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Interactions between Grapefruit Juice and HMG-CoA Reducatase Inhibitors

Permanent Link: http://ufdc.ufl.edu/UFE0021699/00001

Material Information

Title: Interactions between Grapefruit Juice and HMG-CoA Reducatase Inhibitors
Physical Description: 1 online resource (144 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: atorvastatin, damage, exposure, grapefruit, humans, juice, muscle, myopathy, pharmacokinetics, rats, simvastatin, statins
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Grapefruit juice (GFJ) has been shown to increase the plasma concentrations of several drugs including HMG-CoA reductase inhibitors. The predominant mechanism for this interaction is the irreversible inhibition of intestinal drug metabolizing enzymes, mainly Cytochrome P450 3A4 (CYP 3A4). CYP 3A4 metabolizes roughly 60% of all drugs available to patients to a greater or lesser extent. Changes in exposure of HMG-CoA reductase inhibitors have been shown to reach increases of up to 16-fold. However, the long-term and clinical effects of the interaction remain unclear. The purpose of the presented work was to investigate the potential clinical relevance of the interaction between GFJ and HMG-CoA reductase inhibitors using GFJ quantities comparable to human consumption (5mL/kg). For this purpose data from animals experiments and a clinical trial have been analyzed. During the animal experiment male Sprague-Dawley rats were dosed with either 20mg/kg or 80mg/kg simvastatin concomitantly with regular (RS) and double strength (DS) GFJ. The primary outcome parameters were defined as the changes in exposure of simvastatin lactone and simvastatin acid levels. Our findings suggest that grapefruit juice increases the exposure only minimally when administered with 20mg/kg simvastatin. Exposure increased significantly when double strength GFJ was dosed concomitantly with 80mg/kg simvastatin in laboratory animals. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK), total cholesterol (tCHOL) and changes in muscle histology were analyzed as secondary parameters. Changes of the primary parameters however did not result in an increase in the incidence of muscle damage, changes in other secondary parameters or decrease in survival rate. Additionally our results suggest that GFJ is capable of lowering cholesterol by 19% and 15% when administered as RS and DS respectively. The data from the clinical trial suggest that steady state atorvastatin plasma concentrations do not change significantly over 90 days when the drug was coadministered with 10oz of GFJ once daily. Our results indicate that GFJ consumption in moderation is unlikely to result in clinically relevant interactions. Further research in humans however is needed to confirm this hypothesis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Derendorf, Hartmut C.
Local: Co-adviser: Butterweck, Veronika.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021699:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021699/00001

Material Information

Title: Interactions between Grapefruit Juice and HMG-CoA Reducatase Inhibitors
Physical Description: 1 online resource (144 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: atorvastatin, damage, exposure, grapefruit, humans, juice, muscle, myopathy, pharmacokinetics, rats, simvastatin, statins
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Grapefruit juice (GFJ) has been shown to increase the plasma concentrations of several drugs including HMG-CoA reductase inhibitors. The predominant mechanism for this interaction is the irreversible inhibition of intestinal drug metabolizing enzymes, mainly Cytochrome P450 3A4 (CYP 3A4). CYP 3A4 metabolizes roughly 60% of all drugs available to patients to a greater or lesser extent. Changes in exposure of HMG-CoA reductase inhibitors have been shown to reach increases of up to 16-fold. However, the long-term and clinical effects of the interaction remain unclear. The purpose of the presented work was to investigate the potential clinical relevance of the interaction between GFJ and HMG-CoA reductase inhibitors using GFJ quantities comparable to human consumption (5mL/kg). For this purpose data from animals experiments and a clinical trial have been analyzed. During the animal experiment male Sprague-Dawley rats were dosed with either 20mg/kg or 80mg/kg simvastatin concomitantly with regular (RS) and double strength (DS) GFJ. The primary outcome parameters were defined as the changes in exposure of simvastatin lactone and simvastatin acid levels. Our findings suggest that grapefruit juice increases the exposure only minimally when administered with 20mg/kg simvastatin. Exposure increased significantly when double strength GFJ was dosed concomitantly with 80mg/kg simvastatin in laboratory animals. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine phosphokinase (CPK), total cholesterol (tCHOL) and changes in muscle histology were analyzed as secondary parameters. Changes of the primary parameters however did not result in an increase in the incidence of muscle damage, changes in other secondary parameters or decrease in survival rate. Additionally our results suggest that GFJ is capable of lowering cholesterol by 19% and 15% when administered as RS and DS respectively. The data from the clinical trial suggest that steady state atorvastatin plasma concentrations do not change significantly over 90 days when the drug was coadministered with 10oz of GFJ once daily. Our results indicate that GFJ consumption in moderation is unlikely to result in clinically relevant interactions. Further research in humans however is needed to confirm this hypothesis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Derendorf, Hartmut C.
Local: Co-adviser: Butterweck, Veronika.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021699:00001


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1 INTERACTIONS BETWEEN GRAPEFRUIT JUICE AND HMG-COA REDUCTASE INHIBITORS By IMMO ZDROJEWSKI 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 2008

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2 2007 Immo Zdrojewski

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3 To my wife, my parents and my brother.

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4 ACKNOWLEDGMENTS I would like to thank Dr. Hartmut Derendorf fo r the opportunity to work on this exciting project, his guidance and invalu able advice over the years. I w ould also like to thank Dr. Veronika Butterweck as co-chair of my committee, for her invaluable assistance and supervision during this project. I would lik e to thank especially Dr. Regi nald Frye for the guidance and patience during the mass spectrometric analysis. My gratitude also goes to Cherryl Galloway for her greatly appreciated contri bution during the method developm ent and validation. I would like to express my gratitude to Dr. Guenther Hochha us and Dr. Sunny Yoon for their invaluable help and advice during this project. I would like to thank Dr. J ohn Roberts and Shawn Leslie for their advice and guidance during the exploration of the hist ological changes in the rats. I would like to thank Merck & C o., Inc. for the greatly appreciated gift of simvastatin and Steigerwald Arzneimittelwerk GmbH for the greatly appreciated gift of the Vitalab Selectra II without which this project would not have been possible. I express my deepest gratitude for all the support, advice and friendship to all my lab companions, especially Whocely Victor de Cast ro for his help during the grapefruit juice analysis, Oliver Grundmann for all the enlightenment into statistics and fo r the great friendship and Stephan Mathew Phipps for all the help in chemistry and for his friendship. I will not forget to thank all th e interns without whom this project would not have ended in the determined time frame, Christofer Paul Ku rz, Antje Pommerenke, Marisol Gonzales, Birgit Hempsch, Nina Schulte-Loebert and Jochen Me yer, for their help and insight into PHP programming. My personal thanks go to my wi fe (Dorys Zadezensky), my parents (Horst and Ursula Zdrojewski) and my brother (Heiko Zd rojewski) for all the patience, support and understanding during this ch allenging time.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ..........9LIST OF FIGURES................................................................................................................ .......10LIST OF ABBREVIATIONS........................................................................................................14ABSTRACT....................................................................................................................... ............16CHAPTER 1 INTRODUCTION..................................................................................................................183-Hydroxy-3-methylglutaryl (HMG) Coen zyme A (CoA) Reductase Inhibitors..................18History of Development and Mechanism of Action........................................................18Statin Metabolism and Pharmacokinetics.......................................................................20Adverse Events................................................................................................................21The Grapefruit ( Citrus paradisi )............................................................................................22Botany......................................................................................................................... .....22Grapefruit Economy........................................................................................................22Health Benefits................................................................................................................23Grapefruit Juice-Drug Interactions.........................................................................................24Discovery...................................................................................................................... ...24Mechanism of Action......................................................................................................24Compounds of Interest....................................................................................................25Influence of Grapefruit Juice on Statin Pharmacokinetics..............................................26Hypothesis and Objectives.....................................................................................................29Specific Aim 1.................................................................................................................29Specific Aim 2.................................................................................................................29Specific Aim 3.................................................................................................................292 ASSESSMENT OF THE OVERALL INTERACTION POTENTIAL OF GRAPEFRUIT JUICE............................................................................................................41Background..................................................................................................................... ........41Specific Aim................................................................................................................... ........42Material and Methods........................................................................................................... ..42Database Design................................................................................................................ .....43Programming Language..................................................................................................43Database Layout..............................................................................................................43Results........................................................................................................................ .............44Interaction Potential by Drug Category...........................................................................45Antiallergics.............................................................................................................45Antibiotics................................................................................................................45

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6 Anticoagulants..........................................................................................................46Antimalaria drugs.....................................................................................................46Antiparasitic drugs...................................................................................................46Sedative-hypnotics...................................................................................................47Calcium channel blockers........................................................................................47HIV protease inhibitors............................................................................................47HMG-CoA reductase inhibitors...............................................................................48Hormones.................................................................................................................48Immunosuppressants................................................................................................48Antitumor drugs.......................................................................................................48Over the counter drugs.............................................................................................49Beta receptor blocker...............................................................................................49Antiarrhytmics..........................................................................................................49Other drugs...............................................................................................................49Conclusions.................................................................................................................... .503 EFFECT OF LONG TERM INGESTI ON OF GRAPEFRUIT JUICE ON THE PHARMACOKINETICS AND TOXI COLOGY OF SIMVASTATIN................................56Background..................................................................................................................... ........56Specific Aim................................................................................................................... ........58Material and Methods........................................................................................................... ..58Chemicals...................................................................................................................... ..58Stock and Work Solutions...............................................................................................59Material and Methods......................................................................................................59Open field test..........................................................................................................59Grip strength test......................................................................................................60Histological experiment...........................................................................................61Clinical chemistry....................................................................................................61Grapefruit Juice Analysis...................................................................................................... .62HPLC System..................................................................................................................62Determination of Flavonoids (Naringin and Naringenin)...............................................62Determination of Furanocoumarins (Bergamottin and 6',7'-Dihydroxybergamottin).....63Animal Experiment Design....................................................................................................63Pilot Study.................................................................................................................... ...64Final Study.................................................................................................................... ...64Statistical Analysis........................................................................................................... .......65Results........................................................................................................................ .............66Juice Analysis................................................................................................................. .66Survival Rate.................................................................................................................. .66Body Weight....................................................................................................................67Behavioral Experiments..................................................................................................67Open field test..........................................................................................................67Grip strength test......................................................................................................68Extremity temperature..............................................................................................68Histological Experiment..................................................................................................68Clinical Chemistry...........................................................................................................69

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7 Organ weights..........................................................................................................70Conclusions.................................................................................................................... .........714 DETERMINATION OF ACUTE AND CHRONI C EFFECT OF GRAPEFRUIT JUICE ON SIMVASTATIN PLASMA CO NCENTRATIONS IN RATS........................................94Specific Aim................................................................................................................... ........94Material and Methods........................................................................................................... ..94Chemicals...................................................................................................................... ..94Stock and Work Solutions...............................................................................................94Calibration Quality Control Standards............................................................................95Analytical System and Chromatographic Conditions.....................................................95Experiment Design.............................................................................................................. ...96Sample Preparation and Extraction........................................................................................97Pharmacokinetic Analysis......................................................................................................98Statistical Analysis........................................................................................................... .......98Results........................................................................................................................ .............98Acute treatment...............................................................................................................99Simvastatin lactone..................................................................................................99Simvastatin hydroxy acid.......................................................................................100Chronic Treatment.........................................................................................................101Simvastatin lactone................................................................................................101Simvastatin hydroxy acid.......................................................................................102Conclusions.................................................................................................................... .......1025 DETERMINATION OF CHANGES IN S TEADY STATE CONCENTRATIONS OF ATORVASTATIN AFTER LO NG TERM GRAPEFRUIT JUICE CONSUMPTION......112Background..................................................................................................................... ......112Specific Aim................................................................................................................... ......112Material and Methods...........................................................................................................113Chemicals......................................................................................................................113Stock and Work Solutions.............................................................................................113Calibration Standards....................................................................................................114Atorvastatin Quality Control Standards........................................................................114Analytical System and Mobile Phase............................................................................114Analytical Method.........................................................................................................114Experiment Design.............................................................................................................. .115Sample Preparation and Extraction......................................................................................115Statistical Analysis........................................................................................................... .....116Results........................................................................................................................ ...........116Conclusions.................................................................................................................... .......1166 CONCLUSIONS..................................................................................................................119Discussion..................................................................................................................... ........119Conclusions.................................................................................................................... .......126

