 274 (35) 2.0 0.8 (0.1) Control without PPG 538 (36) 558 (36) 294 (29) 1.9 (0.2) 1.0 (0.1) Verapamil (4mg/kg) 827 (133) 856 (132) 385 (84) 2.7 (0.2) b 1.0 (0.2) White GFJC 451 (64) 486 (68) 206 (30) 2.1 (0.1) 1.0 (0.2) WhiteGFJNS 684 (102) g 698 (102)g 337 (56) 2.1 (0.2) 0.7 (0.07) Ruby Red GFJ (acute) 249 (43) 259 (43) 121 (21) 2.2 (0.2) 0.9 (0.2) Ruby Red GFJ (chronic)* 735 (99) d 749 (98)d 442 (54) d 1.8 (0.2) 1.0 (0.1) OJ 530 (70) 545 (68) 279 (42) 1.9 (0.1) 1.0 (0.2) NAG (500 M) 801 (78) 812 (77) 493 (66) 1.7 (0.2) 0.8 (0.1) NAR (813 M) 906 (72) a 937 (70) b 482 (59) 2.1 (0.1) 1.2 (0.3) NAR (3238 M) 771 (81) e 797 (83) e 430 (64) 2.0 1.0 (0.1) BG (32 M) 403 (42) 429 (45) 222 (30) 2.0 1.6 (0.2) BG (128 M) 866 (108) f 896 (102) af 650 (91) cf 1.8 (0.2) 1.3 (0.3) NAR (3238 M) + BG (128 M) 302 (17) 323 (17) 166 (12) 2.0 1.6 (0.1) The juice was given twice daily during one week before drug administration. a p<0.05, b p<0.01, c p<0.001 when compared with the respective control; d p<0.01 when compared with Ruby Red GFJ (acute); e p<0.01 when compared with NAR (3238 M) + BG (128 M); f p<0.001 when compared with NAR (3238 M) + BG (128 M);. g p<0.01 when compared with Ruby Red GFJ (acute). Discussion For the first time in vivo studies involving the effects of pure GFJ compounds and a comparison of different brands and strengths of GFJ on P-gp activity is reported. Most of reports in the literature have focused in the interaction between GFJ and its components with CYP3A4 or tested the effects of only one juice on the activity of P-gp (40, 134, 137, 214).

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109 Similar as observed by other authors, the terminal half-lives of talinolol after oral administration in absence or presence of potential P-gp inhibitors was not affected, suggesting that any effect on talinolol pharmacokinetics parameters can be attributable to processes that occur in the gut rather than to modification of systemic clearance (134). Absence of significant effect of verapamil on talinolol disposition may be dose-related since this drug is also subjected to pre-systemic metabolism by intestinal CYP3A4 (216) and the augment on t max promoted by VER may be due to its inhibitory activity on the gastric emptying rate (217). Orange juice has been described to be a potent inhibitor of P-gp in vitro, modifying the permeability of saquinavir (62). Additionally, Lilja and co-workers (154) have showed that OJ was able to reduce the C max and AUC of celiprolol in humans by 89% and 83%, respectively. According to the authors, modulation of the intestinal pH and the function of some transporters may be responsible for this interaction. However, we found that the effect of OJ on talinolol pharmacokinetics was negligible and that GFJNS promoted an increase on C max and AUC (0-) of talinolol, but in a lesser extent than previously reported (134). Differences in the concentrations of drug-interacting compounds in the juices, variability on P-gp expression in the intestine, presence of multiple binding sites on P-gp, and the interplay of distinct transporters present in the enterocytes may have contributed to this discrepancy of results. On the other hand, the decrease on C max and AUC (0-) values by GFJC compared to the normal strength may be accounted at least in part by the relative higher viscosity of the concentrated juice, which can form a physical barrier and impair the absorption of talinolol in the GI tract.

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110 However, the effect observed with GFJC was not significantly different from the control group. Different effects were obtained after co-administration of talinolol with Ruby Red GFJ compared with GJNS, suggesting that additional constituents seem to be involved in the interactions, affecting preferentially the intestinal uptake rather than P-gp (128). Recent studies also have demonstrated a remarkable decrease of the oral bioavailability of fexofenadine when co-administrated with GFJ. Because this drug is a substrate for both P-gp and OATP, these results indicate that the juice can inhibit preferentially the influx transporter rather than P-gp (79, 90). Conversely, the continued intake of the juice significant increased the C max and AUC (0-) compared with the acute treatment. One hypothesis for this observation may be an over expression of some intestinal uptake transporter such as OATP. Contradictory results regarding the acute and repeated ingestion of GFJ for about one week on the disposition of P-gp substrates have been reported. One study using rat everted ileum sac suggested that co-treatment with GFJ inhibits while chronic administration activates P-gp (214). On the other hand, a recent human trial determined that the C max and AUC of talinolol were reduced in both cases by half indicating that this drug might also be a substrate for some influx transporters (137). Therefore, more information about which transporters are involved with the absorption of talinolol and which are subjected to interact with GFJ is required to better determine the mechanism and relevance of this interaction. Regarding the flavonoids, it seems the maximum effect was reached with NAR (813 M), which increased significantly the AUC (0-) from 512 to 937 ng h/mL. This

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111 NAR concentration is similar of that found in the GFJNS used in this study. Although the aglycone NAG is not present in the juice it may also contribute to the overall inhibitory effect of GFJ, since this flavonoid can be formed as a result of the metabolism of the glycoside NAR by intestinal microflora (211). Among the components of GFJ tested, the furanocoumarin BG (128 M) was the most potent inhibitor of P-gp activity. Studies in vitro have reported this compound as a potent inhibitor of P-gp activity (IC 50 = 40 M) (61). Interestingly, the inhibitory effect of BG (128 M) was not observed when it was combined with NAR (3238 M). Although there is lack of information regarding the effect of multiple GFJ components on the activity of P-gp, this approach is an important tool to better understand such interactions allowing identify possible synergism or antagonism among GFJ components. The NAR to BG ratio of 25 employed in this study was the same found in the White GFJ but differed from that in the Ruby Red GFJ ( 38BGNAR ), suggesting that the relative concentrations of these components may also play an important role on the mechanism of grapefruit juice-drug interaction. Further investigations using different ratios, additional compounds, such as the dimers of furanocoumarins, and new combinations of GFJ components are necessary to clarify this issue. Conclusions In this work we validated a sensitive, accurate and precise HPLC method with ultraviolet detection to quantify talinolol in rat serum. We demonstrated that outcomes obtained during this food-drug interaction study was dependent on the type of the juice, duration of juice intake and the relative concentration of the potential interacting compounds. Although some differences were observed on talinolol disposition among all

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112 treatments, the overall effect of grapefruit juice or OJ was not statistically significant. Even in the cases when a statistical difference was detected the increase on the pharmacokinetic parameters of talinolol was around 2-fold compared with the control, which according to Bjornsson et. al (218) is considered a weak interaction and unlikely to be clinically relevant. However, this study only focused on the pharmacokinetic interaction rather than pharmacodynamics. Hence, physicians should be aware that ingestion of GFJ with other drugs substrates for P-gp with a narrow therapeutic index may not be free of side effects, especially considering that most of the drugs available are also metabolized by CYP3A4. Additionally patients should always report to their physician or pharmacist when they decide to take any drug with GFJ or whenever they experience any abnormality after concomitant drug intake with GJF.

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CHAPTER 5 CONCLUSIONS Fresh fruits, juices and vegetables are considered part of a healthy diet. Their health benefits have been demonstrated by many studies and are mainly due the presence of polyphenolic compounds which act as antioxidant preventing the formation of oxygen radical species, lipid oxidation, or decreasing risk of cardiovascular disease and certain types of cancer. However, concomitant administration of drug and food is not free of risk since it creates an opportunity for interactions that may change the drug disposition resulting in undesired effects. In fact, it is well recognized that grapefruit juice (GFJ) can increase the oral bioavailability of distinct classes of drug. Although the main mechanism of GFJ-drug interaction is currently known to involve the inhibition of the intestinal drug metabolizing enzyme CYP3A4, other drug-absorption pathways, such as the intestinal P-glycoprotein (P-gp), have also been suggested to be affected by GFJ. However, the GFJ constituents and the clinical relevance of the interaction between GFJ and P-gp is still a controversial issue and may depend on the intrinsic bioavailability of the drug, on the expression of the intestinal CYP3A4 and P-gp among subjects, and on the amount and type of GFJ ingested together with the drug. Therefore, further investigations are required to clarify this issue. The study presented here demonstrated a high variability regarding the concentration of the potential drug-interacting compounds present in different brands and lots of GFJ commercialized in our community, which may be responsible for the inconsistent results obtained in GFJ-drug interaction trials. The flavonoid naringin was, 113

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114 by far, the most abundant compound found in all lots and brands of GFJ analyzed followed by the furonocoumarins bergamottin and 6,7-dihydroxybergamottin. On the basis of the demonstrated variability and lack of information regarding the profile of flavonoids and furanocoumarins on the juices used for human trials, a direct comparison between different studies is impaired. Therefore, to minimize prevalent variability in GFJ-drug interaction trials, it is important to correlate measured endpoints to the concentrations of those grapefruit components that are considered to be relevant for the interaction with drugs. In future studies investigating grapefruit-drug interactions, concentrations of compounds with a suspected interaction should be considered. When possible, juices of very similar composition should be used for studies designed for direct comparison. In addition, for animal studies the concentrations of critical compounds may be adjusted by supplementation to normalize the administered juices. Our in vitro studies with Caco-2 cells as an intestinal barrier model and talinolol as a marker compound for P-gp transport suggest that among the GFJ constituents tested the furanocoumarins are much more potent P-gp inhibitors than the flavonoids. These compounds are present in the juice at high enough concentrations to significantly modify the activity of P-gp. Additionally, GFJ strongly inhibited the secretion of talinolol across the Caco-2 cells. In contrast, combination of both a flavonoid (naringin) and a furanocoumarin (6,7-dihydroxybergamottin) increased the secretion of talinolol, suggesting an activation of P-gp activity or inhibition of uptake transporters. It seems that the relative concentrations of the interacting compounds may also play an important role on the outcome suggesting that the overall modulation of GFJ on P-gp activity is the result of a more complex interaction than a simple additive effect. Therefore, further

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115 studies testing simultaneously of two or more grapefruit juice constituents are required to elucidate this question. Regarding the in vivo studies, co-administration of talinolol (10mg/kg) with bergamottin (128 M) or naringin (813 M) increased significantly the AUC of the drug. However, the effects of GFJ and OJ were generally minor. Similar to the in vitro study, combination of one flavonoid (naringin) and one furanocoumarin (bergamottin) promoted an opposite effect suggesting an increase of the intestinal secretion of talinolol due activation of P-gp, or a decrease of its absorption due inhibition of some influx transporter. Albeit Caco-2 is an established model for drug absorption, differences between this in vitro approach and in vivo studies are common and may be expected since the in vitro model does not account for the metabolism of grapefruit juice and its components, gastric emptying time, the presence of mucus layer, and other physiological variables. In summary, our pharmacokinetic results suggest that the interaction of GFJ or OJ with intestinal P-gp is unlikely to be of clinical relevance. Studies combining the pharmacokinetic and pharmacodynamic profiles of drugs co-administered with GFJ will help to a better estimation of the magnitude and the clinical relevance of this interaction.

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134 210. S. Deferme, R. Mols, W. Van Driessche, and P. Augustijns. Apricot extract inhibits the p-gp-mediated efflux of talinolol. J. Pharm. Sci. 91:2539-2548 (2002). 211. B. Ameer, R.A. Weintraub, J.V. Johnson, R.A. Yost, and R.L. Rouseff. Flavanone absorption after naringin, hesperidin, and citrus administration. Clin. Pharmacol. Ther. 60:34-40 (1996). 212. Q. Maoand J.D. Unadkat. Role of the breast cancer resistance protein (abcg2) in drug transport. Aaps J. 7:E118-133 (2005). 213. M.E. Taub, L. Podila, D. Ely, and I. Almeida. Functional assessment of multiple p-glycoprotein (p-gp) probe substrates: Influence of cell line and modulator concentration on p-gp activity. Drug Metab. Dispos. 33:1679-1687 (2005). 214. R. Panchagnula, T. Bansal, M.V. Varma, and C.L. Kaul. Co-treatment with grapefruit juice inhibits while chronic administration activates intestinal p-glycoprotein-mediated drug efflux. Pharmazie. 60:922-927 (2005). 215. R. Oertel, K. Richter, B. Trausch, A. Berndt, T. Gramatte, and W. Kirch. Elucidation of the structure of talinolol metabolites in man. Determination of talinolol and hydroxylated talinolol metabolites in urine and analysis of talinolol in serum. J. Chromatogr. B Biomed. Appl. 660:353-363 (1994). 216. H. Glaeser, S. Drescher, M. Eichelbaum, and M.F. Fromm. Influence of rifampicin on the expression and function of human intestinal cytochrome p450 enzymes. Br. J. Clin. Pharmacol. 59:199-206 (2005). 217. R. Brage, J. Cortijo, J. Esplugues, J.V. Esplugues, E. Marti-Bonmati, and C. Rodriguez. Effects of calcium channel blockers on gastric emptying and acid secretion of the rat in vivo. Br. J. Pharmacol. 89:627-633 (1986). 218. T.D. Bjornsson, J.T. Callaghan, H.J. Einolf, V. Fischer, L. Gan, S. Grimm, J. Kao, S.P. King, G. Miwa, L. Ni, G. Kumar, J. McLeod, S.R. Obach, S. Roberts, A. Roe, A. Shah, F. Snikeris, J.T. Sullivan, D. Tweedie, J.M. Vega, J. Walsh, and S.A. Wrighton. The conduct of in vitro and in vivo drug-drug interaction studies: A phrma perspective. J. Clin. Pharmacol. 43:443-469 (2003).

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135 BIOGRAPHICAL SKETCH Whocely Victor de Castro was born in July 10 th 1970, in Ipatinga, Minas Gerais, Brazil. He obtained his bachelors degree in Pharmacy in 1992 and his Master of Sciences degree in Pharmaceutical Sciences in 2000 from Federal University of Minas Gerais. While doing his Masters, he became a substitute professor at the College of Pharmacy of the Federal University of Minas Gerais, teaching Biological Methods of Quality Control of Pharmaceutical Products and Cosmetics. During 6 years he was the Quality Control manager of the Ezequiel Dias Foundation, a governamental pharmaceutical company, in Belo Horizonte, Brazil. He started his PhD program in August 2002 in the Department of Pharmaceutics of the University of Florida under supervision of Dr. Hartmut Derendorf and Dr. Veronika Butterweck. Whocely received his PhD in pharmaceutics in December 2006.


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Copyright Date: 2008

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Table of Contents
    Title Page
        Page i
        Page ii
    Dedication
        Page iii
    Acknowledgement
        Page iv
    Table of Contents
        Page v
        Page vi
        Page vii
        Page viii
    List of Tables
        Page ix
        Page x
    List of Figures
        Page xi
        Page xii
    Abstract
        Page xiii
        Page xiv
    Introduction
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    Variation of flavonoids and furanocoumarins in grapefruit juices
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    Grapefruit juice and its components inhibit P-glycoprotein mediated transport of talinolol in caco-2 cells
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    Modulation of P-glycoprotein mediated transport of talinolol in rats by grapefruit juice, orange juice, flavonoids and furanocoumarins
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    Conclusions
        Page 113
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    References
        Page 116
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    Biographical sketch
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Full Text












EFFECT OF GRAPEFRUIT JUICE
ON P-GLYCOPROTEIN ACTIVITY
















By

WHOCELY VICTOR DE CASTRO


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Whocely Victor De Castro

































To my parents, my wife and my daughter.















ACKNOWLEDGMENTS

I would like to express my grateful thanks to Dr. Hartmut Derendorf for accepting

me in his team and for his continuous encouragement during my Ph.D. program. My

special gratitude goes to Dr. Veronika Butterweck for her invaluable assistance and

technical support during in vitro and in vivo studies. Many thanks also go to the members

of my supervisory committee Dr. Giinther Hochhaus, Dr. Jeffery Hughes and Dr. Susan

Percival for their guidance and helpful advice throughout the years.

I would like to thank friends and staff in the department of pharmaceutics for their

friendship and support, especially to Sasiporn Sarawek and Immo Zdrojewski for their

suggestions and contributions during the validation of the analytical methods.

I would like to extend my thanks to the secretaries of the department of

pharmaceutics Mr. James Ketcham, Mrs. Andrea Tucker, for their technical support. I

also would like to thank all my assistants (Anke Rubner, Julia Hoevener) and the post-

doc fellows, especially Dr. Vipul Kumar, for his friendship and technical support.

My personal thanks go to my mother and my father for their support, guidance and

encouragement throughout my life. Finally, I would like to express my sincerely

gratefulness to my wife (Enda) and my daughter (Ana) for their support, friendship,

kindness and love.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ......... .................................................................................... iv

LIST OF TABLES ............. ....... ...... ........ ........ .......... ........... .. ix

LIST OF FIGURES ......... ........................................... ............ xi

A B S T R A C T .......................................... .................................................. x iii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

The G rapefruit (Citrus paradise) ........................................... ........................... ..1
The H health B benefits of Polyphenolics .................................. ..................................... 2
G rapefruit Juice-D rug Interactions.......................................................... ..................4
Phenolic Compounds in Grapefruit and Citrus with Potential for Drug Interactions...9
M mechanism of A action ..... ........................................... .... .... .. ........... 13
Cytochrom e P450 Fam ily........................................................ ................ 14
Perm eability-glycoprotein: P-gp ........................................................................20
Organic Anion Transporting Polypeptide: OATP ..........................................26
O ral D rug A b sorption ..................................... .........................................................2 8
Contribution of Small Intestine to Drug Absorption and Metabolism .....................29
Mechanisms of Transport of Molecules across Intestinal Epithelium.....................32
Passive Transcellular D iffusion.......................................... ......... ... ............... 32
P aracellu lar T ran sp ort .............................................................. .....................34
Carrier-M ediated Transport.......................................... ........................... 34
Transcytosis ............................. ..... ................................. 35
In Vitro Methods for Studying Drug Absorption ....................................................35
Talinolol: Model Compound for Drugs Subjected to Intestinal Efflux...................37
Properties of Talinolol ...................................... ................. .... ....... 38
H hypothesis and O objectives .............. ........... .. ......................... ........................ 39
Specific Aim 1: Contents of Flavonoids and Furanocoumarins in Grapefruit
and G rapefruit Juice ............. .............................. .......... ........ ............4 1
Specific A im 2: In Vitro Studies ........................................ ...... ............... 41
Specific A im 3: In Vivo Studies ........................................ ....... ............... 41





v









2 VARIATION OF FLAVONOIDS AND FURANOCOUMARINS IN
GRAPEFRUIT JUICES: A POTENTIAL SOURCE OF VARIABILITY IN
GRAPEFRUIT JUICE-DRUG INTERACTION STUDIES............................... 42

Background ..................................... ................................. ........... 42
S p e c ific A im ...............................................................................................................4 3
M material and M methods ........................................................................... .............43
C h e m ic a ls ................................................................4 3
Sam ple Preparation....................... .. ........ .. .............. 44
Determination of Flavonoids (Naringin and Naringenin) ................................44
Extraction and Determination of Furanocoumarins (Bergamottin and
6',7'-Dihydroxybergamottin) ............................. ... .................. 45
Stock and Work Solutions for the Preparation of Calibration Standards............46
N aringin stock solution ........................................ .......................... 46
N aringenin stock solution....................................... .......................... 46
Bergamottin stock solution.......................... ..................46
6',7'-Dihydroxybergamottin stock solution............................. ...............46
N aringin w ork solution........................................... .......................... 47
N aringenin w ork solution....................................... .......................... 47
Furanocoum arins w ork solution........................................ ............... 47
Standard solutions of flavonoids .............. ............................................. 47
Standard solutions of furanocoumarins ....................... ............... 48
V alid atio n ................................................................4 9
Statistical A analysis ...................................... .............................50
Results and Discussion ...... .................. ......... .........51
L in earity ................................................................... 5 1
Sensitivity ............... ......... .......................52
Specificity .......... .. ........... ..... ...............52
P recision and A accuracy ................................................................................. 52
Recovery ............... ......... .......................57
Stability .............. .... ..... ................................. 57
Quantification of Naringin, Naringenin, Bergamottin, and 6',7'-
Dihydroxybergamottin in Grapefruit Juices. .............................................. 58
Localization of Naringin, Naringenin, Bergamottin, and 6',7'-
Dihydroxybergamottin in Grapefruit. .........................................60
C o n c lu sio n s........................................................................................................... 6 4

3 GRAPEFRUIT JUICE AND ITS COMPONENTS INHIBIT P-
GLYCOPROTEIN MEDIATED TRANSPORT OF TALINOLOL IN CACO-2
C E L L S .................................................................................................................. 6 5

B a c k g ro u n d .....................................................................................................6 5
S p e c ifi c A im ............................................................................................................... 6 6
M material and M methods ..............................................................................66
C h em icals .......................... ................................. ......66
C e ll C u ltu re ................ ........ ................. ............................................. 6 7
Stock, Work Solutions, and Preparation of Calibration Standards ..................68









Transport Study ........................... ..................... ...............74
Sam ple A naly sis .............................................................75
D ata A n a ly sis ................................................................................................. 7 7
Statistical A analysis ...................................... .............................78
R e su lts ................................................................................................................... 7 8
L in earity ................................................................... 7 8
S p e c ifi c ity ..................................................................................................7 9
S e n sitiv ity .................................................................. ..................................7 9
P recision and A accuracy ................................................................................. 80
Stability ............................................. 80
C concentration D ependence............................................................................ 8 1
In h ib itio n S tu d ie s........................................................................................... 8 2
Dose-response Studies........................................85
Effects of NAR and DHB on the Secretory Transport of Talinolol ....................87
D IS C U S S IO N ....................................................................................................... 8 8
C o n c lu sio n s ........................................................................................................... 9 1

4 MODULATION OF P-GLYCOPROTEIN MEDIATED TRANSPORT OF
TALINOLOL IN RATS BY GRAPEFRUIT JUICE, ORANGE JUICE,
FLAVONOIDS AND FURANOCOUMARINS ................................................ 92

B a c k g ro u n d .....................................................................................................9 2
S p e c ifi c A im ............................................................................................................... 9 3
M materials an d M eth od s ......................................................................................... 93
C h em icals ............... ..... .. ..... .......................................9 3
Stock, Work Solutions, and Preparation of Calibration Standards ..................94
T alinolol test solutions ....................................................... 94
Talinolol stock solution I....................................................... 94
Talinolol stock solution II ................................... ..... ............... 95
Talinolol stock solution III ..................................... ............................95
V erapam il test solution I ................................. ............... 95
Naringin test solutions ................................. .......................... ....95
N aringenin stock solution............................................... 96
N aringenin test solution ....................................................... 96
B ergam ottin test solutions .................................................................... 96
T alinolol standard solutions ....................................................... 97
Anim als and Experim ental Protocol ................................................................ 97
Sam ple Preparation............................................. 99
Sam ple A naly sis .............................................................99
V alidation .................................................................................................100
Pharmacokinetic Analysis ..................... ....... ...............101
S statistical A n aly sis ...................................................................................... 10 2
R e su lts ............................ .. .. ........... ... .... .............................................................. 1 0 2
Validation of Analytical Method to Measure Talinolol in Rat Serum ............102
L in e arity ...............................................................10 2
S p ecifi city .................................................... 10 3
Sensitivity .................................................................... .... ......... .................. 104









Precision, accuracy and recovery ............. .......................... .................104
S tab ility ................................................................... 1 0 5
Talinolol Pharm acokinetics ...................................... ...................... ........ 105
D iscu ssion .................................................................................................. .... 10 8
C o n c lu sio n s.................................................... ................ 1 1 1

5 C O N C L U SIO N S ......................................................................... ...... .... 113

L IST O F R E FE R E N C E S ......................................................................... ................... 116

BIOGRAPHICAL SKETCH .............................................................. ...............135















LIST OF TABLES


Table p

1-1 Pharmacokinetics parameters of talinolol after oral administration (50 mg) by
health volunteers (192) ..................................................... .... .. ...... .... 39

2-1 Concentrations of the standard solutions used for the calibration curves and
quality controls (QCs) of naringin (NAR) and naringenin (NAG). .......................48

2-2 Concentrations of the standard solutions used for the calibration curves and
quality controls (QCs) of bergamottin (BG) and 6',7'-dihydroxybergamottin
(D H B ) ................................................................................................ 4 8

2-3 Intra-day (n = 3) and inter-day (n = 9) assay parameters of furanocoumarins
(bergamottin and 6',7'-dihydroxybergamottin) and flavonoids (naringin and
naringenin) ............. ....................... ........... ...... ....... ........ 55

2-4 Recovery values (means + SD) of bergamottin, 6',7'-dihydroxybergamottin,
naringin, and naringenin from the spiked orange juice...........................................57

2-5 Naringin, bergamottin and 6',7'-dihydroxybergamottin content in different
brands of grapefruit juice sold in Florida. .......... ......... ......... ...........................63

3-1 Concentrations of the work solutions of talinolol (TAL) used during the
tran sp ort stu dies.............................................................................. ............... 7 1

3-2 Concentrations of the standard solutions used for the calibration curves and
quality controls (QCs) of talinolol. ........................................ ....... ............... 71

3-3 Concentrations of the inhibitory solutions of Verapamil hydrochloride (VER),
naringin (NAR), naringenin, (NAG), bergamottin (BG),
6',7'-dihydroxybergamottin (DHB), and 6',7'-epoxybergamottin (EPBG) ..............73

3-4 Concentrations of the inhibitory solution of naringin (NAR) plus
6',7'-dihydroxybergamottin (DHB) used during the transport studies.....................74

3-5 Intra-day (n = 3) and inter-day (n = 9) assay parameters of talinolol. Precision
expressed as CV% and accuracy as % of the theoretical concentration ................80

3-6 Concentration range that the grapefruit components are normally found in the
ju ic e .............................................................................. 8 5









4-1 Concentrations of the standard solutions used for the calibration curves and
quality controls (QCs) of talinolol. ........................................ ....... ............... 97

4-2 Regression parameters obtained from calibration curves of talinolol in plasma in
three different days ...................................... ........... .. ................. 103

4-3 Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of
talinolol in rat serum .. .......................................... ........... ..... ........104

4-4 Pharmacokinetics of talinolol in rats after a single dose oral dose (10 mg/kg)......108















LIST OF FIGURES


Figure p

1-1 Chemical structures of phenolic compounds in citrus. ............... ....... ...........12

1-2 M ucosa of sm all intestine............................................... ..............................

1-3 Possible transport routes a drug can follow to cross the intestinal epithelium ........32

1-4 Molecular structure oftalinolol. (*) chiral center. ................................................38

2-1 Mean calibration curves (n = 9) of naringin (A) and naringenin (B) in
methanol/water (1:1), and ofbergamottin (C) and 6',7'-dihydroxybergamottin
(D ) in D M SO /m ethanol (1:3)................................................................................ 5 1

2-2 HPLC separation and absorbance-wavelength spectra of A furanocoumarins:
6',7'-dihydroxybergamottin and bergamottin; B flavonoids: naringin and
naringenin ............... ........ ..............................................................53

2-3 Chromatograms corresponding to the orange juice (OJ), standard solutions (Std)
containing 6',7'-dihydroxybergamottin (DHB) plus bergamottin (BG) (panel
A), and naringin (NAR) plus naringenin (NAG) (panel B). ...............................54

2-4 Absorbance-wavelength spectra of A bergamottin; B 6',7'-
dihydroxybergamottin; C naringin............................................. ...............54

2-5 Results of the stability test after 36 hours at room temperature (A) and after 4
freeze/thaw cycles at -20 OC (B ).. ......................... .............................................58

2-6 Distribution of naringin (A), bergamottin (B) and 6',7'-dihydroxybergamottin
(C) in different tissues of white (open bars) and ruby red (shaded bar)
grapefruits................................... ................................. ........... 62

3-1 Structures of selected flavonoids and furanocoumarins present in grapefruit juice
and tested in this study. ................................... ............ ...... ...... ...... 66

3-2 Mean calibration curve (n = 9) of talinolol in mobile phase. Vertical bars
represent the standard deviations (SD) of the means. ............................................78

3-3 Chromatograms corresponding to the talinolol (100 ng/mL) in HBSS-MES..........79









3-4 Stability of talinolol (low, medium, and high QCs) ..............................................81

3-5 Concentration dependent coefficient permeability (Papp) corresponding to the AP
to BL and BL to AP transport of talinolol across Caco-2 cells monolayers..............82

3-6 Coefficient of permeability (Papp) of talinolol (100[tM) across the Caco-2 cells
m onolayers. ...........................................................................84

3-7 Inhibitory dose-response effects........................................ ........................... 86

3-8 Effect of the combination NAR:DHB (50:1.2; 125:3, 625:15, 1250:30, and
2000:48 [tM) on the secretary transport (BL-AP) of talinolol (100[tM) across the
C aco-2 cells m onolayers.. ............................. .... ....................................... 87

4-1 Comparison of chromatograms corresponding to the talinolol (I) and the internal
standard propranolol hydrochloride (II) after extraction from rat serum (A) with
that obtained after extraction of a blank sample (B). ........................... 103

4-2 Stability of talinolol (low, medium, and high QCs) in rat serum within 24 hours
at room temperature (RT) (A) and during 3 weeks at -70 OC (B). Data represents
means of three replicates and vertical bars the standard deviation of the means...105

4-3 Mean serum concentration-time profiles (n = 8) of talinolol in rats after a single
dose oral dose (10 m g/kg)....................................................................... 107















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFECT OF GRAPEFRUIT JUICE ON P-GLYCOPROTEIN ACTIVITY

By

Whocely Victor de Castro

December 2006

Chair: Hartmut Derendorf
Cochair: Veronika Butterweck
Major Department: Pharmaceutical Sciences

Grapefruit juice (GJF) interacts with several medications increasing their oral

bioavailability and the risk of toxicity. The predominant mechanism is the inhibition of

intestinal CYP3A4 by flavonoids and furanocoumarins present in the juice. Additionally,

it has been suggested that GFJ modulates the activity of the permeability-glycoprotein (P-

gp). In the intestine, this efflux transporter acts as a biological barrier pumping a variety

of drugs back from the enterocytes to the intestinal lumen. Inhibition of this back-

secretion may increase the initial amount of drug that enters the systemic circulation and

the possibility of side effects.

The aim of this study was to compare the ability of GFJ and some of its specific

flavonoids and furanocoumarins to interact with P-gp in vitro and in vivo in order to

estimate the clinical relevance of this interaction. Additionally, the components of these

interacting compounds in commercially available and fresh-squeezed GFJ as well as in









different tissues of grapefruit were quantified and compared in order to assess their

potential influence on the data gained in drug interaction studies.

Flavonoids and furanocoumarins were quantified by high performance liquid

chromatography (HPLC) and considerable variation was observed among the juices

tested. White grapefruit showed the highest concentration of naringin and

furanocoumarins located in albedo (inner peel) and flavedo (yellow layer of the peel)

compared with red varieties. Findings from my study suggest that the concentration of

potentially contributing compounds may crucially influence the magnitude of the

interaction and invalidate direct comparisons of studies where different juices have been

used.

The transport of talinolol, a non-metabolized P-gp substrate, across Caco-2 cell

monolayers, was determined in the absence and presence of distinct concentrations of

grapefruit juice and its constituents. A sigmoid dose-response model was used to fit the

data and to estimate the potencies of the potential inhibitors. Results from this study show

that isolated compounds of GFJ are able to inhibit the P-gp activity, but the overall effect

seems not to be a simple additive effect.

The effects of different types of GFJ and its components on the oral

pharmacokinetics of talinolol in rats suggest that this interaction is unlikely to be of

clinical relevance.














CHAPTER 1
INTRODUCTION

The Grapefruit (Citrus paradisi)

The word citrus is the Latin name of "Ke,\' ,\", a Greek word to designate trees like

cedar, cypress and pine. Because the scent of citrus leaves and fruits was suggestive of

cedar, the name citrus has been linked to the citron (Citrus medical the first citrus with

which the Europeans got acquainted. Later, Linnaeus grouped all other citrus fruits

known to him in the genus Citrus (1). However, the taxonomy and phylogeny have not

been completely elucidated yet and are subject of controversy, mainly, due to sexual

compatibility between citrus and related genera, the high frequency of mutations, long

history of cultivation, and wide dispersion (2).

The grapefruit (Citrus paradisi), specifically, belongs to the Rutaceae family and

received this denomination because the fruits grow in clusters similar to grapes (3).

Studies using genetic markers, together with conventional and taxonomic approaches,

strongly support that the grapefruit originated from an accidental hybrid between

pummelo (Citrus maxima) and sweet orange (Citrus sinensis) (2-4).

Almost all of Citrus species and its wild relatives are supposed to have been

originated in the tropical and subtropical regions of Southeast Asia (5). Conversely, it is

suggested that grapefruit emerged from the island of Barbados in the Caribbean, where

Griffith Hughes, the rector of St. Lucy's parish of Barbados, earliest described it as the

"forbidden fruit of Barbados" in 1750 (3). However, the first scientific description of









grapefruit is attributed to the botanist James Macfadyen in 1837, who named the

forbidden fruit Citrusparadisi Macf. (3).

Grapefruit seeds were probably brought to Florida in the beginning of the

eighteenth century by Counte Odettte Phillippi and distributed to locate growers, where

the settlers began to sow the seeds and acquired taste for the fruits (1, 6). Since that time,

several cultivars have been created, especially in the Indian River citrus region stretching

from Daytona Beach to central Palm Beach County. Today, in a typical season, Florida

accounts for 2 million tons of grapefruit a year corresponding to about 80 percent of the

U.S. total grapefruit production (7).

The Health Benefits of Polyphenolics

The health benefits associated with the consumption of vegetables and fresh fruits

are largely accepted and recognized by many (8-11). They are related to the presence of

polyphenolic compounds, particularly, flavonoids described to act as antioxidants in

different biological systems (12). In fact, Sun et al. (13) demonstrated a linear

relationship between the total phenolic content and the antioxidant activity in

phytochemical extracts of different fruits, including grapefruit (r2 = 0.98, p < 0.01),

indicating that phenols seem to be the major contributor to the total antioxidant activities

in fruits. The antioxidant activity of flavonoids is principally based on their ability to

capture electrons providing great stability to the flavonoid radical and preventing the

propagating chain reactions of the oxygen-free radicals formed during the natural

metabolism of any aerobic cells (12). Flavonoids may also act by chelating iron, which is

thought to catalyze the process leading to the appearance of free radicals (14).

Included in the ordinary diet of many Americans, the grapefruit and the grapefruit

juice are rich in flavonoids, such as narirutin, naringin, hesperidin, neohesperedin,









didymin, quercetin, poncirin and kaempferol. Their total concentration in the grapefruit

juice (GFJ) range from 200 to 840 mg/L (15).

It has been demonstrated that flavonoids have a protective role against many

degenerative disorders, such as cancer and cardiovascular diseases (11, 14, 16, 17). They

can prevent or minimize the oxidation of low- and very low-density lipoproteins,

disrupting a crucial step in the formation of the atherosclerosis lesion and then reducing

thrombotic tendencies (14, 18). The low-density lipoprotein (LDL) can be normally

oxidized or modified within sub-endothelial tissue by cells of the arterial wall including

macrophages, vascular smooth cells, and endothelial cells (19). The modified LDL can

trigger a receptor-mediated process, leading macrophages and smooth muscle cells to

accumulate significant amounts of cholesterol ester (CE). These CE-rich cells or foam

cells are characteristic indicators of early atherosclerotic lesions. There is some evidence

that after consumption of foods rich in flavonoids, such compounds were able to reach

the sub-endothelial space of the arterial wall at concentrations enough to protect

lipoproteins like LDL from oxidation (20).

Le Marchand et al. (21) found decreased rates of lung cancer among the Hawaiian

population whose diet included a large amount of onions and apples (rich in the flavone

quercetin) and white grapefruit (rich in the flavanone naringin). Additionally, Kawaii et

al. (16) assessed the chemopreventive properties of pure flavonoids from citrus fruits

against four human cancer cell lines (lung, melanoma, leukemia and gastric cancer). The

anticarcinogenic properties of flavonoids are related to the suppression of metabolic

activity; antimutagenic, antioxidative, and antiproliferative effects; detoxification of









carcinogens; inactivation of oncogenes; inhibition of DNA adduct formation and the

carcinogenic cell invasion (11-14, 16, 18).

Cerda and co-workers (22) also have shown that dietary supplementation with

grapefruit pectin resulted in the decrease of plasma cholesterol (7.6%), low density

lipoprotein cholesterol (10.8%) and the ratio of low-density to high-density cholesterol

(9.8%) in healthy subjects. Furthermore, orange juice, grapefruit juice, and naringin, a

major grapefruit flavonoid, have been shown to have a beneficial effect on the blood lipid

profile and antioxidant capacity in rats (23, 24).

A case-control study carried out in 299 women (154 had lung cancer and 145 were

the control group) in Athens during a eighteen-month period did not find any correlation

between intake of food with high content of flavonoids with the etiology of lung cancer

(25). However, two distinct epidemiological studies involving around 11,000 volunteers

conducted by different groups in Finland and Netherlands during 1966-1976 and 1985-

1990, respectively, found that regular consumption of a diet rich in flavonoids was

inversely related to coronary heart disease mortality; cerebrovascular disease, lung and

prostate cancer, type 2 diabetes and asthma (26, 27).

Therefore, in vitro, pre-clinical, clinical, and epidemiological studies have

suggested that ingestion of flavonoid-rich foods such as grapefruit juice and grapefruit

juice provide some benefits against heart disease and cancer. However, since the early

nineties these potential health benefits have been over shadowed by possible risks of

interactions between drugs and grapefruit and grapefruit juice.

Grapefruit Juice-Drug Interactions

The first report of grapefruit juice interacting with a drug, altering its

bioavailability was published in 1991. This accidental discovery was made in a study on









ethanol drug-interactions where GFJ was used to mask the taste of the alcohol: the

bioavailability of felodipine, a calcium channel blocker, was increased when subjects

were consuming GFJ concomitantly with the drug. Additionally, the subjects showed a

decreased diastolic blood pressure and an increased heart rate (28).

Subsequent research in the area of fruit-drug interactions focused on grapefruit and

grapefruit compounds of which several were found to affect the absorption or metabolism

of certain drugs. Grapefruit juice was shown to modify greatly the disposition of a variety

of medications taken orally, including some 3-hydroxy-3-methylglutaryl coenzyme A

(HMG-CoA) reductase inhibitors lovastatinn, simvastatin) (29, 30), calcium channel

blockers (felodipine, nicardipine, nisoldipine) (31-33), benzodiazepines (midazolam,

diazepam) (34, 35), and HIV protease inhibitors (saquinavir) (36). Other fruits,

vegetables and dietary supplements also have the potential to cause an adverse interaction

with conventional drugs and are objects of concern (37).

Additionally, consumers have become more aware of the health benefits of

antioxidants, which increased the availability and consumption of dietary supplements,

phytochemical extracts, and foods rich in antioxidant flavonoids, especially, together

with prescription drugs. In fact, over 16% of all prescription drug users reported that they

concurrently take at least one plant-based dietary supplement including grapefruit and

citrus products (38). This may increase the likelihood of an adverse interaction between

foods and certain drugs. For example, the absorption and/or metabolism of a drug may be

harmfully affected, shifting the administered dose outside of the therapeutic range, which

may lead to a lower effectiveness of the drug or to an overdose associated with undesired

or even dangerous side effects (39).









Byproducts from the citrus-processing industry such as grapefruit seed extract,

flavonoids, essential oils from the peel, and pectins may be added to other food products

in order to improve taste, consistency or overall quality. These byproducts also may be

used in the production of dietary supplements (40). Consequently, consumption of food

products containing such additives, which have a potential for an interaction with drugs,

are not free of risk.

Based on the current knowledge, grapefruit compounds interact with drugs, which

are metabolized by cytochrome P450 3A4 (CYP3A4) and also have a low or variable oral

bioavailability. In general, the drugs subjected to GFJ interaction have their area under

the concentration-time curve (AUC) and maximum plasma concentration (Cmax)

increased from 1.2 to 15-fold after oral administration, and the magnitude of the effect

maybe dependent on the brand and amount of GJF ingested, the timing of administration

relative to the food intake and the intrinsic oral bioavailability of each drug (41-44).