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8 LIST OF REFERENCES.............................................................................................................127BIOGRAPHICAL SKETCH.......................................................................................................144

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9 LIST OF TABLES Table page 3-1 Preparation procedure for simvastatin suspensions with respective dosing volume.........903-2 Naringin (NAR) / naringen in (NAG) standard curve........................................................903-3 Bergamottin and dihydroxybergamottin standard curve....................................................913-4 Dosing scheme of the final study.......................................................................................923-5 Muscle damage incidence and severity..............................................................................934-1 Spiking scheme for calibration and quality control standards.........................................1074-2 Pharmacokinetic parameters of simvastatin lactone after a 20 mg/kg oral dose in rats. Data is shown as mean SEM (n=13) (*=p<0.05, **=P<0.01, ***=P<0.001)......1084-3 Pharmacokinetic parameters of simvastatin lactone after a 80 mg/ kg oral dose in rats. Data is shown as mean SEM (n=13) (*=p<0.05, **=P<0.01, ***=P<0.001)...............1084-4 Pharmacokinetic parameters of simvasta tin hydroxy acid after a 20 mg/kg oral dose in rats. Data is shown as meanSEM (n=13) (*=p<0.05)...............................................1094-5 Pharmacokinetic parameters of simvas tatin hydroxy acid after a 80 mg/kg oral dose in rats. Data is shown as m eanSEM (n=13) (**=P<0.01, ***=P<0.001)......................1094-6 Trough(Cmin) and peak(Cmax) concentrations of simvasta tin lactone after a 20 mg/kg and 80 mg/kg oral dose in rats. Data is shown as meanSEM (n=13) (*=p<0.05, **=P<0.01, ***=P<0.001)...............................................................................................1104-7 Trough(Cmin) and peak(Cmax) concentrations of simvas tatin hydroxy acid after a 20 mg/kg and 80 mg/kg oral dose in rats. Data is shown as meanSEM (n=13) (*=p<0.05, **=P<0.01, ***=P<0.001)............................................................................1115-1 Atorvastatin calibration st andards for plasma samples....................................................1185-2 Atorvastatin quality control standards for plasma samples.............................................118

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10 LIST OF FIGURES Figure page 1-1 Causes of death in the year 2004 as reported by the US Census Bureau ..........................31 1-2 Top 10 selling prescription drugs in 2006 (total dollars in billions) .................................31 1-3 Mevalonate pathwa y in mammalian cells .........................................................................32 1-4 Structures of HGM-CoA reductase inhibitors compar ed to mevalonic acid ....................33 1-5 Structure of simvastatin and simvastatin acid ..................................................................34 1-6 Metabolism pathway of simvastatin SV=s imvastatin, SVA=simvastatin acid, I=6OH-SV II= 3-OH-SV, III=3-OH-SV, IV=6-exomethylene SV, V=6-CH2OHSV, VI=6-COOH-SV, VII=1,2,6,7,8,8a -hexahydroxy-2,6-dimethyl-8-(2,2dimethyl-1-oxobutoxy)-1naphthalene-pentanoic acid .....................................................35 1-7 Proposed mechanism of the relactonization of simvastatin acid into simvastatin ............36 1-8 Citrus paradisi Macfaden (grapefruit) ..............................................................................37 1-9 Compounds in grapefruit juice shown to interact w ith CYP 3A4, NAR=naringin, NAG=naringenin...............................................................................................................38 1-10 Compounds in grapefruit juice shown to interact with CYP 3A4, BG=bergamottin, DHB=6,7-dihydroxybergamottin....................................................................................38 1-11 Structures of DHB Tail/Tail dimers identified in grapefruit juice.....................................39 1-12 Structure of BG epoxide and DHB orthospiroester...........................................................40 2-1 Principle of a dynamic website..........................................................................................51 2-2 MySQL table layout for th e drug interaction website.......................................................52 2-3 Sample of the one page summ aries uploaded to the database...........................................53 2-4 Number of interaction drugs in each categ ory: 1=no to weak interaction, 2=moderate interaction, 3=strong interacti on, N/A=not tested in humans............................................54 2-5 Website access statistics per month: A=number of hits, B=number of submitted pages, C=number of files, D=number of visits..................................................................55 2-6 Website access statistics h its-files as rough estimate of returning visitors per month......55 3-1 Schematics of first pass metabolism..................................................................................72

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11 3-2 Open field arena and TopScan software interface.............................................................72 3-3 PANLAB grip strength meter............................................................................................73 3-4 Sampling schedule during the final study..........................................................................73 3-5 Concentration of 6,7-dihydroxybergam ottin (DHB), bergamottin (BG) and Naringin (NAR) in three different juices (mean SD) n=3..............................................74 3-6 Survival rate during both st udies A=pilot study, B=final study........................................74 3-7 Slope of the linear regres sion performed of the body weight of rats in the pilot study from day 0 to day 10 (mean SEM), Control n=7, SV53 n=8, SV200 n=4. (*=P<0.05)..................................................................................................................... ....75 3-8 Slope of the linear regres sion performed of the body weight of rats in the final study from day 0 until day 28 shown as mean SEM (n=13)...................................................75 3-9 Number of line crossings of the open field test in the pilot study (mean SEM), Control n=7, SV53 n=8, SV200 n=4. (*=P<0.05).............................................................76 3-10 Number of line crossings of the open fi eld test in the final study shown as mean SEM (n=13). (**=P<0.01)................................................................................................76 3-11 Distance traveled in the open field test of the pilot study (mean SEM), control n=7, SV53 n=8, SV200 n=4. (*=P<0.05)..................................................................................77 3-12 Distance traveled in the open field test of the final study shown as mean SEM (**=P<0.01).................................................................................................................... ...77 3-13 Grip strength development during the p ilot study mean SE M, control n=7, SV53 n=8, SV200 n=4.................................................................................................................78 3-14 Comparison of the grip strength AUC mean SEM control n=7, SV53 n=8, SV200 n=4............................................................................................................................ .........78 3-15 Grip strength development during the fina l study n=13, data shown as mean SEM......79 3-16 Comparison of the grip strength AUC during the final study, n=13 data shown as mean SEM..................................................................................................................... .79 3-17 Comparison of the extremity temperatur e in the pilot study, mean SEM control n=7, SV53 n=8, SV200 n=4 (** P<0.01)..........................................................................80 3-18 Comparison of the extremity temperatur e in the final study mean SEM n=13............80 3-19 Muscle damage (arrows) observed duri ng the pilotA) contro l group B) SV53 C) SV200.......................................................................................................................... ......81

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12 3-20 Histological changes in the rat muscle during the final study A) water B) SV20 C) SV80 D) RS juice E) DS juice...........................................................................................82 3-21 Histological changes in the rat muscle during the final study A) SV20RS B) SV20DS C) SV80RS D) SV80DS....................................................................................................83 3-22 Cholesterol levels afte r 12 day dosing during the pilo t study mean SEM control n=7, SV53 n=8, SV200 n=4...............................................................................................84 3-23 Cholesterol levels afte r 28 day dosing during the fina l study mean SEM, n=13 (*=P<0.05)..................................................................................................................... ....84 3-24 Alanine amino transferase (ALT) levels af ter 12 days during th e pilot study, mean SEM control n=7, SV53 n=8, SV200 n=4 (*=P<0.05)....................................................85 3-25 Alanine amino transferase (ALT)levels after 28 days during th e final study, mean SEM n=13 (**=P<0.01)...................................................................................................85 3-26 Aspartate amino transferase (AST) levels after 12 days during the pilot study mean SEM control n=7, SV53 n=8, SV200 n=4 (*=P<0.05)....................................................86 3-27 Aspartate amino transferase (AST) levels after 28 days during the final study, mean SEM n=13 (**=P<0.01)...............................................................................................86 3-28 Creatine kinase (CPK) levels be fore and after decapitation n=9.......................................87 3-29 Creatine kinase (CPK) leve ls after 28 days during th e final study, mean SEM, n=13 (**=P<0.01)..............................................................................................................87 3-30 Absolute organ weights after 28 days during the final study, m ean SEM, n=13 A) liver B) liver c) Spleen D) heart E) testis F) adrenal glands G) kidney.............................88 3-31 Organ indices after 28 days during the final study, mean SEM, n=13 A) liver B) spleen C) heart D) testis E) adre nal glands F) kidney G) kidney......................................89 4-1 Plasma concentrations of simvastatin l actone in rat plasma after a 20 mg/kg oral dose. Data is shown as meanSEM (n=13).....................................................................103 4-2 Plasma concentrations of simvastatin l actone in rat plasma after a 80 mg/kg oral dose. Data is shown as meanSEM (n=13).....................................................................103 4-3 Plasma concentrations of simvastatin hydroxy acid in rat plasma after a 20 mg/kg oral dose. Data is shown as meanSEM (n=13)..............................................................104 4-4 Plasma concentrations of simvastatin hydroxy acid in rat plasma after a 80 mg/kg oral dose. Data is shown as meanSEM (n=13)..............................................................104 4-5 Plasma concentrations of simvastatin lact one in rat plasma after a 20 mg/kg oral dose from day 7 to day 28. Data is shown as meanSEM (n=13)...........................................105

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13 4-6 Plasma concentrations of simvastatin lact one in rat plasma after a 80 mg/kg oral dose from day 7 to day 28. Data is shown as meanSEM (n=13)...........................................105 4-7 Plasma concentrations of simvastatin hydroxy acid in rat plasma after a 20 mg/kg oral dose from day 7 to day 28. Data is shown as mean SEM (n=13)..........................106 4-8 Plasma concentrations of simvastatin hydroxy acid in rat plasma after a 80 mg/kg oral dose from day 7 to day 28. Data is shown as mean SEM (n=13)..........................106 5-1 Calibration curve for atorva statin acid 0.25ng/ml to 25 ng/ml........................................117 5-2 Concentrations of atorvastatin from da y 1 to day 90. Data shown as mean SEM (n=121)........................................................................................................................ .....117

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14 LIST OF ABBREVIATIONS HMG-CoA: 3-Hydroxy-3-methyl glutaryl Coenzyme A CNS: central nervous system LDL: low density lipoprotein LDL-C: low density li poprotein cholesterol CYP: cytochrome P450 PGP: p-glycoprotein CK: creatine kinase NLA: national lipid association FDA: Food and Drug Administration apoB: apoliporotein B ACAT: acyl-CoA:cholesterol acyltransferase DS: double strength RS: regular strength DHB: dihydroxybergamottin BG: bergamottin NAR: naringin NAG: naringenin AUC: area under the curve CMAX: peak plasma concentration CMIN: trough plasma concentration TMAX: time of peak plasma concentration T1/2: half life CL/F: clearance over bioavailability Vz/F: volume of distribu tion over bioavailability

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15 GFJ: grapefruit jucie OTC: over the counter PHP: PHP hypertext preprocessor MYSQL: structured querry language RDBMS: relational database management system GNU GPL: GNU general public lisence CMC: carboxymethyl cellulose GGT: gamma glutamyltransferase ALT: alanine aminotransferase AST: aspartate aminotransferase CPK: creatine phosphokinase PVDF: polyvinylidene fluoride DMSO: dimethyl sulfoxide SEM: standard error of the mean SV: simvastatin I.P.: intraperitoneal ACN: acetonitrile H2O: water ESI: electrospray ionization SRM: single reaction monitoring CID: collision induced dissociation

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16 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INTERACTIONS BETWEEN GRAPEFRUIT JUICE AND HMG-COA REDUCTASE INHIBITORS By Immo Zdrojewski May 2008 Chair: Hartmut Derendorf Cochair: Veronik Butterweck Major: Pharmaceutical Sciences Grapefruit juice (GFJ) has been shown to incr ease the plasma concentrations of several drugs including HMG-CoA reductase inhibitors. The predominant mechanism for this interaction is the irreversible inhibition of intestinal drug metabolizing en zymes, mainly Cytochrome P450 3A4 (CYP 3A4). CYP 3A4 metabol izes roughly 60% of all drugs available to patients to a greater or lesser extent. Change s in exposure of HMG-CoA reduc tase inhibitors have been shown to reach increases of up to 16-fold. Howeve r, the long term and c linical effects of the interaction remain unclear. The purpose of the presented work was to investigate the potential clinical relevance of the interaction between GFJ and HMG-CoA reductase i nhibitors using GFJ quant ities comparable to human consumption (5mL/kg). For this purpose da ta from animals experiments and a clinical trial have been analyzed. During the animal experiment male Sprague-D awley rats were dosed with either 20mg/kg or 80mg/kg simvastatin concomitantly with regul ar (RS) and double strength (DS) GFJ. The primary outcome parameters were defined as the changes in exposure of simvastatin lactone and simvastatin acid levels. Our findings suggest that grapefruit juice increases the exposure only

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17 minimally when administered with 20mg/kg simv astatin. Exposure increase d significantly when double strength GFJ was dosed concomitantly with 80mg/kg simvastatin in laboratory animals. Alanine aminotransferase (ALT), aspartate ami notransferase (AST), creatine phosphokinase (CPK), total cholesterol (tCHOL ) and changes in muscle histol ogy were analyzed as secondary parameters. Changes of the primary parameters however did not result in an increase in the incidence of muscle damage, changes in other sec ondary parameters or decr ease in survival rate. Additionally our results suggest that GFJ is capable of lowering cholesterol by 19% and 15% when administered as RS and DS respectively. The data from the clinical trial suggest that steady state atorvastatin plasma concentrations do not change significantly over 90 days when th e drug was coadministered with 10oz of GFJ once daily. Our results indicate that GFJ consumption in m oderation is unlikely to result in clinically relevant interactions. Further research in human s however is needed to confirm this hypothesis.