The enhancement of the oral bioavailability of some drugs such as felodipine,

nicardipine, and halofantrine after consumption of GFJ has been associated with higher

incidence of dose-dependent side effects among the subjects (32, 45, 46). Indeed, one

case-report study depicted a 59-year old patient receiving 60 mg of atorvastatin

(Lipitor) daily who developed rhabdomyolysis after drinking GFJ. Such condition was

reverted and his cholesterol levels normalized after replacing the current drug by

pravastatin, another HMG-CoA inhibitor but not but not metabolized by CYP3A4 (47).

Grapefruit and GFJ have a potential to interact with several oral medications when

consumed in moderate amounts such as 1-2 servings (48). The concern, that the

concomitant administration of grapefruit products with certain drugs may result in









toxicity has lead to the recommendation to avoid consuming grapefruit products in

combination with these medicines. The Food and Drug Administration (FDA) now

requires drugs such as cyclosporine, sirolimus, simvastatin, lovastatin, and felodipine, to

carry a warning label regarding the possibility of an interaction. For example, Neoral,

an immuno-suppressant drug, carries a label stating "...Grapefruit and GFJ affect

metabolism, increasing blood concentrations of cyclosporine, thus should be avoided"

(49). Procardia is labeled "... Co-administration of nifedipine with GFJ resulted in

approximately a 2-fold increase in nifedipine AUC and Cmax with no change in half-life.

The increased plasma concentrations are most likely due to inhibition of CYP3A4 related

first-pass metabolism. Co-administration of nifedipine with GFJ is to be avoided" (50).

Interactions with a number of medications also have been shown for Seville orange juice,

although Seville oranges are usually not processed to juice (51, 52).

For most drugs in question, definitive recommendations regarding their

concomitant administration with GFJ are not available, since conclusions concerning the

clinical significance of the observed or predicted grapefruit-drug interactions are still

limited and many data available are derived from in vitro experiments and not directly

applied to human responses. Furthermore, the determination of the clinical relevance of

observed interactions in human intervention trials is complicated by individual

variability. Additionally, predicting the clinical significance of pharmacokinetic drug

interactions is sometimes difficult especially for drugs where there are no robust methods

to quantify effects or side effects. There has been recent effort in the United States by the

FDA and the Pharmaceutical Research and Manufacturers of America (PhRMA) to

establish some general guidelines to help drug companies, prescribers, and patients









interpret the clinical significance of drug interactions(53). These are the result of clinical

experience gained from some well-known drug interactions, such as the inhibition of

CYP3A4. For this interaction, specifically, it could be shown that the benzodiazepine

midazolam is a reproducible probe that allows quantitative determination of a potential

interaction with an inhibitor of the CYP3A4 enzyme. The degree of interaction can be

measured based on the augmentation in the AUC of the midazolam serum concentrations.

It was recently proposed to classify changes of midazolam AUC being less than 2-fold as

"weak interaction", which is the case observed on midazolam co-administration with

ranitidine, relatively small volumes of GFJ, roxithromycin, fentanyl, or azithromycin(53).

AUC changes that range from 2- to 4.9-fold, which occur on midazolam co-

administration with erythromycin, diltiazem, fluconazole, verapamil, relatively large

volumes of GFJ, and cimetidine are classified as "moderate interaction." Changes that

are 5-fold or higher in midazolam AUC are labeled as "strong interaction." Examples of

drugs demonstrating strong midazolam interactions include: ketoconazole, itraconazole,

mibefradil, clarithromycin, and nefazodone. Strong drug interactions are considered

clinically significant and result in contraindications or warnings on the product label. The

clinical significance of moderate inhibitors may include decisions about dose adjustments

that should be based on the concentration-effect relationship.

It should be noted, however, that almost all drugs, which show an interaction with

grapefruit, can be replaced by alternative drugs belonging to the same therapeutic class

but without a recognized potential for interaction.

The major drug classes for which grapefruit or other citrus interactions include

antiallergics, antibiotics, anticoagulants, antimalaria drugs, antiparasitics, anxiolytics,









calcium channel blockers, HMG-CoA reductase inhibitors, anti-HIV drugs, hormones,

immunossupressants, anti-tumors, and beta blockers (40, 42, 43, 48, 54-57).

Phenolic Compounds in Grapefruit and Citrus with Potential for Drug Interactions

Phenolic compounds represent the major group of citrus compounds. Included are

hydroxycinnamic acids, flavonoids, such as flavanones, flavones, and flavonols,

anthocyanins, and furanocoumarins (Figure 1-1) (58). As previously described, many of

these components have antioxidant properties and may play an important role in

preventing chronic diseases such as cancer, coronary heart disease, diabetes, among

others (26, 27).

GFJ components such as the flavonoids naringin (NAR), naringenin (NAG),

quercetin, kaempferol and the furanocoumarins bergamottin (BG),

6',7'-dihydroxybergamottin (DHB) and its dimers, bergapten, bergaptol, and

6',7 -epoxybergamottin (EPBG) (Figure 1-1) have been suggested to contribute to GFJ-

drug interaction (54, 59-63).

In vitro and in vivo studies have demonstrated interactions between many drugs and

the furanocoumarins, BG and DHB. These GFJ compounds decreased the hepatic

metabolism of saquinavir in vitro and the concentrations necessary to reach half of the

maximal effect (IC5o) was shown to be 0.33 [tM and 0.74 [tM, respectively (64).

Additionally, BG was able to augment the Cmax and AUC of diazepam in dogs (65), of

nifedipine in rats (66), and of felodipine in humans (67). Several human intervention

studies also confirm the contribution of BG and DHB to drug-interactions with GFJ (41,

68-70).









GFJ components have been demonstrated to interact with susceptible drugs by

different mechanisms besides the reduction of metabolism. For example, BG, DHB,

bergaptol and bergapten were shown to elevate the steady state uptake of [3H]-vinblastine

sulfate in Caco-2 cells (71), whereas BG and DHB caused a decrease in the organic anion

transport peptide (OATP)-B-mediated uptake of estrone-3-sulfate into human embryonic

kidney cells (72). Therefore, the effects of grapefruit and GFJ are not restricted on the

activity of the enzyme CYP3A4 but also involve modulation of membrane influx and

efflux transporters such as the permeability glycoprotein (P-gp) and the OATP,

respectively.

Interactions with drugs also have been demonstrated for flavonoids from citrus.

Naringenin and naringin have been shown to inhibit the OATPB-mediated uptake of

estrone-3-sulfate in human embryonic kidney cells (72) and to interact with saquinavir

(64) and simvastatin (73) in vitro. Compared to the control group, NAR increased by 2-

fold the Cmax and the AUC of diltiazem in rats, while there was no significant change in

the time to reach the Cmax (Tmax) and terminal plasma half-life (t 1/2). NAR also increased

the oral bioavailability of quinine in rats by 42% (74). However, this flavoinoid did not

show any effect on the pharmacokinetics and pharmacodynamics of nisoldipine in

healthy volunteers (75).

Quercetin inhibited P-gp-mediated efflux of ritonavir in Caco-2 cells (76),

increased the Cmax and AUC of verapamil in rabbits by 2-fold (77), and inhibited the

metabolism of midazolam and quinidine in human liver microsomes (78). It did not have

an effect on CYP3A4-mediated metabolism and P-gp mediated transport of saquinavir









(64). Nobelitin and tangeretin inhibited OATP-B-mediated uptake of estrone-3-sulfate

into human embryonic kidney cells (72).

For several phenolics from citrus such as eriocitrin, poncirin, and sinapic acid and

anthocyanins, which occur in red grapefruit varieties and blood oranges, no interactions

with CYP3A4 and P-gp have been reported yet.

In vitro studies of single compounds can help to understand the mechanism of

action of each component as well as their relative contribution to the overall effect

observed after ingestion of a food with potential for interacting with drugs. However, the

clinical relevance of data obtained from in vitro studies with single compounds is

questionable, because they have a limited predictability of the effects of the examined

compounds in vivo. Moreover, some phenolics were shown to modify the absorption or

metabolism of drugs only at very high concentrations, which are likely to exceed the

expected in vivo concentration after the consumption of a moderate amount of a

grapefruit/citrus product. Additionally, the overall effect of GFJ in drug interactions

studies is probably the result of the mixture of several compounds present in the juice.

Therefore, the existence of synergism, antagonism or additive effects needs to be

investigated by testing, simultaneously, different combinations of phenolic compounds

normally found in GFJ. Nevertheless, studies with single compounds have demonstrated

that furanocoumarins and their dimers are primarily responsible for the interactions of

GFJ and drugs.

In conclusion, grapefruit, sour orange (Seville), and limes, which contain BG (69)

seem to have the highest potential among the citrus species for interacting with drugs,

whereas other citrus varieties such as sweet orange seem to have an overall low potential











for interfering with medications. However, in studies using juices rather than single


compounds, orange juice (C. sinensis), which does not contain NAR and only small


amount of BG and DHB, has also been shown to interact with some drugs. A more recent


study in rats demonstrated that orange juice (and apple juice) decreased the oral exposure


of fexofenadine, possibly through an inhibition of the influx transporter OATP (79). The


interaction of orange juice and fexofenadine has also been demonstrated in HeLa cells,


where orange juice at 5% of the normal strength potentially reduced the uptake of


fexofenadine, mediated through the human OATPA (80). In general, the effect of orange


juice on CYP3A4 is negligible and in some GFJ-drug interaction studies orange juice was


even used as control (81, 82).


Overall, it can be concluded that orange juice has a minor potential for drug


interactions.

OH O
OH 0 OH 0
S00 HO
H OH
OH 0 OH 0

OHOH OHO


7 Hydryo n .E.m en




beraBer amattn

0 0


Oy O Me O0



OOMe I
MeOA Me OMe 0


Figure 1-1. Chemical structures of phenolic compounds in citrus.









Mechanism of Action

A food-drug interaction can be defined as the alteration of drug exposure and drug

effects by food. The underlying mechanisms can be classified into two broad categories.

The first category is pharmacokinetics, which includes alterations in absorption,

distribution, metabolism, and excretion. The second category is pharmacodynamics,

which describes alterations in the drug concentration-effect relationship (83). Changes in

the pharmacokinetics of drugs are the more common consequences of citrus-drug

interactions, which may shift the effect of the drug outside of its therapeutic window,

possibly leading either to loss of effect or undesired side effects, or even toxicity (48).

Because the majority drugs exhibiting an interaction with GFJ are metabolized

mostly by CYP3A4, which is present not only in the hepatocytes, but also in the

epithelial cells of the intestine, it has been suggested that the effect of GFJ may be due,

primarily, to the inhibition of the activity of this drug metabolizing enzyme (48, 57, 83).

However, differently from the most potent CYP3A4 inhibitors such as ketoconazole,

where the main site of action is the liver, GFJ when consumed in usual volumes, is an

example of inhibitor that appears to act preferentially in the enteric CYP3A4, resulting in

a significant reduction of the presystemic metabolism of drugs without, therefore, modify

their elimination half-life (54, 56). For several drugs such as felodipine, cyclosporine,

midazolam, and nifedipine, the gastrointestinal mucosa has been demonstrated to be a

major metabolic organ, where an inhibition of CYP3A4 caused an increase in oral

bioavailability (84). Additionally, GFJ intake did not change the pharmacokinetics and

pharmacodynamics parameters of midazolam (85), and felodipine (45) after intravenous

administration, but increased their systemic bioavailability from oral dosage forms,









supporting that inhibition of intestinal CYP3A4 by maybe particularly important when

the drug is given orally together with the juice.

Additionally, it has been suggested that GFJ can delay the gastric emptying rate

contributing, in part, to the double peak phenomenon observed after co-administration of

nifedipine with the juice (86). In fact, the lower the pH of the gastric chyme that reaches

the small intestine, the slower will be rate of gastric emptying (87). It is also suggested

that GFJ influences not only the first-pass metabolism of drugs like nifedipine, but also

its secondary oxidative steps (86).

In summary, the major mechanism leading to a grapefruit-drug interaction appears

to be the reduction of the "first-pass" metabolism through the inhibition of intestinal

CYP3A4 (57), although inhibition of secondary metabolic pathways and decrease of

gastrointestinal motility cannot be excluded. In addition, GFJ also can modify the

disposition of several drugs substrates of P-gp and recent reviews compile the effects and

the clinical relevance of such interaction (43, 88). Moreover, GFJ and other fruit juices

have been shown to inhibit in vitro and in vivo a number of organic anion-transporting

polypeptides (OATPs) (72, 79, 80, 89, 90).

Cytochrome P450 Family

The cytochrome P450 (CYP) enzyme family is the major catalyst of phase I drug

biotransformation reactions and accounts for the metabolism, at least in part, of about

60% of all drugs subjected to oxidation in the body (91). CYP enzymes are bound to

membranes of the endoplasmatic reticulum and are predominantly expressed in the liver,

although they are also present in extra-hepatic tissues such as the gut mucosa (84). They

can catalyze a notable number of metabolic processes including aliphatic oxidation,

aromatic hydroxylation, N-dealkylation, O-demethylation, S-demethylation, oxidative









deamination, sulfoxide formation, N-oxidation, N-hydroxylation (83). Such

biotransformation reactions usually lead to inactivation and elimination of most

pharmaceuticals, therefore, ingestion of xenobiotics able to inhibit or activate the CYP

enzymes can affect the disposition of many of CYP-drug substrates.

More than 50 different human CYPs have been recognized to date and their

nomenclature is determined by resemblance of amino acid constitution (83). For instance,

the isoform CYP3A4 belongs to family 3 (matching 40% of amino acid sequencing) and

subfamily 3A (matching 55% of amino acid sequencing) (48). Among the CYP3A

family, the CYP3A4 isoform is the most abundantly expressed and represents

approximately 30% to 40% of the total CYP protein in human adult liver (92). It also can

be found in the enterocytes of the small intestine at levels of 10 to 50% lower than those

found in the liver (84). However, CYP3A4 concentrations equivalent or above of those

found in liver have already been verified in some subjects (93-95), which can explain

much of the inter-subject variability observed during clinical trials studying GFJ-drug

interactions.

The relatively high expression levels in the intestinal mucosa and the broad

substrate specificity may contribute to the high susceptibility of CYP3A4 for citrus-drug

interactions (96). Many drugs for which interactions with citrus have been demonstrated

are metabolized by CYP3A4 (48, 57, 83).

It is currently recognized that the major mechanism for grapefruit-drug interaction

is the inhibition of the drug-metabolizing enzyme CYP3A4 in the small intestine

resulting in a significant reduction of metabolism of drugs during their first passage from

the intestinal lumen into the systemic circulation (54). Types of intestinal CYP3A4









inhibition by GFJ and its components comprise both competitive and mechanism-based,

the latter resulting in accelerated degradation of the enzyme through suicide inhibition

(97, 98). In fact, the amount of CYP3A4 in the small intestine of healthy volunteers has

been shown to decrease by 50-60% after 4-6 h administration of 8 oz (-237 mL) GFJ,

without any change in corresponding messenger RNA levels (60, 97).

Several drug classes such as dihydropyridine calcium antagonists and HMG-CoA-

reductase inhibitors are affected by grapefruit-induced inhibition of CYP3A4. GFJ

increased the AUC and maximal plasma concentration (Cmax) for these calcium

antagonists within an approximated range of 1.5- to 4-fold on average of the values

obtained with water (48).

For the HMG-CoA reductase inhibitor atorvastatin, double-strength GFJ increased

the AUC 2.5-fold but not the Cmax, when the GFJ was administered over three days and

the drug was given on day three (99). In a very similar study design performed by the

same group with simvastatin, double-strength GFJ increased the AUC 16-fold and Cmax

9-fold (100). Additionally, repetitive ingestion of high volumes of grapefruit juice has

augmented the AUC values of lovastatin over 10- fold compared with water (29).

Furthermore, a daily volume of 250 mL of grapefruit juice had a modest effect on

lovastatin, however, in this study grapefruit juice had been taken about 12 h apart from

station (101). All drugs mentioned above are, to a certain extent, subject to CYP3A4

metabolism.

Lundhal et al. (102) compared the acute effect of GFJ intake (200 mL) on the

pharmacokinetics and haemodynamics parameters of the CYP3A4 substrate felodipine

with the interaction followingl4-days intake of drug with juice. The AUC and Cmax of









felodipine after acute treatment with GFJ and on day 14 were similar and around 57% to

73% and 114% to 138% higher than those from the control group, respectively.

Additionally, the authors reported a decrease of heart rate and increase of vascular

adverse events during the GFJ period compared with the control. These results suggest

that one glass of GFJ is enough to produce the maximum inhibitory effect on the

intestinal CYP3A4 activity.

A moderate consumption does not appear to lead to an inhibition of hepatic

CYP3A4 activity (103), however, the repeated consumption (200 mL three times a day)

of double-strength GFJ induced a higher increase in triazolam concentrations and

prolonged its half-life. The repeated consumption of GFJ may have caused an inhibition

of hepatic CYP3A4 (104). In a three day study, GFJ increased the AUC of simvastatin

3.6-fold and that of its active metabolite, simvastatin acid, 3.3-fold. Cmax of simvastatin

and simvastatin acid were increased 3.9-fold and 4.3-fold, respectively, when the GFJ

was administered for three days and simvastatin on day three (30). In an in vitro study

performed with several grapefruit compounds, it was shown that BG, DHB, and the

furanocoumarin dimers GF-I-1 and GF-I-4 inhibited CYP3A4-catalyzed nifedipine

oxidation in a concentration-and time-dependent manner, which is consistent with the

mechanism-based inhibition. DHB was more potent than BG, while the dimers were

more potent than the monomers. Not only CYP3A4 but also CYP2C9, CYP2C19, and

CYP2D6 seem to be affected by citrus compounds. In the same study, the inhibitory

effect of BG was stronger on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on

CYP3A4 (105). In an intervention trial with healthy volunteers, GFJ (twice daily)

decreased the activity of CYP1A2, as determined with caffeine as a probe (106). In









another human study, GFJ and naringenin caused a minor reduction of the activity of

CYP1A2 (107). In addition, ingestion of 8 oz of regular-strength GFJ once or three times

a day did not affect liver CYP3A4 activity, colon levels of CYP3A5, or small bowel

concentrations ofP-glycoprotein, villin, CYP1A1, and CYP2D6 (60, 97). Overall, the

inhibition of CYP enzymes other than CYP3A4 does not appear to be of great magnitude

and may clinically be relevant only for drugs with a narrow therapeutic range (96).

There is a lack of information available regarding the reversible and mechanism-

based inhibition kinetics for grapefruit compounds. In a study conducted with human

intestinal microsomes by Paine and coworkers (103), DHB induced a substrate-

independent reversible and mechanism-based inhibition on CYP3A4. In contrast, BG, a

more lipophilic compound, was a substrate-dependent reversible inhibitor and a

substrate-independent mechanism-based inhibitor. For BG, the inhibition for testosterone

was more potent than for midazolam, possibly due to the higher affinity of BG for the

testosterone-binding site than for the midazolam-binding site. As mechanism-based

inhibitors, BG and DHB are definitely substrates for CYP3A4, but the binding sites are

not known. The authors suggested that both furanocoumarins inactivate CYP3A4 by the

binding of the furanoepoxide to the apoprotein, presumably at or near their respective

substrate domains. The same group determined the onset time of inhibition by both

compounds (63). It was found that DHB inhibited 85% of CYP3A4 activity independent

of substrate within 30 minutes, whereas the onset for BG-induced inhibition was much

later (70% inhibition was reached after three hours). The substrate-dependent inhibition

caused by BG was more than 50% after 0.5 to 3 hours for testosterone 6-hydroxylation,

while midazolam 1'-hydroxylation was unaffected, or activated, within one hour. Both









furanocoumarins caused 40% to 50% reduction of CYP3A4 protein, probably due to

intracellular degradation of the enzyme caused by mechanism-based inactivation. These

data imply that, after the consumption of GFJ, DHB causes the enzyme inhibition earlier

than BG.

Lilja and coworkers (108) conducted a crossover study in which 10 healthy

volunteers ingested 40 mg simvastatin with water (control), or with "high-dose"

grapefruit juice (200 mL double-strength grapefruit juice three times a day for 3 days), or

with water on days 1, 3, and 7 after ingestion of GFJ. When simvastatin was taken with

GFJ, the mean Cmax and AUC of simvastatin were increased 12.0-fold and 13.5-fold,

respectively, compared with control. The Cmax values when simvastatin was administered

with water 24 hours, and 3 days after ingestion of the last dose of GFJ, were increased

2.4- and 1.5-fold, respectively, compared with the control. Similarly, the AUC values

were 2.1- and 1.4-fold higher than those of the control group after 24 hours and 3 days of

GFJ intake, correspondingly. Seven days after ingestion of GFJ, no difference in the Cmax

or AUC of simvastatin were observed. Furthermore, Greenblatt et al. (109) determined,

in a human intervention trial, the time of recovery of intestinal CYP3A4 after the

consumption of 300 mL of regular-strength GFJ and a single dose of midazolam at 2, 26,

50, or 74 hours after administering the juice. After two hours, the AUC was 1.65-fold

increased and after 26, 50, and 74 hours, the AUC was 1.29-, 1.29-, and 1.06-fold higher,

respectively, in comparison to the control. The recovery half-life was estimated at 23

hours. These results indicate that the interaction of grapefruit juice with CYP3A4

substrates dissipates within 3 to 7 days after ingestion of the last dose of GFJ, consistent

with a mechanism-based inhibition.









In summary, the presented in vitro studies confirm the inhibitory effects of

grapefruit compounds on CYP-enzymes, with major effects on CYP3A4, involving both

mechanism-based and reversible inhibition. DHB appears to be more potent than BG;

however, furanocoumarin dimers seem to be more effective than monomers in the

inhibition of CYP3A4. The human intervention trials examining the pharmacokinetic

interaction of GFJ revealed a great inter-individual variability, where subjects with the

highest content of CYP3A4 showed the largest reduction of this enzyme after the

consumption of grapefruit.

Permeability-glycoprotein: P-gp

The interest in transporters as mediators of interactions between grapefruit and

drugs is increasing and one of the most studied drug transporters is the permeability-

glycoprotein or P-gp. This efflux transporter consists of a 170KDa glycosylated plasma

membrane protein that belongs to the ATP-binding cassette (ABC) transporters

superfamily (110, 111).

In humans, the multidrug resistance genes MDR] and MDR3 encode the P-gp

whereas, three members of this family (mdrla, mdrlb, and mdr2) are found in mice (88,

112). The P-glycoprotein encoded by human MDR] and mouse mdrla lb genes are drug

transporters, while human MDR3 and mouse mdr2 P-glycoprotein are thought to be

implicated mostly with the transport of phospholipids (113, 114). Structure-function

analyses revealed that P-gp consists of two homologous halves, each containing six

transmembrane domains where are located the drug-binding sites and a cytoplasmatic

nucleotide-binding domain where the ATP hydrolysis takes place generating the driving

force for the drug efflux (115, 116).









P-glycoprotein was first characterized in tumor cells contributing to multidrug

resistance (MDR) (117), but it is also expressed constitutively at high levels on the apical

membrane of luminal epithelial cells of different tissues, which are involved in drug

absorption and disposition such as hepatocytes cannalicular membrane, renal proximal

tubules, intestinal mucosa and capillaries in the brain (110, 118-120).

One of the most intriguing aspects of P-glycoprotein is its ability to recognize and

transport many drugs with a wide array of chemical structures. So far, a clear structure

activity relationship for predicting P-glycoprotein substrates has not been established and

the only common feature is that most of P-gp substrates are hydrophobic in nature,

suggesting that partitioning within the lipid membrane of cells is the first step for the

interaction of substrate with the active sites of P-glycoprotein (110).

Several models have been proposed to describe the mechanism for drug transport

activity. The more favorable model proposes that P-glycoprotein intercepts lipophilic

drugs either from outer membrane of lipid bilayer, before they enter the cell cytosol, or

from the inner membrane of the bilayer, and extrudes them into the extracellular medium

(115, 116, 121).

The development of mdrla, mdrlb, and mdrla/b knockout mice was a landmark on

P-glycoprotein research (122). In mdr-knockout mice one or both genes responsible for

P-gp expression are deleted, allowing investigation of the function of P-gp in a very clear

experimental design. For example, the AUC of paclitaxel was reported to be 2- and 6-fold

higher in mdrla (-/-) knockout mice than in the wild type animals after intravenous bolus

(i.v.) and oral administration, respectively. The increased AUC of paclitaxel after i.v.

administration in mdrla (-/-) mice was attributed to the decrease in elimination clearance,









whereas the higher AUC after oral administration in mdria (-/-) mice resulted from

combination of a decrease in the elimination clearance and enhancement of drug

absorption. Based on the AUC values after i.v. and oral administration, the bioavailability

of paclitaxel was calculated to be 11 and 35% for wild type and mdrla (-/-) knockout

mice, respectively (123).

Wetsphal et al. (124) demonstrated that rifampin (a P-gp inducer) was able to

reduce the AUC of intravenously (30 mg) and orally administrated (100 mg) P-gp

substrate talinolol in healthy volunteers by 21% and 35%, respectively.

Experiments in mice with cannulated gallbladder revealed that within 90 minutes

after intravenous bolus administration of [3H]digoxin approximately 16% of the dose was

secreted into intestinal contents of wild-type mice, whereas in mdrla (-/-) knockout

animals this value was reduced to only 2% (125).

In summary, the intestinal P-glycoprotein hinder the absorption of distinct classes

of drugs like anti-cancer, anti-HIV, cardiac glycosides, steroids, P-adrenergic agents,

immuno-suppressants, and antibiotics by carrying them from the enterocytes back to the

gut lumen (126, 127). Therefore, inhibition of the intestinal P-gp function may be

expected to be followed by enhanced oral bioavailability of its substrates, increasing the

risk of dose related side effects particularly for drugs with very low therapeutic index.

Due the remarkable overlaps in tissue distribution, substrates and inhibitors

specificity between CYP3A4 and P-gp (128) it is not surprising that GFJ and its

components can also interact with this membrane efflux transporter.

Among the many components of grapefruit juice (GFJ), the flavonoids (naringin,

naringenin, kaempferol, quercetin), the furanocoumarin (bergamottin,









6',7'-dihydroxybergamottin, and 6',7'-epoxybergamottin), and some polymethoxylated

flavones (tangeretin, nobelitin) derivatives (Figure 1-1) are able to modulate the activity

of P-glycoprotein and have been suggested to contribute to grapefruit juice-drug

interaction (56, 62, 71, 129-131).

In contrast to CYP3A4, the levels of P-gp in the small intestine are not affected by

GFJ or its components. Ingestion of 8 oz of GFJ (three times per day for six days) did not

alter the expression of intestinal P-gp in healthy volunteers (97).

Takanaga et al. (132) suggested that GFJ and its components were able to inhibit

the P-gp activity in Caco-2 cells using vinblastine as probe. Since vinblastine also is a

substrate of CYP3A4 (133), the GFJ effects regarding this inhibition were not conclusive.

However, GFJ and phenolic compounds from orange, such as tangeretin, nobiletin, and

heptamethoxyflavone, which have been demonstrated not to alter CYP3A4 activity,

increased the net influx of vincristine into adriamycin-resistant human myelogenous

leukemia cells conclusively, through the inhibition of P-gp (131). Several in vitro studies

with different probes, such as vinblastine (71), talinolol (134), and digoxin (135) also

confirm the findings that GFJ inhibits the efflux of P-gp substrates. In addition to GFJ,

orange juice and pomelo juice also have been shown to inhibit the activity of P-gp in

vitro (135). Flavones from orange juice have been described to be more potent than

compounds from grapefruit in the inhibition of P-gp (132).

In contrast to these findings, Soldner et al. (136) suggested that GFJ significantly

activates the P-gp-mediated efflux of drugs in Madin-Darby canine kidney (MDCK)-

MDR1 cells model.









Five GFJ components quercetinn, naringin, naringenin, bergamottin and

6',7'-dihydroxybergamottin) were screened as modulators of P-gp activity by assessing

the directional transport of saquinavir (a P-gp substrate) across Caco-2 cell monolayers

(64). Only naringin and 6',7'-dihydroxybergamottin had any appreciable effect, reducing

the net secretion of saquinavir transport from 25 to 7.6 and 7.1, respectively.

In order to evaluate the effect of grapefruit juice on P-glycoprotein-related transport

processes, measurements of permeability through Caco-2 cells and in vivo drug

absorption studies in Sprague-Dawley rats were performed using talinolol, which is a P-

gp substrate but not metabolized by CYP3A4, as a model compound (134). Apical to

basolateral talinolol transport across Caco-2 monolayers was increased 3-fold when GFJ

was present. During in vivo study in rats, doubled maximum plasma concentrations,

enhanced AUC values, and decreased apparent oral clearance were found for both

talinolol enantiomers at the same order of magnitude, suggesting an inhibition of P-gp by

GFJ.

A recent clinical trial, however, reported that a single glass of GFJ (300 mL)

reduced the talinolol AUC, and Cmax to 56% and 57%, respectively, of those with water.

Similar effect was observed after repeated ingestion of GFJ (300 mL three times a day for

6 days) (137). These results suggest that constituents in grapefruit juice preferentially

inhibited an intestinal uptake process rather than P-glycoprotein, although it is not clear

yet if talinolol is a substrate of any influx transporter, such as OATP. Therefore,

activation of P-gp cannot be discharged as a grapefruit effect in this study.

Edwards et al. (130) evaluate the effect of GFJ and Seville orange juice on the

disposition of cyclosporine, a P-gp substrate, in man. Compared with the control









experiment, the AUC and peak concentrations of cyclosporine were increased to 55% and

35%, respectively, by GFJ, whereas Seville orange juice did not have an influence on

cyclosporine, while both juices reduced enterocyte concentrations of CYP3A4. The

authors additionally demonstrated that DHB did not inhibit P-gp in vitro. In parallel,

Malhotra et al. (138) through a randomized three-way crossover intervention trial studied

the effect of Seville orange juice, diluted GFJ (normalized to contain equivalent total

concentration of BG and DHB as in Seville orange juice), and sweet orange juice on the

disposition of felodipine in healthy volunteers. Seville orange juice and GFJ increased the

AUC of felodipine. Considering both studies, while Seville orange juice and GFJ

probably interact with felodipine through inactivation of intestinal CYP3A4, the lack of

interaction between Seville orange juice and cyclosporine indicates that grapefruit may

cause interactions also through the inhibition of intestinal glycoprotein. In addition, these

data imply that the inhibition of P-gp activity by other compounds in GFJ (different from

those present in Seville orange) may be responsible for the increased bioavailability of

cyclosporine.

When a tablet containing 0.5 mg of digoxin was co-administrated with GJF in

healthy subjects the pharmacokinetics parameters were slightly increased (1.2- and 1.1-

fold increase in AUC and Cmax, respectively) (139), or not affected at all (140). However,

in both studies it should not be concluded that there is no interaction between GFJ and P-

gp since digoxin has a high oral bioavailability (70-80%) and even a total inhibition

would be expected to enhance its oral bioavailability by only 1.2-1.3-fold (141).

Similarly, the pharmacokinetics of indinavir, another known P-gp substrate was not

altered by GFJ (52), suggesting that possible other unknown mechanisms and factors









such as strength of the administered juices and length of consumption are relevant for the

interactions of citrus with drugs (142).

Overall, it can be stated that grapefruit and other citrus may interact with several

drugs through the combined inhibition of CYP3A4 and P-gp. Additionally, there are

significant differences among the published papers depending on the animal (rats, mice,

dogs) or cell model (Caco-2 or MDCK cell lines) used as well as culture conditions,

study design, and clinical relevance. Furthermore, the magnitude of interaction may

strongly depend on the variations in the polyphenolic profile of grapefruit juice, which is

related to the origin, quality of raw material, the manufacturing procedure, and storage

conditions (40).

In conclusion, the modulation of P-gp activity by grapefruit juice or its components

and the clinical relevance of such interaction is still unclear and is a controversial issue,

since some authors have reported activation (136, 143), others inhibition of P-gp (61, 71,

144), or absence of effect (52, 139, 140). Therefore, additional studies are required in

order to clarify the P-gp-related interactions between drugs and GFJ.

Organic Anion Transporting Polypeptide: OATP

The organic anion-transporting polypeptides (OATPs) are sodium-independent

influx membrane transporters belonging to the superfamily of solute carriers (SCL) (145).

So far, 39 members of the OATP superfamily have been identified in mammalian species

such as human, rat and mouse (146, 147) all of them with broad substrate specificity

(148).

OATP transporters are composed by 12 transmembrane domains and a large

extracellular region between the helices 9 and 10, which is believed to have a important

role on solute transport (149). Most of OATP members are expressed in the basolateral









membrane of polarized cells of multiple tissues, exceptions are the OATP1B1 and

OATP1B3 present predominantly on human hepatocytes (145, 150, 151) and the

OATP2B 1 that has been detected in the apical membrane of enterocytes (152).

The whole superfamily is classified into families and each family into subfamilies.

The individual families contain proteins with not less than 40% in amino acid sequence

identity and are indicated by numbers (e.g., OATP1, OATP2, OATP3, etc.). Subfamilies

contain proteins with amino acid sequence identities equal or higher than 60% and are

designated by letters (e.g., OATP1A, OATP1B, OATP1C, OATP2A, etc.). Individual

paralogues within a subfamily are indicated by different final numbers (e.g., OATP1B1,

OATP1B3) (152).

Considering the co-localization of these absorptive transporters and the secretary P-

gp at the apical plasma membrane of the enterocytes, their potential significance in the

oral absorption cannot be neglected and might be related with a number of drug-drug and

food-drug interactions (120, 127).

GFJ and orange juice (OJ) have been reported to decrease the AUC and Cmax values

of fexofenadine to 30 and 40%, respectively, of those when the drug was taken with

water (80, 90). Similar results were also find when celiprolol was co-administrated with

GFJ or OJ (153, 154). Both drugs are substrates for P-gp and OATP, but not CYP3A4

(155, 156). If P-gp had played a major role in the observed interactions, the

bioavailability would have been increased instead of decreased. This led to the

conclusion GFJ and orange juice preferentially decreased the OATP-mediated transport

of fexofenadine and celiprolol, than the transport carried-out by P-gp.









In a study with human embryonic kidney cells expressing OATP1B, different citrus

juices were tested in their effect on the uptake of estrone-3-sulfate. GFJ, orange juice,

BG, DHBG, quercetin, naringin, and naringenin significantly inhibited OATP1B-

mediated uptake of estrone-3-sulfate. The citrus compounds DHB and tangeretin

significantly inhibited OATPlB-mediated influx of the probe glibenclamide (72). The

effects of fruit juices on the oral availability of fexofenadine also have been tested in rats

(79). In this study, orange juice decreased the oral bioavailability of the drug to a lesser

extent than that observed in humans. Overall, it has to be considered that genetic

differences in the OATPs between humans and other species may contribute to

differences in the susceptibility to grapefruit-induced inhibition, which also is true for

other transporters and enzymes. In summary, OATPs appear to play an important role in

the influx of a number of drugs into enterocytes and hepatocytes. The inhibition of OATP

activity has been demonstrated for orange and GFJ in in vitro and in vivo experiments.

The clinical relevance of this mechanism remains to be investigated in further human

intervention trials.

Oral Drug Absorption

Most of prescription drugs are administered orally due to convenience of

administration, patient compliance, safety, ease of production and distribution. However,

this administration route is frequently associated with issues of low bioavailability,

defined as the rate and extent that a drug is absorbed from its dosage form and becomes

available at the site of action (157, 158).

Among the factors with a potential to influence the bioavailability are the physical-

chemical characteristics of the drug molecules (partition coefficient, ionization constant,

solubility, polymorphism, particle size and particle shape distribution, stability at various









pH values and against gastrointestinal enzymes), the pH and physiological conditions of

the absorption's site, gastric emptying, intestinal motility, absorption windows,

transporters, biotransformation and the manufacturing factors as excipients and

pharmaceutical technology (87, 157).

Additionally, the active ingredient of the dosage form should be appropriately

released, solubilized in the GI fluids, and should cross the epithelial barriers in order to

be absorbed throughout the gastrointestinal (GI) tract and reach the systemic circulation.

In such scenario, the term "release" may include several processes like disintegration,

deaggregation, diffusion, and dissolution of the drug in the GI fluids (87, 157, 159).

Contribution of Small Intestine to Drug Absorption and Metabolism

In a very simplistic point of view, the human gastrointestinal tract can be

functionally divided into a preparative and primary storage region (mouth and stomach),

a secretary and absorptive region (small intestine), a water reclamation system (ascending

colon), and finally, a waste-product storage system (the descending and sigmoid colon

regions and the rectum) (160).

The small intestine, comprising of duodenum, jejunum and ileum, has two major

functions: it can efficiently absorb nutrients, fluids, electrolytes, and many of the drugs

already developed, and simultaneously act as a physical and metabolic barrier against of

potentially toxic xenobiotics and pathogens (161). Such peculiarity can be explained by

its morphology and physiology, which provide an enormous surface area with ability to

absorb liquids and solutes selectively. The mucosa of small intestine is consisted of three

groups of projections: circular folds, villi and microvilli (Figure 1-2). The absorptive cells

or enterocytes are elongated, polarized and intimately connected by tight junctions,

forming a physical barrier between the contents of GI tract and the intercellular space









(87). Another physical barrier located just above the enterocytes is the mucus, a viscous

and elastic gel layer consisted of water, electrolytes, and high-molecular-weight (2 x 106

Da) glycoproteins, produced by the goblet cells (162, 163). The basolateral and apical

membrane of the enterocytes are markedly distinct conferring a polarized feature due the

presence of a striated end (brush border) at the top of the cells (apical side) towards to the

interior of the tract (Figure 1-2). These microvilli multiply by a factor of 20 the surface

area of the small intestine and are consisted, especially, of protein fibers of actin and

myosin (87, 162).

Additionally, most of functionally active transporters protein systems (sugar

transporters, P-gp, OATPs) and metabolizing enzymes (peptidases, esterases, CYP3A

isoforms, N-acetyl transferases and glutathione transferases) are expressed in the mature

enterocytes (163, 164) affecting, therefore, the fraction of the drug initially administrated

available for absorption.

The cellular membrane contains fundamentally a phospholipid bilayer, in which

glycoprotein molecules are located. The phospholipids are arranged such that the

hydrophilic ends face the two membranes surfaces, inner and outer, and the hydrophobic

tails are directed towards the membrane center (87, 162). Such characteristic makes

lipophilic compounds be rapidly and completely absorbed, while slowly and

incompletely passively absorbed drugs are, in general, hydrophilic and with poor

distribution into the cell membranes (165).

Although the intrinsic selectiveness of the small intestinal may impair the

bioavailability of many drugs, it is still the principal target for drug absorption following

an oral administration due to its vast surface area. Physicochemical properties of drug










molecules and formulations parameters can be modified in order to obtain a better

absorption profile. However, co-administration of two or more drugs subjects to similar

metabolic pathways or sharing the same transporter systems increase the likelihood of

drug-drug interaction. Similarly, the ingestion of dietary supplements and foods, like

grapefruit juice, able to affect the normal physiology of the small intestine, the activity of

the metabolizing enzymes, and carriers have a potential to modify markedly the

disposition of drugs taken orally.




Lrrnc of small in tsine












phi -- ..a MUL'









Capillary EI~WOr^L




circular folds, villi "finger-like" structures covered by a monolayer of
epithelial cells separating the lumen from the blood capillary network, and
microvilli the "brush board" projections located at the apical side of the
enterocytes

Source: http://instct.cit.coell.edu/courses/biog 05/pages/demos05/unit6/media/villus.
structure.jpg.









Mechanisms of Transport of Molecules across Intestinal Epithelium

Orally administrated drugs can enter the systemic circulation by crossing the

intestinal epithelium via two major routes, the paracellular or the transcellular pathways

(164). Paracellular can be further divided in passive transcellular diffusion, carrier-

mediated transport or transytosis (Figure 1-3). The importance of each route for drug

transport depends on the physicochemical properties of the compound being transported

such as lipophilicity, size, pKa, hydrogen bond potential, affinity for transport proteins

(166).