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18 CHAPTER 1 INTRODUCTION 3-Hydroxy-3-methylglutaryl (HMG) Coen zyme A (CoA) Reductase Inhibitors Despite the increased effort, coronary heart diseases remain the main cause of death in the western world (Figure 1-1) [1], even though the main risk factors have long been identified. These risk factors include smoking, hypertensio n, lipid disorders and diabetes mellitus. In 1984 the Lipid Research Clinic Coronary Primary Pr evention Trial (LRC-CPPT) concluded that there is a positive relationship between the reduction of plasma choles terol and decrease of myocardial infarction [2]. Subsequently numerous drugs have b een developed to treat lipid disorders such as hypercholesterolaemia. The drugs include bile acid sequestrants, fibrates, selective cholesterol absorption inhibitors, and HMG-Co A reductase inhibitors. One of the most prescribed class of drugs in this category are the HMG-CoA reducta se inhibitors or st atins [3]. Lipitor (atorvastatin) and Zocor (simvastatin) were th e number 1 and 7 prescr iption drugs in the USA in 2006 generating a sales volume of 11.6 billio n dollars according to IMS National Sales Perspectives [3] (Figure 1-2). History of Development and Mechanism of Action The biosynthesis of cholesterol from acet yl-CoA accounts for 60-70% of the total cholesterol available to the human body. This high amount of endogenous production makes this pathway the perfect target for cholesterol loweri ng therapy [4]. The first inhibitors of the cholesterol biosynthesis trip aranol (MER-29) and AY-9944 act ed on a later step of the biosynthesis pathway [5-7]. AY-994 prevented the conversion from 7-dehydrocholesterol to cholesterol whereas MER-29 inhibits the convers ion from 24-dehydrochole sterol (desmosterol) to cholesterol [8]. However the i nhibition of the later stages of th e cholesterol synthesis resulted in the increase of desmosterol in plasma [5] and researchers believe that the accumulation of this

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19 sterol led to ichtyosis [9] and posterior lenticular cataracts [ 10]. The effects of the compounds resulted in the withdrawal of triparanol fr om the market in 1962 and reduced the initial enthusiasm for the inhibition of the chol esterol pathway as a therapeutic target. Later in 1968 Dietschy et al. [11] reported that the endogenou s biosynthesis of cholesterol in the liver was nearly completely inhibited wh en high amounts of choles terol are added to the diet. Further investigation revealed that this feed back mechanism is due to the changes in activity of the HMG-CoA reductase [12] These findings encouraged Endo and Kuroda that the concept of inhibition of the HMG-Co A reductase could be a potentia l target for treatment of hypercholesterolemia in humans [13]. In 1971 Endo and Kuroda began their search for inhibitors of the HMG-CoA reductase isolated from bacteria, assuming that some mi croorganisms would use this pathway a defense mechanism against other microbes. Both researcher s tested close to 6000 ba cterial strains and in 1973 isolated the first irreversib le inhibitor of the HMG-CoA reductase, citrinin, an antibiotic compound from Pythium ultimum [14]. By the end of 1973 another promising structure was isolated from Penecillinum citrium Mevastatin (ML-236B) was show n to inhibit the cholesterol synthesis in the early stages of the biosynthesis pathway from both [14C]acetate and [14C]HMGCoA, but showed no significant effect on the conversion of [3H]mevalonate into successive sterols [13]. Subsequent search for additional inhibitors of the HMG-CoA reductase led to several synthetic and semi-synthe tic compounds currently marketed including atorvastatin and simvastatin, which are the number 1 and 7 best selling prescription drugs in the USA in 2005 [15]. All of the chemical moietie s work by the same mechanism of action: they inhibit the rate limiting step of the endogenous cholesterol bios ynthesis, the conversion from HMG-CoA in

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20 mevalonate by the HMG-CoA reductase (Figure 13), resulting ultimately in the reduction of overall produced cholesterol [13, 16]. Statin Metabolism and Pharmacokinetics Simvastatin and lovastatin are administered as the inactive prodrug which must be hydrolyzed in vivo to the corresponding -hydroxy acid form to achieve activity. All other statins are dosed as the active open hydroxy acid form [4, 16]. Diffe rences also exist in their physical-chemical properties. Atorvastatin, lova statin and simvastatin are lipohilic drugs, whereas pravastatin and fluvastatin are hydroph ilic drugs (Figure 1-4). Lennernaes and Fager reported in 1997 that simvastatin and lovastatin cross the blood brain ba rrier in th eir inactive lactone form [17]. The distribut ion of statins into the CNS is depent on lipophilicity as reported by Sirtori et al. [18]. Furthermore Vickers et al. [19] were able to measure brain simvastatin levels both after intravenous and oral administration. The pharmacokinetic properties of statins show a high degree of variation when administered via the oral route. Therapeutic doses range from 5-80 mg/day (simvastatin), 1080mg/day (atorvastatin, lovastatin), 10-40mg/da y (pravastatin) to 20-40 mg/day (fluvastatin).h Mean LDL cholesterol lowering ranges from 2142% (lovastatin), 22-25% (fluvastatin), 22-34% (pravastatin), 26-47% (simvastatin) up to 43-60% (atorvastatin) [20] The inactive lactone forms of simvastatin and lovastatin are readily converted in vivo into the active open acid form (Figure 1-5). Gender differences exist in the conversion rates of lactone to acid in rats for simvastatin [19]. Th e same has been shown in healthy volunteers for simvastatin and lovastatin [21]. Both the lactone [22, 23] and the acid [24] form of simvastatin undergo extensive metabolism (Figure 1-6) by CY P 3A resulting in more than 10 metabolites. Furthermore lovastatin and ator vastatin are known CYP3A substrat es [4]. The acid form of simvastatin can further undergo beta-oxidation [19, 25]. Lovastatin [26, 27] and pravastatin [28-

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21 30] are known p-glycoprotein substrates. Du e to its extensive fi rst pass metabolism, bioavailability of simvastatin and lovastatin is below 5%. Pravastatins low bioavailability of 18% despite the lack of CYP3A metabolism can be explained by its poor mucosal absorption [4]. Additionally, the acid form can relactonize in vivo (Figure 1-7) [31, 32] Metabolism of both active and inactive form takes place in the enterocy te as well as in the liver and can lead to a variety of active and inactive metabolites [19]. The small intestine is therefore a potential site for drug-drug or drug-food interactions. Adverse Events As mentioned before, the mean decrease of LDL cholesterol can be as high as 60%. The large number of statin prescriptions [15], revealed more markedly the potential of this drug class for a rare but severe adverse event. Statins can be associated with diffu se myalgia, myopathy and rhabdomyolysis. Myalgia is defined as muscle co mplaints without creatine kinase (CK) level elevation. Myopathy is traditi onally defined as muscle pain or weakness accompanied by creatine kinase levels 10 times above the uppe r limit of normal. M yopathy can progress to rhabdomyolysis (CK levels higher than 10 times the normal range) and resu lt in renal failure but rhabdomyolysis does not have to be preceded by myopathy. Determination is complicated since definitions for myopathy and rhabdomyolysis vary. The National Lipi d Associations (NLA) Muscle Safety Expert Panel has been charged to examine the definitions and causative factors of statin myopathy [33]. These seve re side effects are estimated to occur in less than 1 per 100,000 prescriptions [20]. Thompson et al. [34] found a total of 3339 cases of rhabdomyolysis when reviewing the FDA database fr om 1990 to 2002. Interestingly 57% of the cases where assigned to cerivastatin, 18% to simvasta tin and 12% to atorvastatin. It is also noteworthy that approximately half of the cases occurred in 51-75 year old patie nts, with an additional 17% occurring in patients older th an 75 years. According to Dresser et al. myalgia and

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22 rhabdomyolysis seems to occur in conditions wher e the plasma concentrations of both parent drug and metabolite are elevated [35] and is ge nerally dose related. Conversely cases have been reported where myopathy occurs without elev ated plasma levels of creatine kinase [36]. The Grapefruit ( Citrus paradisi ) Botany The grapefruit tree (7-10m la rge) belongs to the genus Citrus Rutaceae. It bears white flowers and its fruits are a modified berry (F igure 1-8) [37]. Even though much controversy exists regarding the clas sification of the genus Citrus the main focus here should not be the history of the Citrus genus, but the origin of the grapefru it and appearance in Northern America. Research suggests that centuries after the first me ntioning of the citrus genus in south-east Asian culture, the oranges, lemons and sweet orange s reached Europe [38]. It is believed that Columbus brought the first Citrus biotypes to parts of the Caribbean. Furthermore the EnglishDutch speaking territories of the Caribbean seem to have undertaken the cultivation of another plant of the Citrus genus, the Citrus grandis [39] Biochemical [40] and genetic analysis [41] suggests indeed, that the grapefruit originated as a cross between Citrus grandis and Citrus sinensis Furthermore, Scoras analysis of the amylase patterns in C. paradisi strengthens the assumption that it is a cross between C. grandis and C. sinensis [38] These results seem to match the historical descriptions from Jame s Macfayden who in 1837 first assigned the name C. paradisi to the forbidden fruit described prior by Griffith Hughes in 1750 and Patrick Browne in 1789. Even though some contradiction exists between Hughes and Brownes description of the fruit, the colloquial name Barba dos grapefruit indicated clearly the origin of this fruit. Grapefruit Economy It is unclear as to when the grapefruit was in troduced to the northern part of the American continent, but indicators exist that Citrus fruits were brought to Florida, most likely St.