1 2 3 4 5











Figure 1-3. Possible transport routes a drug can follow to cross the intestinal epithelium:
(1) passive transcellular transport, (2) passive paracellular transport, (3) active
and/or carrier mediated transport, (4) transcytosis. In addition, drug transport
can be modulated by active efflux carrier mechanisms such as P-gp (5) (167)

Passive Transcellular Diffusion

The majority of drugs given orally reaches the systemic circulation by passive

transcellular diffusion (164). This process consists on permeation of the apical membrane

of the enterocytes, movement across the cytoplasma, and permeation of the lateral or

basolateral membranes (87). Since lipid barriers need to be crossed, passive diffusion of

drugs through the small intestine depends to a large degree on their lipophilicity. The

diffusion process can be mathematically described by the Fick's first law (equation 1-1):









J = P.A.(C-C2) (eq. 1-1)

where J is the flux or mass of transport over time, P is the permeability coefficient, A is

the surface area of the absorptive membrane, and (Ci C2) is the concentration gradient

of the uncharged from of the solute over the membrane. Assuming that the drug does not

accumulate in any region at the basolateral membrane of the enterocytes because the

blood and lymph flow provide a sink condition, the C2 becomes insignificant and the flux

can be expressed just in term of the apical concentration, Ci. Therefore, the driving force

of diffusion is mainly controlled by the concentration of drug remaining to be transported

from one to the other side of the enterocytes (87).

The permeability coefficient across the membrane, defined as the velocity of the

drug transport (cm/sec) into the body (127), can be defined by the equation 2.


K. D,
P = (eq. 1- 2)


where K is the partition coefficient between the water and the membrane, Dm is the

membrane diffusion coefficient and \ is the thickness of the of the membrane (162).

Transport of ionized compounds rely on some the physical-chemistry properties

such as the pKa of the drug molecule and pH of the absorption site, since these

parameters will define the equilibrium of the charge-state of the compound (162).

In general, these principles are followed by many drugs with the exception of

highly branched molecules, which are less permeable than expected due stereochemistry

issue since branched molecules results in greater disturbance in the arrangement of the

lipid layer (87).









Paracellular Transport

This transport route takes place via the tight-junctions between the enterocytes and

works as a semipermeable barrier, which protects against xenobiotics and controls the

passage of ions, water and small hydrophilic molecules (168). The apically located tight-

junctons restrict the paracellular transport of molecules that are larger than a certain

threshold size (molecular weight 350 Da) (164). Additionally, the surface area for the

paracellular transport has been estimate to be about 1000-fold smaller than the total

intestinal surface, suggesting a low efficiency of this pathway on drug transport (165).

Such characteristic has stimulated investigations to enhance the permeability of this route

and, therefore, increase the bioavailability of very hydrophilic drugs (165, 166, 169). The

tight-junctions are also a fence separating lipids and proteins of the apical and basolateral

domains of the enterocytes (168).

The tight-junctions become progressively tighter and the surface area decreases

from the small intestine to the colon, which can explain why the permeability of

hydrophilic compounds decrease in the same direction (170, 171).

Experiments using Caco-2 cells demonstrated that paracellular route can contribute

significantly to the absorption of many drugs including: atenolol, pindolol, cimetidine,

metformin, didanosine, and acyclovir (172). These molecules have a moderate molecular

weight and are relative hydrophilic.

Carrier-Mediated Transport

Generally, carrier-mediated transport processes consist of a reversible interaction

between the solute and a membrane protein, which acts as a transporter. Such transport

has unique characteristics such as specificity, competition, saturation kinetics, and energy

requirement (87). Transporters that use the energy of ATP hydrolysis to maintain a









gradient across the membrane are named primary active transporters, whereas, secondary

active transporters utilize the ion gradient across the membrane generated by the primary

active carriers to transport their substrates.

These carriers can be responsible for the uptake (influx transporters) or secretion

effluxx transporter) of many drug molecules across the intestinal epithelium and in both

cases it may result in non-linear pharmacokinetics and dose-dependent absorption. The

permeability-glycoprotein is a primary active efflux transporter, whereas the organic

anion transport peptide (OATP) is an example of secondary active influx carrier.

The relevance of a carrier-mediated transport for drug absorption depends on the

selectivity, capacity, direction, distribution, and expression of the transporters along the

intestine (127).

Transcytosis

Transcytosis is a specialized pathway where particles and macromolecules are

entrapped in vesicles due to invagination of the apical membrane before to be released

into the cytoplasma (87). Characteristics of this route include a very low capacity of

transport and the large amount of proteolytic enzymes inside the vesicles, which can

hydrolyze many of the solutes. The transport of vitamin B 12 is the best example of

transport mediated by transcytosis (165).

In Vitro Methods for Studying Drug Absorption

Intestinal drug permeability is considered to be one of the two major barriers to

intestinal drug absorption, solubility being the other. For assessment of the mechanistic

aspects of intestinal drug permeability, several in vitro systems have been developed.

Advantages of using in vitro models include small sample volumes, low variability

between replicates, the fact that many external factors that influence transport can be









manipulated easily, and, in general, the outputs obtained by the in vitro assessment can be

extrapolated to the in vivo scenario (165, 173, 174).

Epithelial cell culture models such as Caco-2 are commonly used (165). This

human colon adenocarcinoma cell line undergoes enterocytic differentiation in culture

allowing studies not only of passive diffusion processes but also, active drug uptake,

efflux carrier systems and presystemic drug metabolism, since they are able to naturally

express transporters like P-gp, sugar uptake transporters, multi drug resistance protein,

and metabolizing enzymes such as CYP3A4, N-acetyl transferases, and glutathione

transferase (89, 163, 167, 173, 175-177). The ability of the cells to form and maintain a

confluent monolayer, with characteristics similar to the in vivo barrier being modeled, on

some type of semipermeable support is a prerequisite for transport study.

Despite of the good versatility some potential weakness of the Caco-2 cell culture

model should be considered. For instance, the Caco-2 form very tight monolayers

compared to the human small intestine, which has been explained by the colonic origin of

the cell line (165, 173). Also, a unexpectedly large percentage of the compounds are

found to be substrates for P-gp, whereas the in vivo relevance of these findings remains

unclear, probably because the higher P-gp expression observed in this cell line compared

to the small intestine (163, 165).

The Madin Darby canine (MDCK) and porcine (LLC-PK1) kidney epithelial cell

lines consist of fast and simple additional in vitro models mostly used for screening tests

and measurement of passive diffusion, since they do not express transporters under the

normal conditions. They also can be used to study the effect of one single transport

system or the interplay of efflux and influx carriers, having a untransfected wild-type cell









line as control (62, 71, 76, 178). In contrast to Caco-2 cells, they do not need to be kept 3

weeks in culture before use. Limitations include the fact that MDCK and LLC-PK1 cells

are non-intestinal and non-human cell models (163, 179).

Excised segments of human or rat intestines properly positioned in an Ussing

chamber and the everted gut sac of rat small intestine placed in a oxygenated tissue

culture media are sensitive detection methods to study mechanisms of drug absorption at

different sites in the intestine, to evaluate intestinal metabolism of xenobiotics, to

quantify the paracellular transport of hydrophilic molecules, and to estimate the effects of

potent enhancers on their absorption (171, 180-183). Potential disadvantages of these

approaches are the presence of muscularis mucosa which may lead to underestimation of

compounds with propensity to bind to muscle cells, short duration of the experiments due

to limited cell viability, and the availability of human tissue is scarce (163).

Therefore, distinct in vitro techniques are accessible for the prediction of the

relevant parameters of drug absorption and their advantages, applicability, and limitations

should be carefully considered.

Talinolol: Model Compound for Drugs Subjected to Intestinal Efflux

Talinolol, a highly selective pl-adrenoceptor antagonist, was introduced into

clinical practice in 1975 in the former German Democratic Republic by AWD-Pharma

GmbH & Co. KG (Dresden, Germany) under the trade mark Cordanum. It is used in the

therapy of arterial hypertension, angina pectoris, ischemic heart disease, and rhythmic

disturbances of the heart (184, 185).

Studies in vitro (186, 187) and in vivo (188-190) have demonstrated that talinolol is

a substrate for the P-glycoprotein efflux system. Additionally, this drug is subjected to









minor metabolism in humans, dogs, and rats with less than 1% of the administered dose

being excreted into urine in form of hydroxylated metabolites (124, 191-193). Due the

remarkable overlaps in tissue distribution, substrates, inhibitors, and inducers specificity

between CYP3A4 and P-gp (128), talinolol is considered a good probe for mechanistic

studies involving interactions of drugs or food constituents, like grapefruit juice, with P-

glycoprotein. The intestinal efflux of talinolol can be estimated without an interference of

any biotransformation processes, and the variability of its oral pharmacokinetics should

reflect the variability of the P-gp function (193).

Additionally, talinolol has a moderate passive membrane permeability, which

prevents rapid passive absorption of the drug (187).

Properties of Talinolol

The chemical structure of talinolol ((1-(4-cyclohexylureidophenoxy)-2-hydroxy-3-

tertbutylaminopropane) (molecular weight = 363.5 Da) and its pharmacokinetics

parameters after oral administration are presented on Figure 1-4 and Table 1-1,

respectively.

0


H H OH H
Figure 1-4. Molecular structure of talinolol. (*) chiral center.

The basicity of the weak base talinolol (pKa = 9.4) refers to a secondary amino

functional group located close to the chiral center. Since the nitrogen of this amino group

can be protonated, talinolol features a pH-dependent solubility, which decreases with

increasing pH values (162). The log P of 3.2 indicates a moderate lipophilicity, compared

to other common P-adrenoceptor antagonists. It is less lipophilic than propranolol (log P:

4.6) and more lipophilic than atenolol (log P: 0.2) (194). Solubility in water (pH = 7.0)









have been reported to be 0.2 mg/mL and recommended daily doses range from 50 to 300

mg, in healthy volunteers safety of the drug was proven up to single doses of 400 mg

(186, 192).

Table 1-1. Pharmacokinetics parameters oftalinolol after oral administration (50 mg) by
health volunteers (192).
Parameters Values SD

AUC 0-36 1180 337 ng.h/mL

Vdss* 3.3 0.5 L/kg

Cltot 11.9 2.4 mL/min.kg

Cmax 168 47 ng/mL

Tmax 3.2 + 0.8 h

Elimination half life (ti/2) 11.9 + 2.4 h

Biotransformation < 1%

S60 %
Renal Excretion
of the bioavailable dose
S40%
Extrarenal excretion
of the bioavailable dose

Bioavailability 55 22%; dose dependent

* Vdss was calculated after a constant rate intravenous infusion of 30 mg of talinolol over
30 min
Hypothesis and Objectives

Grapefruit juice has been demonstrated to inhibit the metabolizing enzyme

CYP3A4 in the small intestine via suicide inhibition mechanism. As the result of such

inhibition, the oral bioavailability of many drugs of different therapeutic classes is

increased and in some cases dose dependent side effects are observed.









Because CYP3A4 and P-glycoprotein share many substrates, inhibitors, and

inducers, ingestion of GFJ is likely to modify the disposition of drugs transported by this

efflux system. However, the influence of different GFJ compounds on P-gp is still

unclear and is a controversial issue particularly, regarding the in vivo relevance of this

potential interaction. Therefore, we will test the hypothesis that grapefruit juice and its

constituents change significantly the disposition of talinolol, a substrate of P-

glycoprotein.

Concentrations of potential compounds able to interact with P-gp will be

determined in different brands and lots of grapefruit juice in order to assess the potential

influence of the variability of these juice components on the variability of data gained in

drug interaction studies.

Additionally, experiments using Caco-2 cells and talinolol as a probe compound

will be performed with the purpose of study the mechanistic aspects of the interaction of

grapefruit juice and its components with P-gp.

Furthermore, the effect of GFJ and some of its constituents on the

pharmacokinetics of talinolol in male Sprague-Dawley will be evaluated and the data

obtained from both in vitro and in vivo experiments will be correlated.

We expected that this study will aid to clarify the predominant mechanisms and the

major compounds contributing to the overall effect of grapefruit juice on drug

disposition.

Therefore, to test the hypothesis of this study the following specific aims were

proposed:









Specific Aim 1: Contents of Flavonoids and Furanocoumarins in Grapefruit and
Grapefruit Juice

1. To measure selectively, using validated and sensitive HPLC methods, the contents

of the specific flavonoids (naringin and naringenin) and furanocoumarins

(bergamottin and 6',7'-dihydroxybergamottin) in commercially available and

fresh-squeezed GFJ as well as in different tissues of grapefruit.

Specific Aim 2: In Vitro Studies

1. To determine the permeability of talinolol across Caco-2 cell monolayer

cultures in the absence and in the presence of different concentrations of

grapefruit juice and several of its components.

2. To estimate the kinetic parameters Km and Pmax of P-glycoprotein regarding

the transport of talinolol.

3. To calculate concentrations of grapefruit juice and its components able to

produce a half maximum inhibition (IC50) of the P-gp-mediated transport of

talinolol across the Caco-2 cell monolayers.

Specific Aim 3: In Vivo Studies

1. To evaluate the effect of grapefruit juice and its components on the

pharmacokinetics of talinolol in male Sprague-Dawley rats.














CHAPTER 2
VARIATION OF FLAVONOIDS AND FURANOCOUMARINS IN GRAPEFRUIT
JUICES: A POTENTIAL SOURCE OF VARIABILITY IN GRAPEFRUIT JUICE-
DRUG INTERACTION STUDIES

Background

The amount of active ingredients of grapefruit juice ingested may be an important

factor influencing the mechanism, magnitude, and reproducibility of the grapefruit-drug

interaction (48), compromising the comparison of data gained in different clinical studies.

A clinical trial with healthy subjects has revealed that the AUC and the Cmax of felodipine

were increased by about 73 and 138% compared with water, respectively, when a single

oral dose (10 mg) of the drug was administrated with 200 mL of GFJ (prepared by

diluting 50 mL of frozen concentrate with 150 mL of tap water) (102). However, in a

similar study co-administration of the same oral dose of felodipine and 250 mL of a

commercial GFJ increased the AUC and Cmax values by 305 and 228%, respectively (68).

Although the variability in magnitude of the grapefruit juice-drug interaction might be at

least partially explained by inherent differences in enterocyte CYP3A4 content (68, 97),

it is also possible that more concentrated or larger volumes of grapefruit juice caused a

more pronounced interaction. Furthermore, differences in the concentrations of drug-

interacting compounds in the juices used in both studies may have contributed to the

discrepancy of results. This points out the potential role of the concentration of

components causing drug interactions in juices used in clinical trials.









Specific Aim

The objective of this study was to compare the contents of the specific flavonoids

(naringin and naringenin) and furanocoumarins (bergamottin and

6',7'-dihydroxybergamottin) (Figure 1-1) in commercially available and fresh-squeezed

GFJ as well as in different tissues of grapefruit in order to assess the potential influence

of the variability of these juice components on the variability of data gained in drug

interaction studies. These compounds have been already described as the most abundant

present in GFJ and play a role in the GFJ-drug interaction (98, 195, 196).

Material and Methods

Chemicals

Naringin (NAR) and naringenin (NAG), both > 95% pure, were from Roth GmbH

& Co. (Karlsruhe, Germany), bergamottin (BG) (> 98% purity) was bought from

Indofine Chemical Company, Inc. (Somerville, NJ, USA), 6',7'-dihydroxybergamottin

(DHB), was kindly supplied by Dr. John Manthey at the U.S. Department of Agriculture

(USDA), Citrus and Subtropical Products Laboratory, Agricultural Research Service,

Winter Haven, FL. Upon isolation, the compound identification and purity (> 98%) was

measured by analytical thin layer chromatography, HPLC-MS and melting point

comparisons with authentic standard at the USDA laboratories. Ethyl acetate was bought

from Sigma Chemical Company (St. Louis, MO, USA), methanol HPLC grade,

dimethylsulfoxide, and orthophosphoric acid were acquired from Fisher Scientific (Fair

Lawn, NJ, USA) and purified water was obtained using a NANOPure system from

Barnstead (Dubuque, IA, USA).









Sample Preparation

Twenty-nine commercially available GFJ samples and two types of fresh grapefruit

(white and red) were purchased at local supermarkets. Concentrations of flavonoids

(naringin and naringenin) and furanocoumarins (bergamottin and

6',7'-dihydroxybergamottin) in fresh grapefruit were determined in the flavedo layer (0.1

g), pulp (0.1 g), albedo (1.0 g), seeds (1.0 g), and fresh-squeezed juice. Fruit tissues were

first homogenized with 6 mL (flavedo and pulp) or 12 mL (albedo and seeds) of purified

water using a PowerGen homogenizer (Fisher Scientific, Pittsburgh, PA) prior to the

extraction step. The freshly squeezed juices were obtained by using an HJ 29 handy

juicer (Black & Decker, Towson, MD). Samples were analyzed by a Shimadzu VP series

HPLC system (Kyoto, Japan) equipped with an SPD-MlOAvp diode array detector, an

LC-O1ATvp solvent delivery unit, an SIL-10AF autosampler, a CTO-O1Avp column

oven, an SCL-1OAvp system controller, a DGU-14A on-line degasser, an FCV-1OALvp

low-pressure gradient unit, and Class VP 7.2 SP1 chromatographic software.

Additionally, the peak purity software (Class VP 7.2 SP1 chromatographic software,

Shimadzu) was applied to the diode array data to test for impurities in all of the

chromatographic peaks of interest. The same software was used to determine the

similarity of the ultraviolet (UV) spectra of the target compounds present in the grapefruit

with those from the respective standard compounds.

Determination of Flavonoids (Naringin and Naringenin)

The GFJ and the homogenate of tissues (200 [tL) were mixed with cold methanol

(400 [tL), vortexed for 1 min, and centrifuged at 2500 g for 15 min, as previously

described (197). After filtration through a 0.45 |tm PVDF membrane filter (Millipore









Corp., Bedford, MA), the supernatant (25 [tL) was injected and analyzed at 285 nm. The

flow rate and the temperature were set to 0.5 mL/min and 35 C, respectively. Mobile

phases A and B consisted of water (pH 2.4) (adjusted with orthophosphoric acid) and

water (pH 2.4) (adjusted with orthophosphoric acid)/ methanol (40:60), respectively. The

250 x 4.6 mm i.d., 5 |tm, Lichrospher RP-18 column and Lichrospher 100 RP-18 guard

column (Merck KGaA, Darmstadt, Germany) were initially equilibrated during 30 min

with solvent A. After sample injection, an initial isocratic run for 5 min was followed by

a linear gradient from 100% of A at 5 min to 100% of B at 55 min. This condition was

maintained until 70 min and then returned to 100% of A, which was kept constant during

5 min before proceeding to the next injection.

Extraction and Determination of Furanocoumarins (Bergamottin and
6',7'-Dihydroxybergamottin)

GFJ and the homogenate of tissues (3 mL) were mixed with ethyl acetate (2 mL).

The extraction was performed by shaking the mixtures four times over 30 min. The

mixture was centrifuged at 3200 g for 20 min; the organic phase was collected and

evaporated under vacuum. The residue was reconstituted with 600 ptL of a

DMSO/methanol solution (1:3 v/v). The reconstituted residues were filtered through a

0.45 ptm PVDF membrane filter (Millipore Corp.). Volumes of 25 ptL of each sample

were injected and analyzed at 310 nm. The flow rate and the temperature were set to 1

mL/min and 35 C, respectively. Solvents A and B consisted of water and methanol,

respectively. The column and guard column (as used for flavonoids) were initially

equilibrated with mobile phase consisting of a mixture of solvents A and B (45:55),

respectively. Twenty minutes after injection, solvent B was increased linearly from 55 to

100% in 20 min. This condition was maintained for 5 min, after which the system









returned to the original mobile phase and was equilibrated for a further 5 min before the

next injection.

Stock and Work Solutions for the Preparation of Calibration Standards

All stock solutions were prepared in dimethylsulfoxide (DMSO) in order to obtain

a final concentration of 50 mmol/L. The stock solutions were kept at -20 oC and used

within 4 weeks.

Naringin stock solution

The amount of 58.5 mg of naringin (MW = 580.53 g/mol) was accurately weighed,

and transferred quantitatively to a 2.0 mL volumetric flask. The standard was then

dissolved in DMSO, the volume was completed with the same solvent, and the final

solution mixed thoroughly.

Naringenin stock solution

The amount of 27.2 mg of naringenin (MW = 272.26 g/mol) was accurately

weighed and transferred quantitatively to a 2.0 mL volumetric flask. The standard was

then dissolved in DMSO, the volume was completed with the same solvent, and the final

solution mixed thoroughly.

Bergamottin stock solution

The amount of 33.8 mg of bergamottin (MW = 338.42 g/mol) was accurately

weighed and transferred quantitatively to a 2.0 mL volumetric flask. The standard was

then dissolved in DMSO, the volume was completed with the same solvent, and the final

solution mixed thoroughly.

6',7'-Dihydroxybergamottin stock solution

The amount of 37.2 mg of 6',7'-dihydroxybergamottin (MW = 372.42 g/mol) was

accurately weighed and transferred quantitatively to a 2.0 mL volumetric flask. The









standard was then dissolved in DMSO, the volume was completed with the same solvent,

and the final solution mixed thoroughly.

Naringin work solution

Volume of 1 mL from the naringin (NAR) stock solution was accurately transferred

to a 5 mL volumetric flask, the volume was completed with a solution of methanol/water

(1:1) and mixed thoroughly. The final concentration of NAR was 10 mmol/L.

Naringenin work solution

Volume of 100 ptL from the naringenin (NAG) stock solution was accurately

transferred to a 5 mL volumetric flask, the volume was completed with a solution of

methanol/water (1:1) and mixed thoroughly. The final concentration of NAG was 1

mmol/L.

Furanocoumarins work solution

Volumes of 250 ptL and 100 ptL from the bergamottin (BG) and

6',7'-dihydroxybergamottin (DHB) stock solutions, respectively, were accurately

transferred to a 5 mL volumetric flask, the volume was completed with a solution of

DMSO/methanol (1:3) and mixed thoroughly. The final concentrations of BG and DHB

were 2.5 mmol/L and 1 mmol/L, respectively.

Standard solutions of flavonoids

From the NAR and NAG work solutions, five different concentrations of standard

solutions of naringin and naringenin and three quality controls (QC) were prepared in

methanol/water (1:1) according Table 2-1. All solutions were filtered through a 0.45 |tm

PVDF membrane filter (Millipore Corp.) before analysis.









Table 2-1. Concentrations of the standard solutions used for the calibration curves and
quality controls (QCs) of naringin (NAR) and naringenin (NAG).
Concentration
NAR work NAG work methanol/water Co
Standard (Clmol/L)
solution ([tL) solution ([tL) (1:1) q.s.p. (mL) (mol/L)
NAR NAG
1 50 100 10 50 10
2 150 200 10 150 20
3 250 300 10 250 30
4 350 400 10 350 40
5 450 500 10 450 50
QC1 50 150 10 50 15
QC2 250 250 10 250 25
QC3 450 450 10 450 45

Standard solutions of furanocoumarins

From the furanocoumarins work solution, six different concentrations of

bergamottin and 6',7'-dihydroxybergamottin and three QCs were prepared in

DMSO/methanol (1:3) according Table 2-2. All solutions were filtered through a 0.45

|tm PVDF membrane filter (Millipore Corp.) before analysis.

Table 2-2. Concentrations of the standard solutions used for the calibration curves and
quality controls (QCs) of bergamottin (BG) and 6',7'-dihydroxybergamottin
(DHB).
Sta d Furanocoumarins DMSO/methanol (1:3) Concentration ([tmol/L)
Standard
work solution([tL) q.s.p. (mL) BG DHB
1 10 10 2.5 1
2 25 10 6.25 2.5
3 100 10 25 10
4 250 10 62.5 25
5 500 10 125 50
6 1000 10 250 100
QC1 50 10 12.5 5
QC2 125 10 31.25 12.5
QC3 750 10 187.5 75









Validation

An external standardization method was validated over the range of concentration

of the target compounds present in the grapefruit juice and tissues. The validation

parameters of linearity, sensitivity, specificity, precision, accuracy, recovery and stability

were determined.

For each level of concentration, standards solutions and QCs were injected in the

HPLC in triplicate in three different days. GraphPad Prism version 4.0 (GraphPad

Software Inc, San Diego, CA, USA) was used to construct the calibration curves obtained

by plotting the mean area versus the corresponding concentration of the each standard

solution. The linearity of the standard curves was determined by least-squares linear

regression method and expressed in terms of coefficient of determination (r2). The intra-

and inter-day precision and accuracy of the quantification were measured by replicate

analyses of three different concentration levels (low, medium and high QCs) on the same

day and on alternate days. The precision was based on the calculation coefficient of

variation (CV %), and the accuracy was expressed as percent of the found amount

compared to the theoretical one. The calibration was considered suitable if not more than

1/3 of the quality controls showed a deviation from the theoretical values equal or greater

than 20% at the lower limit of quantification (LLOQ) and within 15% at all other

calibration levels.

The limit of detection (LOD) and LLOQ of NAR, NAG, BG, and DHB were

expressed on the basis of the mean values of the intercept (Ybl), the standard deviation

(Sbl) of the blank responses, and the slope (b) of the original calibration curve (equations

2-1 and 2-2) (198).
Y, +10S Y +3S
LOD= Ybl Ob (eq. 2 -1) LOQ= bI bl (eq. 2 2)
b b









Briefly, a second standard curve for each grapefruit juice component was obtained,

injecting each point in triplicate, but at lower concentrations than those of the used for

quantification. The Ybl values represent the intercepts of these new curves, corresponding

to the output as the concentrations of the analytes are equal to zero. Similarly, the Sbl

values were determined after plotting the standard deviation versus the correspondent

concentration level.

The recovery of each compound was evaluated by adding known amounts of pure

compounds to orange juice, from which they are naturally absent (199), and extracting

them under the same conditions used in the grapefruit samples. The analytical results for

extracted samples at three concentration levels were compared with unextracted standards

that represent 100% recovery.

To assess the flavonoids and furanocoumarins stability at room temperature, three

QCs (low, medium, and high) were prepared using fresh stock solutions and analyzed at 0

and 36 hours, as previously described. The stock solutions were then frozen at -20 OC and

freeze/thaw cycles were carried out once week up to 4 cycles. After each cycle, solutions

of flavonoids and furanocoumarins corresponding to the medium QC level were prepared

and analyzed by the validate HPLC method mentioned formerly. The areas were

compared to those obtained from fresh solutions for each concentration level and results

expressed as % of the control. Three replicates of each concentration level were prepared

for each test performed.

Statistical Analysis

Validation parameters were expressed as mean of three to nine determinations +

SD. The concentration of each compound in the juice and tissues was expressed as the







51



mean of the three determinations + SEM. Asterisks indicate significant differences for


white grapefruit compared to red grapefruit (p < 0.05). Data were analyzed by one-way


analysis of variance (ANOVA) with JMP5 software (SAS Institute Inc., Cary, NC,1996).


Mean separation was conducted using the Tukey-Cramer HSD comparison for all pairs (p


< 0.05). Values with different letters are significantly different (p < 0.05).


Results and Discussion

Linearity

Calibration curves (n = 9) for all four grapefruit juice constituents were linear


within the tested range of concentration with r2 > 0.999 (Figure 2-1), indicating that both


methods showed good linearity.



A B
2.5x107 r2 0.9998 1.3x106 r2 =0.9997
2.0x107 1.0x10 0
1.5x107 7.5x10
< 1.0x107, < 5.0x105
5.0x106- 2.5x105

0 100 200 300 400 500 0 10 20 30 40 50 60
Naringin (pmol/L) Naringenin (pmol/L)




C D
6.0x106 2.5x106
r2= 0.9991 r2= 0.9994
5.0x10' 2.0x106
4.0x10
c 1.5x106
S 3.0x106 -
< < 1.0x106
2.0x10 6
1.0x106 5.0x101
0 .0
0 60 120 180 240 300 0 25 50 75 100 125
Bergamottin (ptmol/L) 6',7'-Dihydroxybergamottin (ptmol/L)

Figure 2-1. Mean calibration curves (n = 9) of naringin (A) and naringenin (B) in
methanol/water (1:1), and ofbergamottin (C) and 6',7'-dihydroxybergamottin
(D) in DMSO/methanol (1:3). Vertical bars represent the standard deviations
(SD) of the means.









Sensitivity

The Ybl values for the blank responses of NAR, NAG, BG, and DHB were 6170.9,

7462.9, 271.1, and 671.6, respectively. In the same way, the Sbl values were 5026, 1790,

6.2, 30.7. Therefore, the LODs values for naringin, naringenin, bergamottin, and 6',7'-

dihydroxybergamottin were 0.47, 0.56, 0.01, and 0.04 [tmol/L, respectively, whereas the

LLOQs were 1.25, 1.11, 0.02, and 0.05 [tmol/L.

Specificity

The methods provided good resolutions between naringin and naringenin as well as

between bergamottin and 6',7'-dihydroxybergamottin. Peaks of the flavonoids and

furanocoumarins had similar retention times and the UV spectra (200-400 nm) in all

samples when compared to the standards. The wavelengths 285 and 310 nm used to

quantify flavonoids and furanocoumarins at their maximum absorption, respectively,

were confirmed by their UV spectra (Figure 2-2). There was no endogenous interference

from GFJ or orange juice (Figure 2-3) in either assay, indicating specificity of both

methods to the tested compounds. The results of the analysis of the peak purity software

suggested the absence of impurities for all compounds (purity > 95% for each individual

peak). Additionally, the UV spectra of all tested compounds showed more than 99% of

similarity with those obtained using the respective standard compounds (Figure 2-4).

Precision and Accuracy

The precisions intra- and inter-day for both flavonoids and furanocoumarins were

satisfactory with CV values between 0.55 and 6%. Similarly, the accuracy of the assay

was between 93 and 114.8% for all compounds tested at three different concentrations.

The results are summarized in Table 2-3.

















800



700



600



500


E
400



300



200



100


0 10 20 30 40 50 6(
Time (min)

180
B

naringin
160



140 loo0 2



120
o -- ----------
20D 2 2M 3 3 4

100


E
80



60
214


40



20
2 naringenin




0 10 20 30 40 50 60 7
Time (min)

Figure 2-2. HPLC separation and absorbance-wavelength spectra of A -

furanocoumarins: 6',7'-dihydroxybergamottin and bergamottin; B -

flavonoids: naringin and naringenin.


6',7'-dihydroxybergamottin


rrr

r~o
i


~arrrrrP1I*oss















A

Me |


Figure 2-3. Chromatograms corresponding to the orange juice (OJ), standard solutions
(Std) containing 6',7'-dihydroxybergamottin (DHB) plus bergamottin (BG) -
(panel A), and naringin (NAR) plus naringenin (NAG) (panel B).


RlOte Riton Tim: 32.05 mn


l00




40g


100
100



200 220 240


200 220 240 260 280 300 320 340 360 380 400
nm
Figure 2-4. Absorbance-wavelength spectra of A bergamottin; B 6',7'-
dihydroxybergamottin; C naringin. (1) represents the spectra of the standard
compound and (2) represents the spectra of the peak with same retention time
of the corresponding standard but obtained after injection of the grapefruit
sample.


I ill ii 10 U ~ ii Il II ~D )1 ln I
14*n


nm


nm












Table 2-3. Intra-day (n = 3) and inter-day (n = 9) assay parameters of furanocoumarins (bergamottin and 6',7'-dihydroxybergamottin)
and flavonoids (naringin and naringenin). Precision expressed as CV %and accuracy as % of the theoretical concentration.
Bergamottin
QC1-12.5 [tmol/L QC2-31.25 [tmol/L QC3-187.5 [tmol/L
Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3
Precision 0.7 0.6 3.8 1.0 1.8 0.9 1.1 2.3 0.7
Accuracy 107.4 109.4 108.4 105.0 108.4 106.2 104.4 108.0 103.4
Inter-day QC1-12.5 [tmol/L QC2-31.25 [tmol/L QC3 187.5 [tmol/L
Precision 2.4 1.3 1.5
Accuracy 108.6 106.5 105.3
6',7'-Dihydroxybergamottin
QC1-5 [tmol/L QC2-12.5 [tmol/L QC3-75 [tmol/L
Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3
Precision 1.5 1.2 1.9 1.2 4.9 1.7 5.2 0.8 0.9
Accuracy 110.3 114.8 114.2 106.0 107.0 99.0 94.6 107.4 105.0
Inter-day QC1-5 [tmol/L QC2-12.5 [tmol/L QC3-75 [tmol/L
Precision 2.9 4.1 4.03
Accuracy 113.1 104.0 102.3
Naringin
QC1-50 [tmol/L QC2-250 [tmol/L QC3-450 [tmol/L
Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3
Precision 4.3 0.6 0.8 0.6 1.2 1.5 3.2 2.1 1.6
Accuracy 98.2 95.1 93.0 104.7 106.0 100.9 98.3 98.3 100.8
Inter-day QC1-50 [tmol/L QC2-250 [tmol/L QC3-450 jtmol/L
Precision 6.1 2.0 5.0
Accuracy 95.4 103.9 99.1












Table 2-3 Continued
Naringenin
QC1-15 [tmol/L QC2-25 [tmol/L QC3-45 [tmol/L
Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3
Precision 2.4 0.8 3.6 2.3 1.2 0.7 1.3 0.6 5.9
Accuracy 100.9 96.6 96.6 106.1 103.1 96.2 100.8 99.5 99.8
Inter-day QC1-45 [tmol/L QC2-25 [tmol/L QC3-15 [tmol/L
Precision 3.0 5.8 4.5
Accuracy 98.1 101.8 102.7









Recovery

The recoveries from orange juice spiked samples were between 97.7 and 106%

(Table 2-4). Concentrations were back calculated using the equation obtained from linear

regression of the standard curves.

Table 2-4. Recovery values (means SD) of bergamottin, 6',7'-dihydroxybergamottin,
naringin, and naringenin from the spiked orange juice. Values in the
parenthesis represent the mean % recovered from each added concentration.

GFJ components added to the Theoretical Concentration Mean measured
orange juice ([tmol/L) Concentration (n = 3)
6.25 6.1 0.3 (97.7)
Bergamottin 25 24.8 1.7 (99.2)
150 156.6 + 12.4 (102.0)

5 5.2 0.1 (103.7)
6',7-Dihydroxybergamottin 25 24.9 0.8 (99.8)
100 101.3 + 2.8 (101.3)

50 52.2 + 3.3 (103.4)
Naringin 250 265.9 25.6 (106.2)
450 475.5 + 32.3 (105.9)

15 15.4 0.4 (102.7)
Naringenin 25 25.6 1.5 (102.5)
45 44.2 + 1.2 (98.4)


Stability

The standard solutions of flavonoid and furanocoumarins were found stable at

room temperature within 36 hours, whereas the stock solutions showed adequate stability

during at least 1 month after freeze/thaw cycles at -20 OC. In both cases, the shifting of

the areas of each sample tested was less than 5% of those obtained from a fresh solution

at the same level of concentration (Figure 2-5).










A
125-
S Low QC
100- = -- = = Medium QC
-I High QC
I 75-
0
50-

25-


BG DHB NAR NAG
B 125-
OI1BG (31.25 pmol/L)
100n n n DHB (12.5 pmol/L)
o C NAR (250 pmol/L)
S75- / NAG (25 pmol/L)
50
O 50-

25-


1 2 3 4
Cycles

Figure 2-5. Results of the stability test after 36 hours at room temperature (A) and after 4
freeze/thaw cycles at -20 OC (B). Data represents means of three replicates and
vertical bars the standard deviation of the means.


Quantification of Naringin, Naringenin, Bergamottin, and 6',7'-Dihydroxybergamottin
in Grapefruit Juices.

The values found for all compounds (Table 2-5) are in the same general range as

those reported in previous publications except for naringenin, which was absent in all

samples tested. In contrast to a previous study in which concentrations of naringenin were

found from 19.5 to 595 [pmol/L in 8 different brands of GFJ (196), we did not detect this

flavonoid in any of the 14 brands of juices analyzed, which was also in agreement with









other authors (41). However, a great variability of naringin (from 174 to 1492 [tmol/L),

bergamottin (from 1.0 to 36.6 [tmol/L), and 6',7'-dihydroxybergamottin (from 0.22 to

52.5 [tmol/L) contents was observed among all tested brands of GFJ. Although Bronner

and Beecher (195) found similar concentrations of naringin (850 [tmol/L) in two different

brands of GFJ (Giant and Minute Maid), a large variability of naringin, bergamottin, and

6',7'-dihydroxybergamottin in GFJ has also been confirmed by different authors with

ranges of 218-2062 [tmol/L (196), 2.0-28.3 [tmol/L (71), and 9.1-42 [tmol/L (71),

respectively. In the fresh-squeezed GFJ the highest concentration of naringin (312.6

[tmol/L) was measured in the white variety, whereas the red fruit provided the highest

contents of bergamottin (3.1 [tmol/L) and 6',7'-dihydroxybergamottin (10.3 [tmol/L). In

the pink GFJ (A-C) the concentrations of 6',7'-dihydroxybergamottin were not

statistically different intra- and inter-lot (p > 0.05) and brand B showed a higher content

of naringin when compared with the other two brands, although the variability of this

flavonoid between the lots was not significant. Additionally, the content of bergamottin

in lot 2 of brand C was 2.4-fold higher than in lot 1. Intra-lot comparisons revealed that

the content of naringin was significantly different (p < 0.05) in all brands of white GFJ

used in this study. The white brands D and E showed similar concentrations of

bergamottin within lots. In red juices only brand H showed significant intra-lot variability

of naringin and 6',7'-dihydroxybergamottin. The concentrations of bergamottin and

6',7'-dihydroxybergamottin in brand E (frozen from concentrate) were significantly

higher when compared to all other juices. Although there is a lack of detailed information

regarding the stability of furanocoumarins (bergamottin and 6',7'-dihydroxybergamottin)

present in GFJ, brand E is sold as a frozen product, which may possibly have prevented









extensive degradation of furanocoumarins compared to juices stored at room temperature.

In general, the white GFJ showed the highest levels of naringin, bergamottin, and

6',7'-dihydroxybergamottin with mean concentrations of 1010 87, 24.5 2.3, and 14.5

+ 6.7 tlmol/L, respectively. Additionally, the red variety showed higher amounts of

bergamottin (9.5 + 1.8 [tmol/L) and 6',7'-dihydroxybergamottin (5.6 + 1.9 [tmol/L)

compared to the pink variety, although the mean concentration of naringin was -1.7- fold

lower.

Localization of Naringin, Naringenin, Bergamottin, and 6',7'-Dihydroxybergamottin
in Grapefruit.

A comparison of concentrations and distribution of the target compounds between

fresh white and red grapefruit varieties (Figure 2-6) revealed that extracts from white

grapefruit showed higher concentrations of naringin (3152 [tg/g), bergamottin (19.8

[tg/g), and 6',7'- dihydroxybergamottin (106 [tg/g) located in the albedo and flavedo. The

lowest concentrations were found in the seeds and pulp of red grapefruit. These results

are in agreement with the values found in the commercial juices where, in general, the

white grapefruit juices showed the highest contents of naringin, bergamottin, and

6',7'-dihydroxybergamottin compared to the pink or red varieties. A similar study

was conducted to determine the content of the dimers of bergamottin and

6',7'-dihydroxybergamottin in the white and red grapefruits (200). Bergamottin and

6',7'-dihydroxybergamottin were able to inhibit the human microsomal CYP3A-mediated

testosterone 63-hydroxylation by half at 22 and 2 [tmol/L, respectively (98). With regard

to their effects on P-gp activity, bergamottin (10 [tmol/L) and 6',7'-dihydroxybergamottin

(33 [tmol/L) have been reported to reduce the activity of P-gp in vitro by 58 and 50%,









respectively (41, 80). In addition, 6',7'-dihydroxybergamottin has been shown to be a

potent inhibitor of rat OATP3 (IC50 = 0.28 [tmol/L) (80). Although the flavonoid naringin

has been excluded as the principal CYP3A4 inhibitor in GFJ (41, 75, 201), it promoted a

significant inhibition on P-gp and OATP activity in vitro at concentrations of 1000 and 5

[tmol/L, respectively (80, 202). Naringin has also been shown to be hydrolyzed into the

more potent CYP3A4 inhibitor naringenin by the gastrointestinal microflora located

preferentially in the distal part of the small intestine and in the colon (203). The clinical

relevance of this interaction is uncertain because most of the drug absorption takes place

in the small intestine. Overall, the concentrations of naringin, bergamottin, and

6',7'-dihydroxybergamottin in the samples analyzed in this study appear to be high

enough to considerably decrease CYP3A4, P-gp, and OATP activities. Other GFJ

constituents (not tested in this study) have been shown to inhibit the CYP3A4 activity,

such as the dimeric compounds (IC5o < 1 [tmol/L) (98) and epoxybergamottin (IC5o = 4.2

[tmol/L) (94). Additionally, the flavonoids quercetin and kaempferol have been shown to

inhibit the organic cation transporters (OCT) by half at 32 and 38 [tmol/L, respectively

(202). In general, the GFJ-drug interactions studies are characterized by a wide variability

in the pharmacokinetics and pharmacodynamics data among patients from the same

study, as well as between different studies. Variations in the intestinal concentrations of

CYP3A4 among individuals seem to contribute to the differences in the intensity of this

interaction (94). In addition, it was speculated that the high variability of components in

GFJ may also increase the risk of an interaction in patients who drink GFJ habitually and

appear to be equilibrated during a drug therapy, if the brand or even the lot is switched

during drug therapy with a susceptible drug (43). However, further studies comparing the











effect of different brands on the magnitude of the GFJ-drug interaction are needed to

clarify this issue. Additionally, the correlation between the content of the interacting

compounds in a GFJ preparation and their inhibitory effects would help to estimate the

role of each component on the overall GFJ-mediated inhibition and also to predict and

circumvent such interactions (109).