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23 Augustine, around the year 1565 laying the founda tion for the Florida Citrus economy [38]. Other researchers believe that the grapefruit was brought to Flor ida by Count Odette Phillip, who settled close to Tampa Bay [37]. Florida seems to be one of ideal regions for the Citrus cultivation, since times where temperatures r each the fruit freezing temperature are rare and temperatures in general are in the interval of optimal growth. Citrus trees do not grow when the air temperature reaches lower than 50oF and growth is imperceptible when the air temperature is above 95oF [42]. It is noteworthy, that in case of gr apefruit marketing the Duncan grapefruit is predominately sold in the first five months of the harvesting period, however sales are shifted towards the Marsh Seedless grapefruit in later time points [43]. The main percentage of the Florida grapefruit harvest is processed to frozen c oncentrate or chilled juice. In total, the United States of America produce a quarter of the wo rlds grapefruit produc tion of which Florida contributes 13.5% of the world grapefruit crop. Despite the increase in personal disposable income however, the per capita grapefruit cons umption has decreased by 50% since 1999[44]. Health Benefits Already in the year 1928 Bertha M. Wood included a pear and gr apefruit salad in her Dietetic treatment of Hypertension [45]. Even t oday consumers of grapef ruit and grapefruit juice believe in the beneficial attri butes for their health. In fact 14% of grapefruit juice consumers believe that grapefruit juice can prevent heart disease and 21% of the consumers believe that grapefruit juice can lower cholesterol [46]. In 2006 Gorinstein et al. investigated the effect of red grapefruit on serum cholesterol levels and found th at the consumption of grapefruit over thirty days can lower serum total cholesterol, LDL-choles terol and triglyceride levels in hyperlipidemic patients [47]. Intake of 200 mL juice daily for four weeks lowered cholesterol by 9%, low density lipoprotein cholesterol (LDL-C) by 21% and triglycerides by 25% [48]. It could furthermore been shown that a diet supplemente d with grapefruit or flavonoids contained in

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24 grapefruit can increase the plasma antioxidant potential [49, 50], effects which have also been attributed to sweetie [48, 51]. Re search suggests, that the flav onoid naringenin, which is also contained in grapefruit, inhibits the apolipoprotein-B (apoB) s ecretion in cell models [52-54], whereas naringin inhibits 3-hydroxy-3-methylgl utaryl-CoA (HMG-CoA) reductase and acylCoA:cholesterol acyltransferase (ACAT) in ra ts [55]. Furthermore the grapefruit industry cooperated with the American Heart Association and launche d the heart healthy campaign certifying grapefruit juice with the heart check mark for certain nutritional levels. Grapefruit Juice-Drug Interactions Discovery The combination of facts about grapefruit related and drug rela ted health benefits can be intriguing for the consumer, who might consume his drug together with grapefruit juice or consume grapefruit juice during the day to achieve maximum beneficial effect, because of the 1989 finding that grapefruit juice ca n increase the oral bioavailability of certain medications. This was originally discovered by Bailey et al [56] who were inves tigating the interaction between the calcium channel antagonist felodipine and ethanol. In this study grapefruit juice was used to mask the taste of ethanol. After the discov ery of this interaction more than 160 scientific papers have been published concerning grapefruit juice drug interactions. Mechanism of Action It has been shown that the predominant mechanism for this interaction is the irreversible inhibition of intestinal Cytochrome P450 3A4 (C YP 3A4) enzyme with a marked decrease in presystemic metabolism [57]. This is of great interest to the co mmunity of health professionals since roughly 60% of the drugs available to patien ts are metabolized to a greater or lesser extent by CYP 3A [58, 59]. In addition, another mechanism has been proposed, potentially leading to

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25 increased fractions of drug absorbed by inhibi tion of the enterocytic efflux transporter Pglycoprotein (Pgp) [60]. The increase in fraction absorbed however is limited to presystemic metabolism in the enterocyte. In most cases, studies have shown th at half-life of the drug under investigation does not change and the interaction doe s not change liver metabolism. This is further confirmed by a study comparing the raise in felodipine concen trations after intrav enous and oral dosing concomitantly with grapefruit ju ice. No significant increase in exposure could be found after intravenous dosing of felodipine with oral consumption of grap efruit juice [61]. The aforementioned effects seem to be depende nt on individual pati ent variability, batch, amount, and type of grapefruit jui ce, as well as dosing schedule [62]. This can be shown when comparing two studies, investigated by the same group of researchers. Lilja et al. [63-65] performed two clinical trials assessing the interac tion of simvastatin and gr apefruit juice. In the first study, double strength (DS) gr apefruit juice (grapefr uit juice frozen conc entrate diluted with half the regular amount of water) wa s given three times a day for three days. This resulted in a13 fold increased plasma levels of simvastatin. Years later a similar study performed with regular strength (RS) grapefruit juice resulted in only a 3.5 fold increase in simvastatin plasma concentrations. The effect of the interaction can last up to 3 days and has been extensively studied after single dose administra tion of drugs. Available sources for long term effect studies after multiple drug administration however are limited. Compounds of Interest Furanocoumarins and flavonoids ar e currently suggested to pl ay an active role in the inhibition of intestinal CYP 3A4 by grapefruit juice. 6,7-Dihydroxybergamottin (DHB) and a furanocoumarin dimer have been identified to be i nhibitors of this CYP en zyme [57]. In vitro experiments also confirmed the potential of naringin (NAR) and naringenin (NAG) as an

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26 inhibitor of CYP 3A4 in humans (Figure 1-9). Concentrations of 50 M showed a significant decrease in simvastatin intrinsic clearance in rat hepatocytes [66]. Bergamottin (BG) has also been reported to have the same inhibitory activity as naringenin [67]. Furthermore some studies with furanocoumarin free grapef ruit juice have shown that furanocoumarins account for some but not all of the inhibitory eff ect [68]. Roughly one third of the in hibition could be attributed to the concentrations of DHB and BG (Figure 1-10). Further inhibito ry potential is attributed to DHB dimers, epoxides and orthospiroesters. Tassaneeyankul et al. [69] determined the content of the two tail/tail dimers (Figure1-11) to range from not detectable to 0.39 ppm for GF-I-1 also called Paradisin A and 0.08-0.44 ppm for GF-I-4 Paradisin B. Guo et al. [70] quantified the contents of the BG epoxide and the DHB orthospiro ester. Concentrations of the epoxide resulted to vary from 0.17-0.27 ppm and the orthospiroeste r ranged from not detectable to 6.31 ppm. The dimers have great potential for CYP 3A4 inhibition. GF-I-1 and GF-I-4 seem to be potent inhibitors in human liver microsomes, with KI of 40.0 M and 5.56 M respectively [71]. When compared to DHB, GF-I-1 seems to be a far more potent inhibitor of CYP 3A4 with IC50 values of 0.075 M compared to 0.45 M [72]. Epoxiberg amottin showed slightly greater inhibition potential that DHB with a reported IC50 of 0.33 M [73]. No definitive data about the inhibition potential of grapefruit spiroesters has been publ ished so far, however in 2000 Bioavailability Systems,LLC received a patent for the develo pment of CYP 3A4 inhibi tors that resemble orthospiroesters from grapefruit juice to increase the bioavailability of CYP 3A4 substrates [74]. Influence of Grapefruit Juice on Statin Pharmacokinetics The effects of high doses of statins regarding mu scle toxicity have b een extensively studies and the risk has been identified [34]. It is pa rticularly worrying that patients and consumers attribute cholesterol lowering a nd cardio-protective properties to grapefruit juice consumption and take their cholesterol loweri ng drug and grapefruit ju ice concomitantly at breakfast in order

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27 to maximize the effect. The Food and Drug Administ ration (FDA) classifies grapefruit juice as a moderate inhibitor of CY P 3A metabolizing enzymes which can lead to a 2 to 5 fold increase in drug concentrations. However the FDA also notes th at the effects of grapefruit juice vary widely [75]. Interestingly recent experiments have shown that components of grap efruit juice also seem to exhibit an esterase inhibi ting effect. Influence on lovastat in metabolism has already been shown [76, 77] and might also be true for simvastatin. The effects of grapefruit juice on the pharm acokinetic properties of statins have been shown by a battery of clinical tr ials. The degree of increase in plasma AUC correlates with the bioavailability of the respective drug. Simv astatin and lovastatin, both exhibiting a bioavailability lower than 5%, show the greatest degree of interaction. The highest degree of interacti on was discovered when healthy subjects were administered double strength grapefruit juice three times a day over a period of three days. The AUC after a single dose of 60 mg simvastatin and simvastatin acid increased 16 fold and 7 fold respectively. Changes in Cmax were 9 fold and 7 fold respectively. An assessment of active and total inhibitors in the plasma revealed an increase of 2.4 fold for the former and 3.6 fold for the later [63]. A later study performed with regular strength grapef ruit juice and 40mg of simvastatin resulted in smaller increases in AUC and Cmax. Changes were 3.6 fold and 3.9 fold for simvastatin and 3.3 fold and 4.3 fold for simvastatin acid. Active or to tal inhibitors were not assessed in this trial [65]. The half life decreased slightly in the later but not in the former clinical trial. Significant changes after a single do se of grapefruit juice were measured up to 3 days. Changes in half-life however could also be attributed to simvasta tins own potential to i nhibit CYP 3A4 metabolism [78].

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28 The extent of increase in pharmacokinetic pa rameters of lovastatin when taken with grapefruit juice varied greatly with the type of juice administ ered. Double strength grapefruit juice resulted in increases of 12 fo ld and 15 fold in lovastatin Cmax and AUC respectively after an 80mg dose. Changes for lovastatin acid were 4 fold and 5 fold for Cmax and AUC respectively [79]. Regular strength juice and a dose of 40mg led to approx imately 2 fold increases in lovastatin Cmax and AUC and to an increase of 1.6 fold in lovastatin acid Cmax and AUC. The AUC of both active and total inhibito rs increased around 1.3 fold [80]. Changes in atorvastatin aci d, which has a bioavailability of 12% [4] were far less pronounced. Increases in atorvasta tin acid plasma exposure (AUC) range from 1.4 fold [81] and 1.8 fold [82] with single stre ngth grapefruit juice to 2.5 fold when given with double strength grapefruit juice [83]. Statistically significan t though small increases in AUC were also found with pitavastatin [82]. As expected, grapefruit juice had no effect on pravastatin, since pravastatin is not a substrate for CYP 3A [81, 83]. Company information and trials with other CYP 3A inhibitors suggest that fluvastatin [84] and rosuvastatin[85, 86] are not substrates for th is metabolizing enzyme. It is therefore unlikely that they might interact with grapefruit juice. Since only the enterocytic metabolism is aff ected by the grapefruit juice-drug interaction, metabolic ratios might be shifted and might lead to different concentrations of active and inactive metabolites. This has been shown by assessing the amount of active inhibitors in human samples after grapefruit juice and statin administration. Levels of active inhibitors were far less elevated as one would expect from the increase in simvastatin levels [63].

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29 Hypothesis and Objectives Grapefruit juice has been shown to increase ex posure of some drugs. The effect however varies greatly. Furthermore, the po tential for clinically significan t drug interaction is most likely limited to low bioavailability drugs or drugs give n at the higher end of the therapeutical range. We hypothesize, that most of the investigated dr ugs do not interact in a clinically relevant manner with grapefruit juice (GFJ) and that th e degree of the interaction is dependent on the batch and strength of the grapefruit juice. Regard ing the interaction with the cholesterol lowering drug we hypothesize, that the interaction of grapef ruit juice with simvasta tin is not clinically relevant and in low doses does not lead to gr eater muscle damage than simvastatin alone. Furthermore we expect that grapefruit juice ca n lower cholesterol as it has been shown with pomelo juice. To test these hypotheses, the follo wing specific aims were proposed: Specific Aim 1 Create an online database summarizing all published clinical trials to assess the overall interaction potential. A comprehensive, dynamic, and expandable database was created covering prescription and OTC drugs with ta ilored information for patients and health care professionals. Specific Aim 2 Assess the magnitude to which GFJ changes the simvastatin plasma levels in rats and atorvastatin levels in humans after long term us e. The magnitude of the interaction was assessed in a time and dose dependent manner. The stud y was performed over a 4-week period using two different strengths of grap efruit juice and two differe nt doses of simvastatin. Specific Aim 3 Assess the potential risk of myopathy devel opment in rats due to the grapefruit juicesimvastatin interaction. A mid-term study over four weeks was performed, using behavioral,

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30 physiological, biochemical, and histological mark ers to assess the pot ential development of myopathy after the alteration of the metabolic profile.