3000- A




zl .


Flavedo Albedo Pulp Seeds
Grapefruit tissues



S20 B



0,




Flavedo Albedo Pulp Seeds
Grapefruit tissues



O 100-
E




0 -


Flavedo Albedo Pulp Seeds
;o Grapefruit tissues

Figure 2-6. Distribution of naringin (A), bergamottin (B) and 6',7'-dihydroxybergamottin
(C) in different tissues of white (open bars) and ruby red (shaded bar)
grapefruits. Values are means + (SEM) (n = 3). Asterisks indicate significant
differences between values of white compared to red grapefruit.









Table 2-5. Naringin, bergamottin and 6',7'-dihydroxybergamottin content in different
brands of grapefruit juice sold in Florida.
Grapefit Concentration ([lmol/L)
G rapefruit i -------------------
peuit Variety Lots 6',7'-Dihydroxy-
Juices Naringin Bergamottin Beramottin
Bergamottin
Brand A L1 777.6 (4.3)a h-kb 9.76 (0.5) klm 0.44 (0.01) hij
L2 772.3 (10.1) h-k 9.03 (0.2) Imn 0.44 (< 0.01) hij
Brand B* L1 878.9 (32.4) gh 8.79 (1.0) Imn 0.65 (0.04) hij
Pink
L2 939.3 (34.1) fgh 12.13 (0.6) jkl 1.07 (0.02) hij
Brand C L1 668.5 (28.1) i-1 5.98 (0.02) mno 0.35 (< 0.01) hij
L2 654.6 (6.3) i-1 14.24 (0.1) h-k 0.76 (0.02) hij
Brand B* L1 1,262 (42.1) bc 18.81 (1.3) fgh 1.14 (0.1) hij
L2 1,492 (44) a 25.86 (1.0) de 2.21 (0.1) hij
Brand C L1 820.4 (43.9) ghi 21.95 (0.5) ef 1.03 (0.03) hij
L2 589.9 (14) Im 28.04 (0.3) cd 2.29 (0.3) hij
Brand D* White L1 915.6 (8.8) fgh 15.54 (0.9) g-j 3.37 (0.2) g-j
L2 651.8 (15.4) i-1 14.71 (0.7) g-j 1.34(0.2) hij
Brand E ** L1 810.9 (58.5) hij 31.77 (1.1) bc 45.04 (1.8) b
L2 1,056 (22.6) def 34.37 (1.6) ab 49.01 (2.5) ab
L3 1,318 (36.3) b 36.34 (1.6) a 52.51 (2.8) a
Brand F L1 1,216 (57) bcd 17.14 (0.4) ghi 0.73 (0.05) hij
L2 980.6 (7.8) efg 25.42 (0.7) de 1.15 (0.03) hij
Brand C L1 174.1 (2.8) o 2.95 (0.03) op 0.28 (< 0.01) ij
L2 238.9(3.8)0 5.06 (0.1) nop 0.28 (0.01) ij
Brand F L1 234.0 (3.1) o 3.59 (0.3) op 0.22 (< 0.01)j
L2 303.6 (12.6) no 4.02 (0.1) op 0.25 (< 0.01) ij
Brand D R L1 667.5 (15.7) i-1 13.56 (0.2) g-j 14.57 (0.4) c
Red L2 648.4 (14.8) jkl 14.16 (0.20) g-j 17.92 (0.7) c
Brand G L1 303.9 (5.1) no 5.17 (0.3) m-p 0.25 (< 0.01) ij
L2 291.5 (2.8) o 2.58 (0.2) op 0.24 (0.02) ij
Brand H L1 469.4 (18) mn 12.19 (0.5) i-1 4.55 (0.05) j-i
L2 1,147 (97) cde 12.73 (0.2) jkl 7.38 (0.02) efg
Brand I L1 584.4 (10) Im 19.18 (1.3) fg 8.74 (0.4) def
L2 613.4 (18.8) klm 18.92 (0.9) fg 12.10 (0.8) cd
Fh White 312.6 (9.5) no 0.97 (< 0.01) qr 4.71 (0.1) fgh
Fresh
squeezed Ruby
squeezed Red 245.7 (28.3) o 3.10 (0.1) p 10.28 (0.6) cde

aValues are means (n=3) with SEM in parenthesis. Values with same letters) are not
significantly different (Tukey-Cramer multiple comparison, p<0.05). = from
concentrate; ** = frozen from concentrate. Brands: A Blue Bird; B Thirft Maid; C
Ocean Spray; D Publix; E Minute Maid; F Win Dixie; G Sam's Choice;
H Florida's Natural; I Indian River.









Conclusions

The analytical methods applied were suitable to quantify the predominant

flavonoids and furanocoumarins in the juice and in different tissues of the grapefruit.

On the basis of the demonstrated variability and lack of information regarding the

profile flavonoids and furanocoumarins of juices used for human trials, a direct

comparison between different studies is difficult. Therefore, to minimize prevalent

variability in GFJ-drug interaction trials, it is important to correlate measured endpoints

to the concentrations of those grapefruit components that are considered to be relevant for

the interaction with drugs. In future studies investigating grapefruit-drug interactions,

concentrations of compounds with a suspected interaction should be considered. When

possible, juices of very similar composition should be used for studies designed for direct

comparison. In addition, for animal studies the concentrations of critical compounds may

be adjusted by supplementation to normalize the administered juices.














CHAPTER 3
GRAPEFRUIT JUICE AND ITS COMPONENTS INHIBIT P-GLYCOPROTEIN
MEDIATED TRANSPORT OF TALINOLOL IN CACO-2 CELLS

Background

Due to the striking overlaps in tissue distribution, substrates and inhibitors

specificity of CYP3A4 and permeability-glycoprotein (P-gp) (128) it is not surprising

that grapefruit juice (GFJ) and its components can also interact with this membrane

efflux transporter, which inhibition would be expected to be followed by augment of the

oral bioavailability of its substrates. However, modulation of P-gp activity by GFJ and

the clinical relevance of such interaction is still unclear and is discussed controversially,

since some authors have reported activation (136, 143) and others inhibition of P-gp (61,

71, 144). Recently, the P-blocker talinolol, which is a non-CYP substrate, yet a P-gp

substrate, showed an increase in bioavailability when associated with GFJ in both in vitro

and in vivo models (134). However, a clinical trial had shown an opposite outcome, i.e.

reduction of talinolol bioavailability (137) following its co-administration with GFJ in

healthy subjects.

Many GFJ compounds have been proposed to interact with CYP enzymes and P-

gp. These include both flavonoids (eg. naringenin, naringin, quercetin and kaempferol)

and nonflavonoids (eg. bergamottin, 6',7'-dihydroxybergamottin). However, the influence

of different GFJ constituents on P-gp is uncertain, particularly regarding the in vivo

relevance of this potential interaction mechanism.










Specific Aim

It was the aim of this study to systematically investigate in vitro, using a Caco-2

cell model and talinolol as a marker compound, the potential drug interaction between

selected ingredients of GFJ (Figure 3-1) alone and in combination- and the P-

glycoprotein transporter system, which controls many barriers in the body. Additionally,

the concentration dependent transport of talinolol was demonstrated and its kinetics

parameters estimated.

O O 0 O C


O O O



H HO OH H

Bergamottin 6',7'-Dihydroxybergamottin 6',7'-Epoxybergamottin


OOH
OH \H O
S OH OH O
HO ~O NO
HO OOH

OH 0
OH 0 O
Naringenin Naringin
OHOH
Figure 3-1. Structures of selected flavonoids and furanocoumarins present in grapefruit
juice and tested in this study.

Material and Methods

Chemicals

The following materials were used: (rac)-verapamil hydrochloride, Hank's

balanced salt solution (HBSS), 2-(N-morpholino)ethanesulfonic acid solution (MES);

sodium pyruvate solution (1 M), Lucifer yellow dipotassium salt, all of which were from









Sigma Chemical Company (St. Louis, MO, USA); Dulbecco's modified Eagle's medium

(1X) high glucose without sodium pyruvate (DMEM), nonessential aminoacids, trypsin

(0.05%)-EDTA (0.02%) solution, Fungizone antimycotic solution containing 250 |tg

of amphotericin B and 205 |tg of sodium deoxycholate per mL as solubilizer, penicillin G

(10,000 units/mL) and streptomycin sulfate (10,000 tlg/mL) solution,

(N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] buffer solution (1 M)

(HEPES) were obtained from Gibco BRL Life Technology (Grand Island, NY, USA);

fetal bovine serum heat inactivated and Dulbecco's phosphate buffered saline (DPBS)

(pH 7.4) were purchased from Mediatech Inc. (Herdon, VA, USA); talinolol (99.9%

purity) was gently provided by AWD-Pharma GmbH & Co. KG (Dresden, Germany),

naringin (NAR) and naringenin (NAG), both > 95% pure, were from Roth GmbH & Co.

(Karlsruhe, Germany), bergamottin (BG) (98% purity) was bought from Indofine

Chemical Company, Inc. (Somerville, NJ, USA), 6',7'-dihydroxybergamottin (DHB), and

6',7'-epoxybergamottin (EPBG) were kindly supplied by Dr. John Manthey at the U.S.

Department of Agriculture (USDA), Citrus and Subtropical Products Laboratory,

Agricultural Research Service, Winter Haven, Florida. Upon isolation, the compounds

identification and purity (> 98%) were measured by analytical thin layer chromatography,

HPLC-MS and melting point comparisons with authentic standards at the USDA

laboratories. Triethylammonium phosphate buffer (1 M) was obtained from Fluka

(Buchs, Switzerland). All others reagent were analytical grade.

Cell Culture

Caco-2 cells were obtained from ATCC (Rockville, MD, USA) and maintained as

stock cells in T flasks of 150 cm2 (Corning Costar Corp., Cambridge, MA, USA). The









growing medium consisted of DMEM containing 10% of fetal bovine serum, 1% of

nonessential amino acids, 100U/mL penicillin G, 100 tlg/ml streptomycin, 1.25 tlg/mL

amphotericin B, and 10 mM sodium pyruvate. The atmosphere was kept with 90-95% of

relative humidity, 37 C, 5% C02, and 95% air. The cells were passage upon reaching

80-90% confluence using trypsin-EDTA ( 5mL) and plated at densities of 1:5 1:7 in

new T flasks. After reaching passages 20-40 the Caco-2 cells were seeded at density of

60,000 cells/cm2 onto each 12-mm transparent polyester cell culture insert 12 well

plates with area of 1.13 cm2 and pore size of 0.4 |tm (Transwell, Corning Costar

Corporation, Cambridge, MA, USA). The DMEM medium was added in both apical (AP)

and basolateral (BL) compartments and changed 4 days after seeding, and every 2 days

thereafter. The cells were used on days 18 to 25 post-seeding to obtain differentiated

monolayers and higher expression of P-glycoprotein (173, 174). Transepithelial electrical

resistance values (TEER) were monitored with EndOhm Voltohmmeter equipped with a

STX-2 "chopstick" electrode (World Precision Instruments Inc, Sarasota, FL, USA) in

order to check cell confluence and integrity. Confluent Caco-2 monolayers with TEER

values >350 f cm2, after correction for the resistance obtained in control blanks, were

used in the transport experiment. All volumes amounted to 0.5 mL at the apical side and

1.5 mL at the basolateral side.

Stock, Work Solutions, and Preparation of Calibration Standards

The stock solutions at 50 mmol/L of NAG, BG, and DHB were prepared in

dimethylsulfoxide (DMSO) as previously described in Chapter 2. Talinolol stock solution

I was prepared in ethanol, verapamil hydrochloride (HC1) in buffer pH 6.0, NAR and

EPBG in DMSO.









Talinolol stock solution I

The amount of 36.3 mg of talinolol (MW = 363.5 g/mol) was accurately weighed,

and transferred quantitatively to a 2.0 mL volumetric flask. The standard was then

dissolved in ethanol, the volume was completed with the same solvent, and the final

solution mixed thoroughly in order to obtain a final concentration of 50 mmol/L

Talinolol stock solution II

The amount of 25 mg of talinolol (TAL) was accurately weighed and transferred

quantitatively to a 25 mL volumetric flask. The standard was then dissolved in ethanol,

the volume was completed with the same solvent, and the final solution mixed thoroughly

in order to obtain a final concentration of 1 mg/mL.

Talinolol stock solution III

Volume of 1 mL from the talinolol stock solution II was accurately transferred to a

100 mL volumetric flask, the volume was completed with a solution of 0.025 mol/L

triethylammonium phosphate buffer (pH 3.0 adjusted with 1 M NaOH) and acetonitrile

(77:23) and mixed thoroughly. The final concentration oftalinolol was 10 [tg/mL.

Talinolol stock solution IV

Volume of 1 mL from the talinolol stock solution III was accurately transferred to a

10 mL volumetric flask, the volume was completed with a solution of 0.025 mol/L

triethylammonium phosphate buffer (pH 3.0 adjusted with 1 M NaOH) and acetonitrile

(77:23) and mixed thoroughly. The final concentration oftalinolol was 1000 ng/mL.

Verapamil hydrochloride stock solution

The amount of 49.1 mg verapamil HC1 (MW = 491.05 g/mol) was accurately

weighed, and transferred quantitatively to a 2.0 mL volumetric flask. The standard was

then dissolved in buffer pH 6.0, the volume was completed with the same solvent, and the









final solution mixed thoroughly. The final concentration of verapamil HC1 was 50

mmol/L.

Naringin stock solution I

The amount of 145.1 mg of naringin (MW = 580.53 g/mol) was accurately

weighed, and transferred quantitatively to a 5.0 mL volumetric flask. The standard was

then dissolved in 1 mL of DMSO, the volume was completed with HBSS-MES (pH 6.0)

and mixed thoroughly in order to obtain a final concentration of 50 mmol/L.

6',7'-Epoxybergamottin stock solution

The amount of 35.4 mg of 6',7'-epoxybergamottin (MW = 354.41 g/mol) was

accurately weighed, and transferred quantitatively to a 2.0 mL volumetric flask. The

standard was then dissolved in DMSO, the volume was completed with the same solvent

and solution mixed thoroughly in order to obtain a final concentration of 50 mmol/L

Talinolol work solutions

For the inhibitory study, a dosing solution of talinolol (100 pM) was prepared in

HBSS-MES transport medium (HBSS buffered with 10mM MES and adjusted to pH 6

with 1 M NaOH) or in HBSS-HEPES transport medium (HBSS buffered with 10 mM

HEPES and adjusted to pH 7.4 with 1 M NaOH). The concentration-dependent transport

oftalinolol was studied in the concentration range of 25 to 1000 [LM (25, 50, 200, 400,

600, 800 and 1000 [LM). Solutions were prepared according Table 3-1 using the talinolol

stock solution I. In all cases the ethanol concentrations of the final solutions did not

exceed 2 %. Yamashita et al. (204) already reported that ethanol (< 5 v/v %) did not

affect the permeability of dexamethasone and the integrity of Caco-2 cells monolayers,

suggesting its usefulness as a solubilizing agent of drugs during in vitro experiments.









Table 3-1. Concentrations of the work solutions of talinolol (TAL) used during the
transport studies.
So n TL sk s n () Buffer pH 6.0 or pH 7.4 Talinolol
Solution TAL stock solution I (tL) (mo/
q.s.p. (mL) ([lmol/L)
1 5 10 25
2 10 10 50
3 20 10 100
4 50 10 250
5 80 10 400
6 120 10 600
7 160 10 800
8 200 10 1000

Talinolol standard solutions

From the talinolol stock solutions III and IV, nine different concentrations of

standard solutions and three quality controls (QC) were prepared in 0.025 mol/L

triethylammonium phosphate buffer (pH 3.0 adjusted with 1 M NaOH) and acetonitrile

(77:23) according Table 3-2. All solutions were filtered through a 0.45 |tm PVDF

membrane filter (Millipore Corp.) before analysis.

Table 3-2. Concentrations of the standard solutions used for the calibration curves and
quality controls (QCs) of talinolol.
TAL stock TAL stock Buffer/ACN Talinolol
Standard
solution III (|tL) solution IV (tL) (77:23) q.s.p. (mL) (ng/mL)
1 5 10 5
2 10 10 10
3 25 10 25
4 50 10 50
5 100 10 100
6 250 10 250
7 500 10 500
8 750 10 750
9 1000 10 1000
QC1 5 10 5
QC2 100 10 100
QC3 1000 10 1000









Inhibitory solutions

For the inhibitory experiments solutions ofverapamil HC1 (50, 100, 250, 500, and

1000 LM) (positive control) or NAR (100, 250, 500, 625, 1250, 2500 [LM), NAG (10, 50,

100, 250, 500 [LM), DHB (0.5, 10, 25, 50, 500 [LM) and EPBG (0.5, 1, 5, 10, 50 [LM)

were prepared in HBSS-MES in the presence or absence of the marker compound

talinolol (100 ptM), according shown in Table 3-3. BG was tested only in two

concentrations (1 and 10 ptM) due to solubility issues. Similarly, solutions containing a

fixed ratio of the flavonoid NAR to the furanocoumarin DHB (50:1.2, 125:3, 625:15,

1250:30, and 2000:48 ptM) were prepared considering the range these compounds are

found in the juice (Table 3-4). The effect of the combination of these major compounds

present in the GFJ on the P-gp mediated transport of talinolol was evaluated. The white

GFJ frozen concentrated (Minute Maid) was bought in the local market and diluted

with HBSS-MES to 0.5, 1, 5, 10, 25 and 50% in the presence or absence oftalinolol (100

[tM). The GFJ solutions were previously passed to a 45 |tm filter PVDF membrane filter

(Millipore Corp., Bedford, MA) before addition of talinolol. Concentrations of NAR,

NAG, BG, and DHB in the juice were 811 [LM, < 0.56 [LM, 31.8 [LM, 45 [LM,

respectively, as previously reported (205). The concentration of DMSO in these

inhibitory solutions was < 1%.