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31 Figure 1-1. Causes of death in the year 2004 as reported by the US Census Bureau [1]. Figure 1-2. Top 10 selling prescription drugs in 2006 (total dollars in billions) [3]

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32 Figure 1-3. Mevalonate pathwa y in mammalian cells [16] Acetyl-CoA Acetoacetyl-CoA HMG-CoA Mevalonate Mevalonate-PP Isopentenyl-PP Geranyl-PP Farnesyl-PP Squalene Cholesterol Dimethylallyl-PP Isopentenyl-tRNA cis -geranylgeranyl-PP Dolichol trans -geranylgeranyl-PP Protein isoprenylation Ubiquinone HMG-CoA reductase Statins

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33 COO-Na+ N OH O H F CH(CH3)2 OH COOH O H C H3 N N H O COOO H O H F 2 Fluvastatin Mevalonic acid Ca2+ Atorvastatin O H O O O H O Simvastatin O H O O O H O H Mevastatin (Compactin) O H O O O H O H OH H O H O O H O COO-Na+ Pravastatin Lovastatin (Mevinolin) Figure 1-4. Structures of HGM-C oA reductase inhibitors comp ared to mevalonic acid [4]

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34 O H O O O H O H O O H O OH COOH Lactone form Hydroxy acid form SV SVA Figure 1-5. Structure of simvastatin and simvastatin acid [23]

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35 O H O O H O O O H O O H O O C H3 O H O H O O H O O O H H O O H O OH COOH H O O COOH O H O O H O O OHC IV O H O O H O O HOOC O H O O H O O CH2OH O H O O H O O C H3 OH V VI VII SVA III I II SV Figure 1-6. Metabolism pathway of simvastatin SV=simvastatin, SVA=simvastatin acid, I=6OH-SV II= 3-OH-SV, III=3-OH-SV, IV =6-exomethylene SV, V=6-CH2OH-SV, VI=6-COOH-SV, VII=1,2,6,7,8,8a-hex ahydroxy-2,6-dimethyl-8-(2,2dimethyl-1-oxobutoxy)-1naphtha lene-pentanoic acid [23]

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36 OH COO-glucuronide O H R O H O O R OH O H COOHR Metabolite glucuronide Metabolite lactones Metabolite open acids CYP P450 CYP P450 oxidation CoASH Hydrolysis (chemical / esterase) Spontaneous Glucuronidation(UGT) -glucuronidase Statin lactone Statin acid Statin glucuronide Figure 1-7. Proposed mechanism of the relactonization of simvastatin acid into simvastatin [32]

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37 Figure 1-8. Citrus paradisi Macfaden (grapefruit)

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38 OH O O OH O OH O OH O OH OH OH OH O O OH OH O O H Naringin (NAR) Naringenin (NAG) Figure 1-9. Compounds in grapefru it juice shown to interact with CYP 3A4, NAR=naringin, NAG=naringenin O O O O O O O O OH OH Bergamottin (BG) 6',7'-dihydroxybergamottin (DHB) Figure 1-10. Compounds in grapefruit juice show n to interact with CYP 3A4, BG=bergamottin, DHB=6,7-dihydroxybergamottin

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39 O O O O O O O O OH O Paradisin B (GF-I-4) O OH O O O O O O O OH O Paradisin A (GF-I-1) Figure 1-11. Structures of DHB Tail/Tail dimers identified in grapefruit juice

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40 O O O O O Bergamottin-6',7'-epoxide (GF-I-5) O O O O O O O O H O OH OH GF-I-6 Figure 1-12 Structure of BG e poxide and DHB orthospiroester

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41 CHAPTER2 ASSESSMENT OF THE OVERALL INTERACTI ON POTENTIAL OF GRAPEFRUIT JUICE Background So far there have only been two case reports involving statins and potentially severe consequences of grapefruit consumption [87]. As of now, there is only one case report involving a woman that developed severe muscle damage after starting to consume one grapefruit a day [88]. Her simvastatin dose was on the higher en d of the therapeutic spectrum. Another case report involved atorvastatin [87]. This patient reported severe fatigue and muscle pain after exercising and consumption of grapefruit juice with his cholesterol lowering medication. Case reports like the above mentioned can only provi de limited information regarding the clinical relevance of this interaction. However the mass media impact of those case reports can have a high consumer impact. Regularly public mass media companies report about grapefruit juice drug interactions. However the public awareness inte ntions of multiple major television stations also publicize common misconceptions and mis understandings. To clar ify the interaction potential, a MSNBC reporter said that grapefruit ju ice can double your drug concentration by as much as three times [89]. In contrast to the mass media publication of th e grapefruit juice drug interactions there has been a large quantity of scientific publications that focus on pre clinical, clinical or mechanistic investigation of this inte raction. It is challenging however, to find this information especially for consumers of grapefruit juice who are not also hea lthcare professionals, since for them scientific database are largely unavailabl e or the information presented on the internet is not comprehensive and does not show the overall picture or does not in clude all drugs.

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42 Specific Aim Create an online database summarizing all published clinical trials to assess the overall interaction potential. A comprehensive, dynamic, and expandable database was created covering prescription and OTC drugs with ta ilored information for patients and health care professionals. Material and Methods To create a comprehensive data base of all releva nt publications rega rding the grapefruit juice drug interactions, database searches in Pu bMed, Web of Sci, Physicians Desk Reference, Clinical Pharmacology database, WebSpires a nd Google were performed. The search terms included grapefruit, grapefruit juice, a combin ation of either with d rug interaction or a combination of a drug know to interact or citru s paradisi. Furthermore searches in review articles, books (Drug Interaction Facts 2004 [90]) and other web databases focusing on grapefruit drug interactions were performed. All papers f ound were categorized into in vitro and in vivo experiments, where in vivo experiments were su bcategorized in clinical trials and animal experiments. In cases where no human in vivo da ta was available, animal in vivo data was considered. Each compound was assigne d an interaction level according to Bjornson et al .: weak or no interaction < 2.0-fold, modera te interaction > 2.0to 4.9-fold, strong in teraction > 5-fold increase in plasma AUC after grapefruit jui ce administration [91]. When different studies resulted in a categorization of two categories, th e higher category was chosen for safety reasons. The database was then programmed in My SQL (MySQL AB, Sweden). The programming for the user frontend was performed in PHP4. The one page summaries were written in MS Word and converted to PDF using Adobe Acrobat Professional. Webali zer 2.01 was used to record the websites access statistics.

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43 Database Design Programming Language MySQL is a free, open source Relational Data base Management System (RDBMS), which means that the data is organized and stored in a set of related tables. MySQL is licensed via the GNU GPL (General Public License). In order for a MySQL server to understand a request sent by a user, the command needs to be communicated using the Structured Query Language (SQL). In order to send the commands to the MySQL se rver we use the PHP scripting language. PHP stands for PHP:Hyper Text Preprocessor. PHP however does not understand SQL commands; it just establishes the connection to the MySQL server and sends the SQL message over that connection [92]. Through the combination of My SQL and PHP scripting language it is possible to create truly dynamic websites. This means that not every user will s ee the same website and website are tailored towards the users need. This enables us to create differentiated websites for health care professionals or for grapefruit ju ice consumers out of the same database. The principle of a dynamic website is illustra ted in figure 2-1. The visitor of the website requests a certain dataset. The MySQL server receiving that re quest via PHP then accesses the database to gather the requested data. The data is then merged with a previously determined website template and sent to the user computer The advantages of this principle are clearly visible. Dynamic web programming enables us to update the database without changing the overall website template, or to change the webs ite without having to reprogram the dataset. Database Layout As mentioned before, MySQL is a Relational Database Management System, meaning that the data is stored and organized in sets of re lated tables. A graphical illustration of the Table layout can be found in figure 2-2. The database layout consists of three different tables. Table 1 contains the information about the drug categories. Each category is assi gned a unique category

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44 identification (ID) number. The drug category ta ble contains the information fields category name (c_name) and category general information (c_info). Table 2 is the table that lists the informa tion for a specific drug in each category. Again, each drug is assigned a drug identifier (ID). Table 2 contains the information fields drug name (d_name), drug brand name (d_product), drug ph armacokinetic information (d_pkinfo), drug pharmacodynamic information (d_pdinfo), a differe ntiated information text for consumers (d_textconsumer) or hea lth care professionals (d_textprofe ssional) and an identifier for the classification in different drug in teraction categories (d_interactio nlevel). In order to link this table to the previously defined drug category, a category reference field (categoryID) is also contained in table 2. To deliver the most comprehensive informati on on grapefruit juice drug interactions, one page summaries of the respective literature were created and publishe d together with the respective pubmed link and fulltext for Center members. Table 3 of the database stores the relevant information of the studies, the study ti tle (s_title) the hyperlink to the study summary (s_summary), pubmed abstract (s_pubmed) a nd fulltext pdf (s_fulltext). The studies are crosslinked to the respective drug in table 2 using a drug identifi cation reference field (drugID). One disadvantage of the RDBMS system is that the table layout has to be implemented before the data is input into the database. Once the dataset is created, changes of the database structure are difficult and may result in manually reentering the data. Results The review of online databases and current lite rature resulted in a dataset of 74 drugs. A total of 163 scientific publications were reviewed and summarized in simple one page summaries, consisting of a refere nce section, a material and met hods sections, a results section and a conclusions section (Figure 2-3). The database is cu rrently online under

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45 www.druginteractioncenter.org Out of the 74 drugs analyzed, 51 (69%) drugs showed a weak or no interaction with grapefru it juice, 14 drugs (19 %) showed a moderate interaction with grapefruit juice and 6 drugs (8%) showed a strong interaction with grapefruit juice. 3 drugs (4%) have not been studied in humans (Figure 2-4). The necessity of a comprehensive online database can be reflected in the great number of hits that the website received. A total of roughly 9 million hits were registered on the website. A total of more than 1.5 million pages were submitted to the users. Results of the access parameters can be found in Figure 2-5. Rough estimation of returning visitors shows a relatively stable number of users accessing the website multiple times (Figure 26). Interaction Potential by Drug Category Antiallergics Several clinical trials have been performed assessing the interaction potential of grapefruit juice with antiallergics. Studied drugs includ e terfenadine [93-96], de sloratadine [97] and fexofenadine [97]. Exposure for desloratadine was not altered when the drug was consumed concomitantly with grapefruit juice, whereas th e AUC of fexofenadine was decreased. Three performed studies showed an increase in terfenadine exposure when administered simultaneousely with grapefruit ju ice[94-96]. The reported effects s eem improbable to be of high clinical relevance. Antibiotics Three antibiotics where studied in the published literature: clarithromycin [98], erythromycin [99] and telithromycin [100]. Overall the clinical relevance of the interaction with grapefruit juice appears to be low.

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46 Anticoagulants The data resulting from 4 published c linical trials is inconsistent. Merkel et al. [101] found the percentage of 7-hydroxycoumarin, a metabolite of coumarin excreted in the urine, to be decreased, controversly in a second study the app earance of the metabolite in urine was delayed, but the recovery remained uncha nged[102]. The delay in appearance of the metabolite has been confirmed in a third study [103] No interaction with warfar in has been reported [104]. Currently, no clinically relevant interactions have been confirmed for this drug class. More extensive clinical studies are necessary to asse ss the overall effect of GFJ on this drug class. Antimalaria drugs Grapefruit juice has been reported to increa se the exposure of artemether [105, 106] and halofantrine [107]. The increased exposure of artemether did not re sult in bradicardia or changes in QTc interval. No changes in pharmacokinetic parameters were regist ered during simultaneous administration of quinidine [108, 109] or quini ne [110]. Though no changes in pharmacokinetic parameters were observed, the QTc interval in the grapefruit juice qu inine group was prolonged at the 1 hour timepoint after drug administra tion. According to the aforementioned drug classification the interactions with quinidine an d quinine would be consid ered weak and unlikely to be clinically relevant, the interaction with halofantrine however might result in a change in clinical effect. Antiparasitic drugs Moderate and weak interac tion were reported for albend azole [111] and praziquantel respectively [112]. A clinical si gnificant interaction with prazi quantel seems unlikely; however grapefruit juice should only be consumed w ith albendazole after a careful risk benefit assessment.