Table 3-3. Concentrations of the inhibitory solutions of Verapamil hydrochloride (VER),
naringin (NAR), naringenin, (NAG), bergamottin (BG),
6',7'-dihydroxybergamottin (DHB), and 6',7'-epoxybergamottin (EPBG) used
during the transport studies.
VER VER stock solution Buffer pH 6.0 or pH VER (
VER VER (amol/L)
([tL) 7.4 q.s.p. (mL)
1 25 25 50
2 50 25 100
3 125 25 250
4 250 25 500
5 500 25 1000
,NAR NAR stock solution Buffer pH 6.0 or pH NAR (
NAR NAR (amol/L)
([tL) 7.4 q.s.p. (mL)
1 50 25 100
2 125 25 250
3 250 25 500
4 312.5 25 625
5 625 25 1250
6 1250 25 2500
NAG NAG stock solution Buffer pH 6.0 or pH NAG
NAG NAG (amol/L)
(tiL) 7.4 q.s.p. (mL)
1 5 25 10
2 25 25 50
3 50 25 100
4 125 25 250
5 250 25 500
BG BG stock solution Buffer pH 6.0 or pH
BG BG (amol/L)
([tL) 7.4 q.s.p. (mL) B tm
1 1 50 1
2 10 50 10
DHB DDHB stock solution Buffer pH 6.0 or pH DHB (
DHB DHB (amol/L)
([tL) 7.4 q.s.p. (mL)
1 0.5 50 0.5
2 5 25 10
3 12.5 25 25
4 25 25 50
5 250 25 500
EPBG stock Buffer pH 6.0 or pH
EPBG EPBG (amol/L)
solution ([tL) 7.4 q.s.p. (mL)
1 0.5 50 0.5
2 0.5 25 1
3 2.5 25 5
4 5 25 10
5 25 25 50









Table 3-4. Concentrations of the inhibitory solution of naringin (NAR) plus
6',7'-dihydroxybergamottin (DHB) used during the transport studies.

NAR:DHB NAR stock DHB stock Buffer pH 6.0 Concentration
inhibitory or pH 7.4 (tlmol/L)
solution solution () solution (L) q.s.p. (mL) NAR DHB
1 25 0.6 25 50 1.2
2 62.5 1.5 25 125 3
3 312.5 7.5 25 625 15
4 625 15 25 1250 30
5 1000 24 25 2000 48


Transport Study

On the study day, the DMEM was removed, the Caco-2 monolayers were rinsed

with DPBS, and incubated for 30 min with the transport medium (TM) under the same

atmospheric conditions described previously. The TM on the AP and BL side consisted

ofHBSS-MES and HBSS-HEPES, respectively.

After 30 min incubation, the drug-free transport medium on the AP side was

replaced by the HBSS-MES medium (pH 6.0) containing talinolol or talinolol plus

inhibitor in order to investigate the AP to BL transport. Alternatively, for the BL to AP

transport assessment, the buffer of the donor chamber was replaced by a solution of

talinolol in HBSS-HEPES medium (pH 7.4), additionally the volume of the acceptor

chamber was replaced by HBSS-MES medium (pH 6.0) containing the inhibitor. For the

control experiments only solutions of talinolol were used. Thereafter, sample aliquots

(200 [tL) were taken at 20, 40, 60, 90 and 120 min from the AP or BL side and the

amount of talinolol transported at each time point was determined by HPLC. Following

the transport experiment, the TEER values were measured again and an additional control

of monolayer integrity was performed using Lucifer yellow (LY) (173)









Sample Analysis

A reversed phase-HPLC method previously described (124) was optimized to

enable talinolol to be quantified. Succinctly, samples from Caco-2 (25 [tL) were directed

injected into a Shimadzu VP series HPLC system (Kyoto, Japan) equipped with a RF-

10AXL fluorescence detector, a LC-1OATvp solvent delivery unit, an SIL-10AF

autosampler, a CTO-1OAvp column oven, a SCL-1OAvp system controller, a DGU-14A

on-line degasser, a FCV-1OALvp low-pressure gradient unit, and Class VP 7.2 SP1

chromatographic software. The excitation and emission wavelengths were set at 252 and

332 nm, respectively, the flow rate at 1 mL/min and the temperature at 40 oC. The

analytical column was a Lichrospher 60RP-Select B column, 250 x 4.6 mm i.d., 5 |tm,

preceded by a Lichrospher 60RP-Select B guard column (Merck KGaA, Darmstadt,

Germany). Talinolol was eluted isocratically using a mobile phase consisted of 0.025

mol/L triethylammonium phosphate buffer (pH 3.0) and acetonitrile (77:23).

The validation parameters of linearity, specificity, sensitivity, precision, accuracy,

and stability of this external standardization method for talinolol were determined. For

each level of concentration, standards solutions and QCs were injected in the HPLC in

triplicate in three different days. GraphPad Prism version 4.0 (GraphPad Software Inc,

San Diego, CA, USA) was used to construct the calibration curves obtained by plotting

the mean area versus the corresponding concentration of the each standard solution. The

linearity of the standard curves was determined by least-squares linear regression method

and expressed in terms of coefficient of determination (r2). Specificity was determined by

comparing the chromatogram of talinolol with that obtained from a blank solution

containing only the inhibitory compounds. The intra- and inter-day precision and









accuracy of the quantification were measured by replicate analyses (n = 3) of three

different concentration levels (low, medium and high QCs) on the same day and on

alternate days. The precision was based on the calculation coefficient of variation (CV%),

and the accuracy was expressed as percent of the found amount compared to the

theoretical one. The calibration model was accepted if the residuals (% difference of the

back-calculated concentration from the nominal concentration) were within 20% at the

lower limit of quantification (LLOQ) and within 15% at all calibration levels. Calibration

curves were rejected if more than 1/3 of the QCs showed a deviation from the theoretical

concentration equal or greater than 20%.

The lower limit of quantification (LLOQ) was established as the lowest

concentration used in the calibration curve.

The stability at room temperature and at 37 C of a solution of talinolol in HBSS-

MES (pH 6.0) and HBSS-HEPES (pH 7.4) transport medium was assessed within 48

hours and 4 hours, respectively. The same samples were frozen at -20 OC during one

week in order to check the freeze/thaw stability. Similarly, the stability of talinolol (low,

medium, high QCs) in mobile phase was determined within 24 hours at room

temperature. The samples were analyzed by the validate HPLC method mentioned

formerly, the areas were compared to those obtained from fresh solutions for each

concentration level, and results expressed as % of the control. Three replicates of each

solution were prepared for each test performed.

An additional stability test was conducted during 3 hours in HBSS-VMES at 37 C

for all grapefruit juice constituents tested in this study. Samples were analyzed as









described in Chapter 2. The 6',7'-epoxybergamottin was analyzed using the same method

as described for furanocoumarins in Chapter 2.

Data Analysis

The coefficients of permeability (Papp) talinolol were calculated by using equation

(1):


P = (1)
app ATxAx Co


where AQ/AT is the amount of drug (ng/min) appearing in the acceptor

compartment as a function of time obtained from the slope of the linear portion of the

amount transported vs time plot., Co is the initial concentration of talinolol in the donor

compartment (ng/mL), and A the surface area of the semi-permeable membrane in cm2

(62, 135). All solutions were kept under 37 C before use.

The passive and the Michaelis-Menten parameters of talinolol were estimated using

Scientist v. 2.0 (MicroMath Inc., St. Louis, MO, USA). To apply the model the equation

(2) was used:

P x C
P = P passive + -max (2)
app (BL-AP) app (BL AP) KM +Co



where the Papp (BL-AP) is the observed permeability (cm/sec) of talinolol from basolateral to

apical side of Caco-2 monolayers. The Papp (BL-AP) passive is the permeability due only to

diffusion, Pmax (cm/sec) is the maximal permeability due to the active transport and the

KM ([LM) is the Michaelis-Menten constant.









The IC5o values (concentration to reach half of the maximum inhibition) of

verapamil, GFJ and its components were then determined using nonlinear regression

according to the Hill equation with GraphPad Prism v.4.0 (GraphPad Software Inc, San

Diego, CA, USA). For NAR and verapamil, the best fit was obtained according to the

sigmoid dose-response model without the variable slope.

Statistical Analysis

The mean of at least three experiments and their standard error (SEM) were used to

express the values of Papp and the kinetics parameters. The confidence interval of each

IC50 value was determined. Statistical analyses were performed with one-way ANOVA

followed by Dunnet's multiple comparison tests as post-hoc analysis. A probability of

less than 0.05 (p < 0.05) was considered to be statistically significant.

Results

Linearity

The range of validation (5 to 1000 ng/mL) showed good linearity with r2 > 0.998

(Figure 3-2).


6.0x106.
1r= 0.9996
5.0x106.
4.0x106-

S3.0x106-
2.0x106-
1.0x106
0
0 250 500 750 1000 1250
Talinolol (ng/mL)

Figure 3-2. Mean calibration curve (n = 9) of talinolol in mobile phase. Vertical bars
represent the standard deviations (SD) of the means.











Specificity

The method provided good specificity since the chromatograms of the blank


solution did not show any peak with similar retention time as expected for talinolol


(Figure 3-3)





90

80

70

60

50

2 40

E
30

20

10

0A


-10o


1 2 3 4 5 6 7 8 9 10 11 12
Minutes


Figure 3-3. Chromatograms corresponding to the talinolol (100 ng/mL) in HBSS-MES
with retention time about 9.3 min (A) and a blank solution (B) containing the
inhibitory compounds NAR, NAG, BG, DHB, EPBG and VER all at 50
[tmol/L in HBSS-MES.

Sensitivity

The LLOQ of 5 ng/mL was accepted as the lowest concentration of the calibration


curve since their concentrations could be determined with acceptable precision (%CV <


20), and accuracy (%error < 20).









Precision and Accuracy

The precisions intra- and inter-day for both flavonoids and furanocoumarins were

satisfactory with CV values between 0.7 and 8%. Similarly, the accuracy of the assay was

between 93 and 113% for all compounds tested at three different concentrations. The

results are summarized in Table 3-5.

Table 3-5. Intra-day (n = 3) and inter-day (n = 9) assay parameters of talinolol. Precision
expressed as CV% and accuracy as % of the theoretical concentration.
Intra-day CQ 5 ng/mL CQ 100 ng/mL CQ 1000 ng/mL

Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

Precision 0.8 0.7 7.9 4.7 4.2 4.8 4.2 0.8 2.9

Accuracy 109.9 105.6 113.1 93.0 93.3 98.3 100.4 100.1 98.9

Inter-day CQ 5 ng/mL CQ 100 ng/mL CQ 1000 ng/mL

Precision 5.3 4.8 2.7

Accuracy 109.9 94.9 99.8


Stability

The standard solutions of talinolol were found stable at room temperature within 48

hours and at 37 C in both transport medium for 4 hours. Additionally, in general, all

grapefruit components tested showed adequate stability under the test conditions for 3

hours. The 6',7'-epoxybergamottin showed a 20% of degradation and a peak at the same

retention time as the DHB was observed concomitantly, indicating that EPBG was

partially converted into DHB. For the others compounds, the shifting of the areas of each

sample tested was less than 5% of those obtained from a fresh solution at the same level

of concentration (Figure 3-4).












A 125


I -


12 h (RT)


- Low QC
I Medium QC
iM High QC


B 125-


C 1251


e
&
\~. QI C
q- C, ~
@


OTalinolol in MES (5.5mnol/L)
TTalinolol in HEPES (5.5mnol/L)


e .Q
0


J 1


(' 'C


Figure 3-4. Stability oftalinolol (low, medium, and high QCs) in mobile phase within 24
hours at room temperature (RT) (A). Stability of talinolol in transport medium
within 4h at 37 C, during 48 h at RT, and after one week at -20 OC (B).
Stability of GFJ components in after 3h in HBSS-MES (pH 6) at 37 C (C).
Data represents means of three replicates and vertical bars the standard
deviation of the means.

Concentration Dependence

The absorptive permeability (Papp (AP-BL)) of talinolol was concentration independent

and significantly lower than the permeability in the secretary direction (Papp (BL-AP)) at all

concentrations (Figure 3-5). On the other hand, the Papp (BL-AP) decreased considerably as


24 h (RT)


U -. . .- -, .- -


-, r'-7 r-


- r ,-









the concentration of talinolol on the basolateral side of the Transwell system was

increased above 200 pLM. The apparent Pmax, Papp (BL-AP) passive, and KM values and their

standard deviations associated with the transport of talinolol in the secretary direction are

(2.2 + 0.33) x 10-6 cm/sec, (2.0 + 0.05) x 10-6 cm/sec, and 737 + 258 jtM, respectively.


25-


20)-
S0, o BLtoAP

S15 AP to BL
E 15
o I



0.




o 20 400 600 800 1000

Talinolol (gM)

Figure 3-5. Concentration dependent coefficient permeability (Papp) corresponding to the
AP to BL and BL to AP transport of talinolol across Caco-2 cells monolayers.
Data are expressed as mean SEM (n = 3-6). Fitting was performed using
Scientist 2.0 as described in "Material and Methods". All points of Papp (BL-
AP) were compared with the talinolol 25 tM and all data of Papp (AP-BL)
were compared with the respective pair representing the secretary transport of
talinolol. p < 0.05; ** p < 0.01;t p < 0.001; p < 0.01.









Inhibition Studies
-O


0 t I It


0 200 400 600 800 1000
Talinolol (pM)

Figure 3-5. Concentration dependent coefficient permeability (Papp) corresponding to the





The Figure 3-6 showsto BL and BL to AP transport of talinolol across the Caco-2 cells monolayers.
Data are expressed as mean SEM (n = 3-6). Fitting was performed using





monolayers in the absence 2.0 as described in "Matof differial and Methods". Allrations of verapamil, GF(BL

and its AP) were comVerapared with the talinhibitor of P-gp, decree a t io Papp (-BL-AP)
to Pappwere compared with to 3.he respective pair representing the attenuation of secretary transport of
(14 x 10talinolol. p 0.05; 10 cm/sec) and to the enhancement of absorptive (1.9 x 10 to 3.1 x
Inhibition Studies

The Figure 3-6 shows the permeability of talinolol across the Caco-2 cells

monolayers in the absence and presence of different concentrations of verapamil, GFJ,

and its components. Verapamil, a known inhibitor of P-gp, decreased the ratio Papp (BL-AP)

to Papp (AP-BL) of talinolol from 7.4 to 3. This effect was due to the attenuation of secretary

(14 x 10'7 to 9.4 x 10'7 cm/sec) and to the enhancement of absorptive (1.9 x 10'7 to 3.1 x









10-7 cm/sec) transport of talinolol. However, compared with the respective controls

reduction of Papp (BL-AP) was more significant than the enlargement of Papp (AP-BL).

Similarly, grapefruit juice (GFJ) reduced the Papp (BL-AP) of talinolol from 22 x10-7 to 4.5 x

10-7 cm/sec but did not affect significantly its absorptive transport. Although NAR, the

main constituent of GFJ, was found in the juice at relative high concentration (811 [tM),

it showed a minor effect on the secretary transport of talinolol. Concentrations higher

than 625 [LM decreased the Papp (BL-AP) to around 1.3-1.5 fold compared to the control.

Likewise GFJ, the effect of NAR on the absorptive transport of talinolol was negligible.

On the other hand, the aglycone NAG, which is not normally found in GFJ (205),

decreased the BL to AP transport more than 2-fold compared to the control as its

concentration was increased beyond 250 [tM. Analogous to 6',7'-dihydroxybergamottin,

bergamottin at 1 [ M and 10 [tM did not change the permeability oftalinolol however, in

vitro studies of further concentrations were prejudiced because solubility issues. The

furanocoumarins 6',7'-dihydroxybergamottin (DHB) at concentrations higher than 10 [tM

and 6',7'-epoxybergamottin (EPBG) exerted a considerably reduction on the BL to AP

transport of talinolol. In fact, the Papp (BL-AP) of talinolol in the presence of DHB (500 [tM)

and EPBG (50 [LM) decreased 5- and 2.5- fold, respectively, when compared with the

controls. The effective concentrations of these inhibitory compounds are in the same

range as they are found in the juice (Table 3-2).














C AP/BL 25-
BL/AP 0 A
S20-
E
S15-
U

*o 10-

5o
I n n (L 0-

0 50 100 250 500 1000
Verapamil (RiM)


0 0.5 1 5 10 25 50
GFJ (%)


25-

S20-

15-
E 10-
x
CL 5-
Q.


I F1 l



0 100 250 500 625 1250 2500
Naringin (tIM)


L~
S20-
E
S15-
F lo
F 10-
x
CL 5-
Q.


0
0 0.5 10 25 50 100 500
6',7'-Dihydroxybergamottin (u.M)


nJ1JniL,


n.


0 10 25 50 100 250 500
Naringenin (pM)












_jI Jj


0
0 0.5 1 5 10 50
6',7' Epoxybergamottin (gM)


Figure 3-6. Coefficient of permeability (Papp) of talinolol (100lM) across the Caco-2
cells monolayers in the absence and presence of distinct concentrations of A:
verapamil HC1 (25, 50, 100, 250, and 500 [LM); B: NAR (100, 250, 500, 625,
1250, 2500 [LM); C: DHB (0.5, 10, 25, 50, 500 [LM); D: GFJ (0.5, 1, 5, 10, 25,
and 50 %); E: NAG (10, 50, 100, 250, 500 [LM); F: EPB (0.5, 1, 5, 10, 50
tLM). Data are expressed as mean SEM (n = 3-6). p < 0.05; ** p < 0.01.


) 20-
E
S15.
A
r- 10'
x
CL 5.
a.
IC.


) 20'
E
U 15.
15-
B lo
x
CL 5m
L 0.






S25
U
) 20.
E
S15-
C o
x
5
C.









Table 3-6. Concentration range that the grapefruit components are normally found in the
juice.
Inhibitor Cone. in GFJ (gM)
Naringin 174 1492 (205)a
Naringenin < 0.56 (205)
Bergamottin 1 36.6 (205)
6',7'-Dihydroxybergamottin 0.22 52.5 (205)
6',7'-Epoxybergamottin 0.1 7.4 (60
a references are in parenthesis

Dose-response Studies

The dose-response curves for the inhibitory effects and the IC5o values of

verapamil, GFJ, and its components are presented in Figure 3-7, respectively. Except for

NAR, the range of concentration tested was enough to obtain a sufficient estimation

(narrow confidence interval range) of the potency and efficacy of all tested compounds

and GFJ. The lower potency of NAR affected the accuracy of its IC50 value (2409 [tM),

since further concentrations (> 2500 [LM) could not be tested because of the negative

impact on the integrity of the Caco-2 monolayers (decreased the TEER values and

increased LY transport) and due to solubility problems. Verapamil, however, showed a

relatively high potency (28 [tM) but did not present a good efficacy reducing the activity

of P-gp by only 35% compared to the control. GFJ (IC50 = 0.6%) diluted in HBSS-MES

buffer solution (pH 6.0) decreased the secretary transport of talinolol to 27% of that

without any inhibitor. Correspondingly, NAG was 10-fold more potent than its glycoside

NAR reducing the P-gp mediated secretary transport of talinolol by 60% when compared

to the control. Interestingly, the furanocoumarin EPBG was about 50-fold more potent

than DHB with IC50 values of 0.7 [tM and 34 [tM, respectively. Additionally, compared







86


at the same concentration (50 ptM), EPBG and DHB showed similar efficacy reducing the


Papp(BL-AP) of talinolol by 55% and 60%, correspondingly.


IC50= 28 pM
"-.. C.I.= 10-77



Y--


101 102
Verapamil (pM)


103


S IC50o= 0.6 %
C.I. =0.5 -0.8






100 101 102
GFJ (%)


C loo.
S90.
80-
0
O 70-
60-


IC50= 2409 pM


A"---.. C.I.=2


'^.


01-1 ..... ....... ... .
0 102.0 102.5 103.0

Naringin (pIM)


E 100 A
S IC50 = 34 pM
g 75 C.I.=24-47

O 50-


0 ..i ........i .......i .......
0 100 101 102 103
6',7'- Dihydroxybergamottin (pIM)


21 -26306 D o100 A -a--. IC5o = 236 pM
9- C.I. =69 -809
2 80-
70-




10"5 0 101 102 103
Naringenin (ipM)


F 100-


IC50o= 0.7 pM
, C.I. = 0.5 -0.9
\,


40-1 -

010-2 10-1 100 101 102
6',7'-Epoxybergamottin (gM)


Figure 3-7. Inhibitory dose-response effects of A: verapamil HC1 (50, 100, 250, 500, and
1000 [tM); B: GFJ (0.5, 1, 5, 10, 25, and 50 %); C: NAR (100, 250, 500, 625,
1250, 2500 [tM); D: NAG (10, 50, 100, 250, 500 [tM); E: DHB (0.5, 10, 25,
50, 500 [tM; F: EPBG (0.5, 1, 5, 10, 50 [tM) on the transport of talinolol in the
secretary (BL-AP) direction. Data are expressed as mean + SEM (n = 3-6).
The IC50 values of each compound and their respective 95% of confidence
interval (C.I.) were estimated by nonlinear regression using GraphPad Prism
4.0 as described in "Material and Methods".


60-
0 10-
0 100