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47 Sedative-hypnotics Concurrent administration of grapefruit juice and alprazol am [113] did not result in changes in pharmacokinetic parameters duri ng a single dose experime nt. Midazolam [114-117], triazolam [117-121] and quazepam [120], when admi nistered concomitantly with grapefruit juice exhibit minor exposure increases. In a recent clinical trial with tr iazolam, the acute and extended exposure to GFJ produced a significant inhibition of enteric, but not hepatic, CYP3A4 and also caused significant pharmacodynamic effects [121]. A pparently clinically re levant interactions have also been observed for buspirone [122] and diazepam [123]. More clinical trials are necessary to definitively assess the extent of the interaction for this drug class. Calcium channel blockers Amlodipine [124, 125], diltiazem [126, 127], nimodipine [128], nifedipine [129-131], pranidipine [132] and verapamil [133-135] were shown to interact weakly with GFJ. However, only for amLodipine, diltiazem, nimodipine and verapamil no changes in pharmacodynamic parameters heart rate and blood pressure were reported. No pharmacodynamic alterations were reported for nifedipine and GF J. Felodipine [61, 124, 133-146], ni cardipine [147], nisoldipine [148, 149], nitrendipine[150] and manidipine [151] moderately interacted with GFJ. Changes in pharmacodynamic parameters were reported after GF J administered with felodipine. Overall, felodipine, nicardipine, nisoldipine and nitr endipine containing pr oducts should only be consumed with GFJ after a cautious risk and benefit assessment. HIV protease inhibitors Amprenavir [152] and indina vir [153, 154] pharmacokinetic pa rameters did not change when the drugs were administered concomita ntly with GFJ. The AUC of saquinavir was increased by GFJ [155, 156]. Howe ver, overall the reporte d interactions can be considered weak and are unlikely to be clinically relevant.

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48 HMG-CoA reductase inhibitors Increased exposures were reported for ator vastatin [81-83], lovastatin [79, 80] and simvastatin [10, 63-65], but not for pravastatin [81, 83]. Small but statistically significant changes were found for pitavastatin [82]. Atorvastatin exhibited a moderate interaction with GFJ regarding the overall ex posure. Lovastatin and simvastatin exhibited a strong interaction. Pravastatin or pitavastatin coul d be chosen as an alternative drug if clinically recommendable and patients want to ensure a lack of interaction. Hormones GFJ did not affect 17-beta estradiol [157] or prednisone [158] pharmacokinetics. AUCs were weakly elevated for ethinylestradiol [159]and methylpredniso lone[160]. A decreased morning cortisol plasma concentrations was f ound after methylprednisolone administration with GFJ. The absorption of levothyroxine was mildly decreased after repeated consumption of grapefruit juice, however here the authors conclude a low clin ical relevance [161]. Anecdotal reports that grapefruit juice may cause contraceptiv es to lose their effect could not be confirmed by scientific studies. Immunosuppressants Eleven out of a total of thirteen studies reported increased cyclos porine exposure up to 2fold [162-172]. Conversely, two st udies reported no GFJ-induced ch ange in AUC. Overall, the clinical significance of interactions is assumed to be low [158, 173]. Antitumor drugs Few studies have been performed with human subjects for this drug class. The mean AUC of etoposide was increased when administered concomitantly with GFJ [174]. However, the report does not state the statistical significan ce of the reported data. In order to develop recommendations for this drug class further research will have to be conducted.

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49 Over the counter drugs Contradicting results were reported when caffe ine was administered with GFJ. Whereas no changes in AUC, blood pressure, and heart rate were reported by Maish et al. [175], a second study found increased AUC and half life, without an assessmen t of pharmacodynamic parameters [176]. Overall, no clinically significan t interactions of an ove r-the-counter drug with GFJ have been reported. Beta receptor blocker AUC and Cmax of celiprolol decreased by 95% when administered concomitantly with GFJ [177], however heart rate and blood pressu re remained unchanged. Conclusively, observed interactions to be clinically re levant. The plasma concentrations of acebutolol [178] were slightly decreased by grapefruit juice. Fu rther human studies will have to be conducted in order to derive reliable conclusions. Currently, clinically si gnificant interactions appear to be unlikely. Antiarrhytmics Coadministration of amiodarone [179] with re gular strength GFJ resu lted in a significant but weak increase in AUC and Cmax, however a decrease in the alteration on the PR and QTc interval was observed. These data imply that not only pharmacokinetic data should be considered when determining the extent of the significance of a given GFJ-drug interaction. Overall, the changes in pharmacodynamic parameters should be taken into account when prescribing this drug to patients who wish to consume GFJ. Other drugs GFJ has shown to increase the exposure of carbamazepine [180], cisapride [181-184], fluvoxamine [185], losartan [186], methadone [1 87], scopolamine [188], sertraline [189] and repaglinide[190]. However, only the interaction of GFJ and carbamazepine and cisapride appear to be clinically relevant. No alteration in exposure was observed for clozapine [191, 192],

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50 theophylline [193] and haloperidol [194], omepr azole [69] and phenytoi n [195]. Contradicting results were reported for itrac onazole [196-198] digoxin [199, 200] and sildenafil [201, 202]. Conclusions The results of the creation of this database support the im portance of a comprehensive online database. Additionally a dynamic, updat eable MySQL database combined with PHP scripting seems to be the ideal tool for pati ent and health care professional information and education. The amount of visitors of the online database strengthens the need for up to date scientific information. The work performed demonstr ates that the current c onception that most of the drugs interact with grapefru it juice cannot hold. On the other side however is it also unsafe and irresponsible to say that pa tients can consume grapefruit ju ice to lower their drug dose and reduce drug costs. Strong indicators are presen t that suggest a need for the measurement of certain furanocoumarins and flavonoids in the juice prior to conducting a study and report those results in the literature to achieve comparable results.

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51 Figure 2-1. Principle of a dynamic website.

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52 Figure 2-2. MySQL table layout fo r the drug interaction website

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53 Figure 2-3. Sample of the one page summaries uploaded to the database

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54 1 2 3 N/A 0 20 40 60Interaction categorynumber of drugs [#] Figure 2-4. Number of interaction drugs in each category: 1=no to weak interaction, 2=moderate interaction, 3=strong interacti on, N/A=not tested in humans

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55 Aug-05 J an -06 J un-06 N ov-06 A pr0 7 S e p -07 0 200000 400000 600000 Hits [#] Au g -05 J a n-06 J u n -06 Nov-06 Ap r0 7 Sep-07 0 50000 100000 150000 Pages[#]A B Figure 2-5. Website access statistics per mont h: A=number of hits, B=number of submitted pages, C=number of files, D=number of visits A ug -0 5 J a n -0 6 J un -0 6 No v -0 6 Ap r-0 7 Se p -0 7 0 50000 100000 150000 Hits-Files [#] Figure 2-6. Website access statistics hits-files as rough estimate of returning visitors per month. Aug-0 5 Jan-06 Jun-06 Nov-06 Apr-07 Sep-07 0 100000 200000 300000 400000 500000 Files [#] Aug-05 Jan-06 Jun-06 Nov-06 Apr-07 S ep-07 0 5000 10000 15000 20000 25000 Visits [#]C D

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56 CHAPTER 3 EFFECT OF LONG TERM INGESTI ON OF GRAPEFRUIT JUICE ON THE PHARMACOKINETICS AND TOXI COLOGY OF SIMVASTATIN Background Besides cholesterol lowering drugs, it seems that consumers also attribute healthy and cholesterol lowering qualities to grapefruit juice. 14% of grapefruit juice consumers believe that grapefruit juice can prevent hear t disease and 21% of consumers are convinced that grapefruit juice lowers cholesterol [46]. Furthermore the grapefruit indu stry cooperated with the American Heart Association and launched the heart healt hy campaign certifying grapefruit juice with the heart check mark for certain nutritional levels. Th is combination of facts can be intriguing for the consumer, who might consume his drug together with grapefruit juice or consume grapefruit juice during the day to achieve maximum benefi cial effect, because of the 1989 finding that grapefruit juice can increase the oral bioavailabil ity of certain medications [56]. As mentioned in the previous chapter, grapefruit juice has been sh own to alter the disposition of simvastatin in healthy subjects. Lilja et al [63] demonstrated in 1998 as well as 2004 [65] that grapefruit juice can increase the AUC of simvastatin by as much as 13 fold. Lower increases have been found in changes in the main active metabolite simvasta tin acid, as well as the influence on active and total inhibitors of the HMG-CoA reductase. Lilja et al. [63] conclude that large amount of grapefruit juice should be avoided, or the dose of simvastatin should be adjusted in order to prevent the increased risk of mu scle toxicity associated with increased serum concentration of simvastatin. It remains unclear how ever if the increase in simvasta tin serum concentrations leads in fact to a higher incide nce of muscle toxicity. In experiments performed by Smith et al. [203, 204] this research group showed that doses of 100 mg/kg x day-1 and 150 mg/kg x day-1 do not lead to any detectab le muscle damage after 24 weeks in Sprague Dawley rats. However a dose of 180 mg/kg x day-1 has been shown to

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57 induce muscle damage ranging from very slight to moderate in 4 out of 15 rats during a subjective evaluation. In contrast to the findings by Smith et al. recently performed experiments by Westwood et al. [205] indicate that muscle damage might be induced in female Wistar Hannover rats with a maximum tolerated dose of 80 mg/kg x day-1. The rats in the dose finding experiment exhibited detectable muscle damage in this dosing group after 10 days ranging from mild to severe depending on the muscle under investigation. A dos e of 60 mg/kg x day-1 however showed no muscle necrosis after 43 days. It is widely believed that the occurrence of myopathy is correlated to the inhibition of th e HMG-CoA reductase. Experiments have shown that a dietary substitution of mevalonate, th e subsequent reaction product of the HMG-CoA reductase can reduce the severity of muscle damage [205]. On the other hand experiments conducted with 3 week old Wistar rats have show n that muscle damage can also occur at a dose of 100 mg/kg x day-1 in seven out of nine rats ranging from moderate to severe without showing significantly different levels of cholesterol compared to the untre ated control [206]. It has also been shown that administration of HMG-CoA reducta se inhibitors in rats might not lead to a cholesterol lowering effect in this species. The rat can upregulate the cholesterol synthesis overcoming the effect of the hypercholesterolem ic drug, to a point where the cholesterol synthesis rate and cholesterol levels seems normal even in the presence of a competitive inhibitor [207, 208]. Many researchers therefore assume that the high simvastatin exposure after concomitant grapefruit juice consumptions exhibit the same muscle damaging potential as the administration of a regular increased dose. That this fact is not necessarily ac curate might be demonstrated by the fact that simvastatin is a prodrug that needs to be metabolized in order to be activated and that the prodrug itself has a low bioavailability [4]. As menti oned before, grapefruit juice only

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58 interacts with the CYP 3A4 in the gut wall and seems to have no influence on liver metabolism (Figure 3-1). Simvastatin however is already metabolized to an extent of almost 95% when reaching systemic circulation to metabolites wh ich account for the gross part of the activity. High doses of simvastatin (as they are reached by increase in simvastatin dosing) thus results in an increased amount of metabolites. High doses of simvastatin that ar e achieved as a result of an enterocyte sided inhibition of the drug meta bolizing enzyme CYP 3A4 however lead to predominantly increased concentrations of the prodrug lactone form of simvastatin which is inactive. It is therefore crucial to investigate the relationship between the frequency of muscle damage after concomitant grapef ruit juice simvastatin administ ration over a increased period of time. Specific Aim The objective of this study was to assess the magnitude by which GFJ changes the simvastatin plasma levels after long term use in rats. The magnitude of the interaction was assessed in a time and dose dependent manner. Th e study was performed in rats over a 4-week period using two different strengths of grapefruit juice and two different doses of simvastatin. Further to assess the po tential risk of myopathy developm ent due to the grapefruit juicesimvastatin interaction. A mid-term study in rats over four weeks was performed, using behavioral, physiological, biochemical, and hi stological markers to assess the potential development of myopathy after the al teration of the metabolic profile. Material and Methods Chemicals Floridas Natural grapefruit juice Ruby Red was purchased at Publix supermarkets (Gainesville, FL, USA) at a to tal amount of 2 Gal. Lot: 98 and lot 2A6 and frozen in 250mL aliquots on 06/21/06.

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59 Minute Maid Premium 100% pure frozen con centrate grapefruit ju ice (with Calcium added) was purchased on 06/23/06 at Publix supe rmarkets (Gainesville, FL, USA) in amount of 4 cans. Lot AD3 1712 and frozen in 125 mL aliquot s after dilution with half the regular amount of water. 1 can was diluted with 532.5 mL regular tap water. Simvastatin (SV), 99.4% pure, wa s a kind gift from Merck & Co., Inc. ( West Point PA., USA). Naringin (NAR) and naringenin (NAG), both > 95% pure, were from Roth GmbH & Co. (Karlsruhe, Germany), bergamottin (BG) (> 98% purity) was obtained from Indofine Chemical Company, Inc. (Somerville, NJ, USA), 6 ,7 -dihydroxybergamottin (DHB) (> 97% purity) was purchased from Sigma-Aldrich (Saint Louis, MO .). Sodium heparin was purchased from SigmaAldrich (Saint Louis, MO). Stock and Work Solutions Solutions of Simvastatin were made by susp ending SV in a 0.8% Carboxymethylcellulose (CMC) solution in water. Suspensions for simvasta tin were prepared daily by the scheme in table 3-1. Solutions for NAR, NAG and BG, DHB were pr epared according to table 3-2 and 3-3. Material and Methods Laboratory animals Male Sprague-Dawley weigh ting 230-270g rats were purchas ed from Harlan Sprague Dawley, Inc. (Indianapolis, IN). The animals were housed in conventional housing, two animals per cage. The experiment was approved by the Univ ersity of Florida Institutional Animal Care and Use Committee (IACUC). Open field test This method is usually used to evaluate pos sible sedative or stim ulating activities of animals [209]. The test is also used as an indi cator for locomotor activit y. The open field consists

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60 of a round grey plastic arena measuring 70 cm in diameter surrounded by a grey plastic wall of 34 cm height and is evenly lighted with three 40 W light bulbs. The floor of the arena is divided into several concentric units by black painted lines, dividing the arena into 19 equally sized fields. Each rat will be placed in the center of the arena and recorded fo r 5 min after the 28 day experiment. The parameters evaluated in this te st include number of fi eld crossings and total distance covered by the animal. We assume that drug effects on the muscle would be present if both number of field crossings and distance are significantly lower in the treatment groups compared to control Open field tests were digitally recorded using a high-resolution video camera WV-CP244 (Panasonic, Secaucus, NJ, U.S.A). The analysis of the videos was performed using TopScan (Figure 3-2), Top View Animal Behavior Anal yzing System (version 1.00, Clever Sys Inc. Preston, VA, U.S.A) by a blinded person. The open field test arena was thoroughly cleane d with a soap solution after each animal, to avoid influencing factors such as odor left of the previous animal. Grip strength test This test is used to assess muscular strengt h in rodents that can be influenced not only by sedative drugs and muscle relaxants but al so by toxic compounds. Reliable assessment of gripping ability requires that anim als are used to handling. Accord ingly, the first 5 sessions of training were limited to handling each animal for 5 min. During the training sessions, animals were held with both hands and placed on the grid of the grip strength meter (Linton grip strength meter for the pilot study, GSM, PANLAB grip strength meter fo r the final study, distributed by Stoelting & Co, IL, USA figure 3-3). When the animals are placed on the grid, the animals readily hold on to the grid as a natural reflex, a nd the animals are then gently pulled away from the device. The GSM then measures the maximal fo rce before the animal releases the grip. GSM

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61 testing (3 trials/animal/session) was carried out once per week for the duration of the entire treatment. The area under the grip strength curve was compared. Histological experiment Muscle biopsies of the gastronemius and the extensor digitorum long muscle were taken on the final day of the experiment. The muscle biop sies were immediately pr eserved in 10% neutral buffered formalin. After completion of both expe riments, the samples were processed to wax blocks and the 5m thick sections were staine d with Haematoxylin & Eosin stain. Transversal and longitudinal sections of the muscle were ev aluated. The occurrence of muscle damage was analyzed objectively by a blinded analyst unde r light microscopy, evaluating eight randomLy chosen sections in the muscle. Total muscle cel ls and damaged muscle cells were counted. The observed sections were equally di stributed between transversal a nd longitudinal sections. The following parameters were used in the assessment of muscular damage: Variability in size and shape of muscle fibers, mu scle fiber striation or loss of striation as well as size and localization of nuclei. The assessment of muscle damage was performed by assigning the observed damaged muscle cells into four categorie s: No damage to Minimal less than 10% damaged fibers, Mild less than 20% damaged fibers, M oderate less than 50% damaged fibers, Severe More than 50% damaged fibers following a similar classification by Westwood et al [205] Clinical chemistry Plasma chemistry parameters were evaluated using the Vitalab Selectra II Autoanalyzer (Vital Scientific NV, Spankeren, The Netherlands). Reagent, Control and Calibrator Kits for total cholesterol, gamma-glutamyltransferase (GGT), alanine amino transferase (ALT), aspartate amino transferase (AST) and Creatine kinase (CPK) were purchased from Clinical Data,

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62 Smithfield USA. The Vitalab Selectra II was a kind gift from Steigerwald Arneimittelwerk GmbH (Darmstadt, Germany). Grapefruit Juice Analysis HPLC System Samples were analyzed by a Shimadzu VP se ries HPLC system (Kyoto, Japan) equipped with a SPD-M10Avp diode array detector, a LC -10ATvp solvent delivery unit, a SIL-10AF autosampler, a CTO-10Avp column oven, a SCL-10Avp system controller, a DGU-14A on-line degasser, a FCV-10ALvp low-pres sure 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 te st for impurities in al l of the chromatographic peaks of interest. Determination of Flavonoids (Naringin and Naringenin) The GFJ and the homogenate of tissues (200 L) were centrifuged at 5000 rpm for 5min and were mixed with cold methanol (1mL for DS juices 600microL for RS juices), vortexed for 1 min, and centrifuged at 2500 g for 15 min, as previously de scribed [210]. After filtration through a 0.45 m PVDF membrane filter (Milli pore Corp., Bedford, MA), the supernatant (25 mL) 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) (a djusted with orthophosphoric acid)/methanol (40:60), respectively. The 250 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 mi n to 100% of B at 55 min. This condition was

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63 maintained until 70 min and then returned to 100% of A, which was kept constant during 5 min before proceeding to the next injection. Determination of Furanocoumarins (Ber gamottin and 6',7'-Dihydroxybergamottin) GFJ and the homogenate of tissues (3 mL) we re mixed with ethyl acetate (2 mL). The extraction was performed by shaking the mixtures four times over 30 min. The mixture was centrifuged at 3200g for 20 min; th e organic phase was collected and evapor ated under vacuum. The residue was reconstituted with 600 L of a DMSO/methanol so lution (1:3 v/v) as described previously [62]. The reconstituted residues were filter ed through a 0.45 m PVDF membrane filter (Millipore Corp.). Volumes of 25 L of each samp le were injected and analyzed at 310 nm. The flow rate and the temperature were set to 1 mL/min and 35 oC, respectively. Solvents A and B consisted of water and methanol, respectively. The column and guard column (as used for flavonoids) were initially equilibr ated with mobile phase consisting of a mixture of solvents A and B (45:55), respectively. Tw enty minutes after injection, so lvent 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. Animal Experiment Design The animal experiment was separated in two parts: Part 1: a pilot study to assess the detectability of muscle da mage in male Sprague Dawley rats after two different doses of Simvastatin Part 2: a major (final) study to assess the influence of two differ ent concentrations of grapefruit juice on two different doses of simvastatin.

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64 Pilot Study A 4 week animal experiment using two doses of simvastatin was performed. Non-fasted male Sprague-Dawley rats weighing 214-245g were used. Simvastatin was given in doses of 53 mg/kgxday-1 (SV53), 200 mg/kgxday-1 (SV200) bot h in 0.8% CMC. Water was given as control also with 0.8% CMC. The aim of this st udy was to assess whether it is possible to detect myopathy after different doses of simvastatin in rats. A total of 24 male Sprague Dawley rats were used. 8 rats were randomly assigned to one of the following three groups and dosed daily (Table 3-1). On each day, we measured the forelimb grip performance using a Linton grip strength meter. Furthermore, the daily body wei ght was recorded. On the last day the animals were sacrificed and the organs (liver, spleen, kidneys, adrenal glands, heart and testes) were weighted. Additional muscle biopsies from the hindlimb were taken. Two muscles were sampled, the gastronemicus and the extensor dig itorum long. These muscles have been shown to be susceptible for simvastatin muscle damage in the literature [203, 205]. As potential parameters, the extremity temperature was meas ured using a non contact infrared thermometer and an open field test (to eval uate potential locomotor impairment) was performed on the last day prior to scarification. Final Study Non-fasted male Sprague-Dawley rats wei ghing 239-346 g were used. The animals were handled daily a couple of days before the study star ts in order to reduce stress and adapt them to the researcher. Body weight was checked daily as well. The male Sprague-Dawley rats had free access to food and water during the experiment except for the first two hours after drug administration. The animals were di vided in the groups shown in table 3-3. On the first day of the experiment the rats were dosed with th eir respective simvastatin dose (SVlowdose = 20 mg/kgxday-1 (SV20) Simvastatin, SVhighdose = 80 mg/kgxday-1 (SV80) Simvastatin) and their

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65 grapefruit juice permutations (s imvastatin and regular strength grapefruit juice; SV20RS and SV80RS, and simvastatin and double strength grapefruit jucie; SV 20DS and SV80DS). The grapefruit juice lot # 98 was used for regular strength dosing and lot # AD3 was used for double strength dosing. To establish a pharmacokinetic baseline profile, blood samples (500 l) were taken from the sublingual vein at 0, 1, 2, 4, 6, 8, 12 hours after drug administration (three blood collections per day; e.g. time points 1, 4, 8; then two weeks washout, then the remaining time points 0, 2, 6, 12 in the same animals). After th e acute phase two blood samples (1000 l) was taken each week for 4 weeks right before dosin g the drug (trough level) and 2 hours after dosing (peak level) figure 3-4. After each blood sample approximately 1000 l of isotonic saline were replaced by i.p. injection in orde r to maintain the blood fluid. The blood samples were analyzed for Simvastatin, Simvastatin acid. Liver enzymes we re also measured to observe the influence of simvastatin on its target organ (the liver) and total cholesterol was measured as an effect parameter. Muscle strength parameters were measured before blood sampling of each of the acute study days and the chronic study phase. The animals were fed grapefruit juice (GFJ) (5 mL/kg) at different concentrations (double and regular strength), or water (5mL/kg) and S imvastatin through an oral feeding needle daily. At the end of the study the rats were sacrifi ced, blood was collected (for determination of simvastatin plasma levels) and muscle samples were taken to assess differences in muscle histology and organs were weight to assess organ changes. Statistical Analysis Pilot study: One-way ANOVA with Dunnetts po st test was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. Differences were consid ered statistically significant at P<0.05.

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66 Final study: Samples were an alysed using SAS version 9 (Cary, NC, USA). All samples were analyzed using a Two Way ANOVA with a B onferroni multiple comparison post hoc test to compare treatments to each other. Differences were considered statistically significant at P<0.05, As set a priori, the following comparisons were evaluated: All parameters except cholesterol: o Water control, SV20, SV20RS and SV20DS o Water control, SV80, SV80RS and SV80DS Cholesterol: o Water control, RS, DS, SV20 and SV80 o Water control, RS, SV20 SV80 SV20RS, and SV80RS o Water control, DS, SV20, SV80, SV20DS and SV80DS Outliers were detected with Grubbs test us ing Graphpad QuickCalcs, GraphPad Software, San Diego California USA, www.graphpad.com. Results Juice Analysis No naringenin could be found in either of the juices analyzed. Conten ts of naringin were 1475 42.3 M and 684 12.0 M for both batches of Floridas Natural Ruby Red, lot 98 and 2A6 respectively. The Minute Maid frozen concentrate contained 1476 34.3 M naringin. Concentrations of bergamottin were 31.64 1.1 M (batch 98) and 19.13 .1 M (batch 2A6); the dihydroxybergamottin content was 6.53 0.3 M and 4.9 1.3 M respectively. The juice concentrate AD3 contained 47.77 1.9 M bergamottin and 39.2 1.6 M dihydroxybergamottin. Values are given as mean (n=3) SD. Figure 3-5 Survival Rate One animal died at the beginning of the Pilot study in the control group due to complications during blood sampling. All animal s survived in the low dose 53 mg/kgxday-1 and four rats died in the 200 mg/kgxday-1 before the end of the study. The study was prematurely aborted due to the strong signs of toxicity in the 200 mg/kgxday-1 group.

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67 The grapefruit juice final study was completed as planned. All animals survived during this study (Figure 3-6). Body Weight During the pilot study, the cont rol group and the 53 mg/kgxda y-1 group show an increase in body weight over 10 days; the 200 mg/kgxda y-1 group shows a significant decrease in body weight (Figure 3-7). During th e final study, all groups showed an increase in body weight over the period of 28 days. There was no significant diffe rence in weight gain comparing the a priori selected groups (Figure 3-8). Behavioral Experiments Open field test The question to be answered was whether simvas tatin in combination with grapefruit juice decreases the locomotor activity as a sign of muscle damage. After the completion of the pilot study, the 53 mg/kgxday-1 group exhibited a signif icantly higher number of line crossing than the control (P<0.05). The number of line crossi ngs was decreased in the 200 mg/kgxday-1 group. These differences however were not statistically significant. Figure 3-9 No differences in the number of line crossi ngs could be found in the SV 20 group. Within the SV80 group, the number of line crossing of the SV80DS was increased compared to the SV80 control and the water control (both P<0.01). Figure 3-10 The total distance travelled by the rat resulted in the following differences. In the pilot study the animals in the 53 mg/kgxday-1 group travel more distance in the recorded 5 minutes than the control group. The 200 mg/kgxday-1 ha d a much lower travelling distance in millimeters than the control group, however the difference was not statistically significant (Figure 3-11).

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68 In the final study, the distance covered by the animals in the SV80DS group was significantly higher than its re spective control SV80 and the water control (P<0.01 and P<0.05 respectively) No differences were observed in the SV20 group (Figure 3-12). Grip strength test Comparisons of the grip strength results were made as area under the grip strength curve. No significant differences were found in the p ilot study (Figure 3-13, 3-14) or in the final study (Figure 3-15,3-16). Extremity temperature The measurements of the paw temperature in the pilot study resulted in significant differences when comparing the 200 mg/ kgxday-1 group to control (Figure 3-17). In the final study, differences were observed in when comparing the SV20ERS group with the water control. No differences were observe d in the SV80 juice combinations with the SV80 control (Figure 3-18). Histological Experiment During the pilot study, all animals in th e control group and the 53 mg/kgxday-1 group exhibited no or only minimal muscle damage; out of the four surviving animals in the 200 mg/kgxday-1 group three showed no to minimal si gn of muscle damage. One rat in this group exhibited clear signs of muscle damage and was cl assified as moderate muscle damage with 32% of damaged muscle fibers. The incidence of muscle damage in the final study was minor. Throughout the groups no damage to minimal muscle fiber damage was pr evalent. Light miscroscopical analysis at different magnifications revealed one animal with mild muscle damage in the regular strength grapefruit juice control group, furthermore 1 one rat in the SV20DS group exhibited mildly damaged muscle fibers (Figure 3-19,3-20,3-21)

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69 Clinical Chemistry Values below 10 mg/dl for Cholesterol were considered measuring errors and were not considered. No significant differences in the cholesterol levels in between the groups during the pilot study were found after 10 days of treatment. Although two rats in the 200 mg/kgxday-1 had slightly elevated cholestero l levels (Figure 3-22). During the final study, we found that the chol esterol levels of RS and DS juice are significantly lower than the water control (P<0.05 for both) and that the cholesterol levels in each was not different from either the SV20 or SV 80 group. The levels for SV20 and SV80 however were not different from the water control. Furthermore SV80RS cholesterol levels were significantly lower than SV20RS or the water co ntrol. Additionally cholesterol levels after SV20DS treatment were significantly higher when compared to a regular dose of DS GFJ (Figure 3-23). ALT values measured in the pilot study were el evated in two rats out of the four surviving rats in the 200 mg/kgxday-1 group. One Way an alysis of variance resulted in a significant difference between this group a nd the control (P<0.05) (Figure 3-24). In the final study no elevated ALT values were identified. The SV80RS group however show ed significantly lower ALT values than the water control group, th e SV80DS group or the SV80 group. (P<0.01) group (Figure 3-25). AST values measured in the pilot study were el evated in two rats, the same rats that had elevated ALT values, out of the four surviv ing rats in the 200 mg/kgxday-1 group. One Way analysis of variance resulted in a significant difference betw een this group and the control (P<0.05) (Figure3-26).

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70 After the final study, AST values of all SV 80 and AV20 groups were significantly lower than the water control (P<0.001) however SV20DS showed highe r AST values than SV20 or SV20RS.(Figure 3-27). CPK values for the pilot study could not be evaluated. The plasma samples for this study were taken after decapitation and additional studies have shown that decapitation dramatically increases CPK values (Figure 3-28). Consequen tly the CPK results for the final study also resulted in higher values for the Control, RS and DS group since these values were also taken after decapitation. Peak values for the other grou ps were taken before sacrificing and are there comparable. CPK values in the SV20DS group were significantly higher than in the SV20 group (P<0.001) or the SV20RS group (P<0.001). No di fferences were observed within the SV80 group (Figure3-29). Organ weights After removal of the organs during the pilo t study, no significant differences could be found in the absolute organ weight of the liver, adrenal gland and testes the absolute spleen, kidney and heart weight was si gnificantly lower in the 200 mg/kgxday-1 group compared to control. The organ index was higher in the 200 mg/kgxday-1. All other or gan indices were not significantly different. During the final study, no difference could be observed in absolute spleen, kidney, testis, and adrenal gland weight. Absolute liver weight was significantly lower in all SV20 and all SV80 groups. Furthermore absolute heart weight was decreased in the SV20Rs group compared to the water cont rol (Figure3-30). Furt hermore no differences were observed in organ indices of heart, testis spleen, liver, and adrenal gla nd weight. Organ indices were increased in SV20RS and SV20Ds compared to water and in SV80, SV80RS and SV80Ds compared to water (Figure 3-31)

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71 Conclusions The results of our study to evaluate the risk of muscle damage after chronic coadministration of grapefruit juice with simvastatin demonstrate that no increased muscle damage can be observed when simvastatin is take n with grapefruit juice than taken alone. We were not able to observe increase d organ toxicity or a decreased survival rate. This study also indicates that grapefruit juice can lower cholesterol in laboratory animals; however this result will need to be confirmed humans.

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72 Figure 3-1. Schematics of first pass metabolism Figure 3-2. Open field arena a nd TopScan software interface.

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73 Figure 3-3. PANLAB grip strength meter Figure 3-4. Sampling schedule during the final study

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74 DHB BG NAR 0 25 50 500 1000 1500 2000AD3 2A6 98 SubstanceConcentration [ M] Figure 3-5. Concentration of 6,7-dihydroxybe rgamottin (DHB), bergamottin (BG) and Naringin (NAR) in three different juices (mean SD) n=3 Figure 3-6. Survival rate during both studies A=pilot study, B=final study

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75 -6 -4 -2 0 2 4Control SV53 SV200 *Slope Figure 3-7. Slope of the linear re gression performed of the body wei ght of rats in the pilot study from day 0 to day 10 (mean SEM), Control n=7, SV53 n=8, SV200 n=4. (*=P<0.05) 0 GF J RS GF J DS GFJ 0 GF J RS GF J D S GFJ 0 GF J RS GF J D S GFJ 0.0 0.5 1.0 1.5 2.00 SV 20 SV 80 SV Slope Figure 3-8. Slope of the linear re gression performed of the body wei ght of rats in the final study from day 0 until day 28 shown as mean SEM (n=13).

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76 Co n trol SV5 3 SV200 0 50 100 150 200*Line crossings [#] Figure 3-9. Number of line cros sings of the open field test in the pilot study (mean SEM), Control n=7, SV53 n=8, SV200 n=4. (*=P<0.05) 0 GFJ RS G FJ D S GFJ 0 G FJ R S G FJ DS GFJ 0 G FJ R S G FJ DS GFJ 0 50 100 150 2000 SV 20 SV 80 SV ** **Line crossings [#] Figure 3-10. Number of line crossi ngs of the open field test in the final study shown as mean SEM (n=13). (**=P<0.01)

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77 Co n trol S V 53 S V 200 0 2000 4000 6000*Distance [mm] Figure 3-11. Distance traveled in the open field test of the pi lot study (mean SEM), control n=7, SV53 n=8, SV200 n=4. (*=P<0.05). 0 GFJ R S GFJ D S G FJ 0 G F J RS GFJ DS GFJ 0 GFJ R S G FJ D S G F J 0 2000 4000 60000 SV 20 SV 80 SV ** **Distance [mm] Figure 3-12. Distance traveled in the open field test of the fi nal study shown as mean SEM (**=P<0.01).

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78 0 5 10 15 0 200 400 600 800 1000 Control SV53 SV200 Time [days]Grip strength [g] Figure 3-13. Grip strength development during th e pilot study mean SEM, control n=7, SV53 n=8, SV200 n=4. Control S V5 3 SV2 0 0 0 2000 4000 6000 8000AUC (grip strength) [g*h] Figure 3-14. Comparison of the grip streng th AUC mean SEM control n=7, SV53 n=8, SV200 n=4

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79 0 10 20 30 1200 1400 1600 1800 2000 2200 2400 Control RS DS SV20 SV20RS SV20DS SV80 SV80RS SV80DS Time [days]Grip strength [g] Figure 3-15. Grip strength development during the final study n=13, data shown as mean SEM. 0 GFJ RS GFJ DS G F J 0 GFJ RS GFJ DS G F J 0 GFJ RS GFJ DS G F J 0 20000 40000 600000 SV 20 SV 80 SV AUC (grip strength) [g*h] Figure 3-16. Comparison of the gr ip strength AUC during the fi nal study, n=13 data shown as mean SEM.

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80 Control SV 5 3 SV 2 00 0 10 20 30Temperature [oC]** Figure 3-17. Comparison of the extremity temper ature in the pilot study, mean SEM control n=7, SV53 n=8, SV200 n=4 (** P<0.01). 0 GFJ R S G F J DS GFJ 0 GFJ R S G F J DS GFJ 0 G F J RS G F J DS G F J 0 10 20 30 400 SV 20 SV 80 SV Temperature [oC] ** Figure 3-18. Comparison of the extremity temp erature in the final study mean SEM n=13

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81 A B C Figure 3-19. Muscle damage (arrows) observed during the pilotA) control group B) SV53 C) SV200

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82 A B C D E Figure 3-20. Histological changes in the rat muscle during the fi nal study A) Water B) SV20 C) SV80 D) RS juice E) DS juice

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83 A B C D Figure 3-21 Histological changes in the rat muscle during the fina l study A) SV20RS B) SV20DS C) SV80RS D) SV80DS

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84 Control SV5 3 SV2 0 0 0 50 100 150Cholesterol [mg/dl] Figure 3-22. Cholesterol levels after 12 day dosing dur ing the pilot study mean SEM Control n=7, SV53 n=8, SV200 n=4. 0 GFJ RS GFJ D S G F J 0 GFJ R S G F J D S G F J 0 G F J R S G F J D S G F J 0 50 100 1500 SV 20 SV 80 SV Cholesterol [mg/dl] 0 G F J R S G F J D S G F J 0 G F J R S G F J D S G F J 0 G F J R S G F J D S G F J 0 50 100 1500 SV 20 SV 80 SV Cholesterol [mg/dl] 0 G F J R S G F J D S G F J 0 G F J R S G F J D S G F J 0 GF J RS G F J D S G F J 0 50 100 1500 SV 20 SV 80 SV Cholesterol [mg/dl] ** ** *** *** Figure 3-23. Cholesterol levels after 28 day dosing during the final study mean SEM, n=13 (*=P<0.05).

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85 0 GFJ RS GFJ DS GFJ 0 GFJ R S GF J D S GFJ 0 GFJ RS GFJ DS GFJ 0 20 40 60 80 100 300 400 500 6000 SV 20 SV 80 SV ALT [U/L] 0 GFJ R S GF J D S GFJ 0 GFJ RS GFJ DS GFJ 0 G F J R S GFJ DS GFJ 0 20 40 60 80 100 300 400 500 6000 SV 20 SV 80 SV ALT [U/L] ** ** Control SV53 SV2 0 0 0 200 400 600*ALT [U/L] Figure 3-24. Alanine amino transferase (ALT) levels after 12 days during the pilot study, mean SEM Control n=7, SV53 n=8, SV200 n=4 (*=P<0.05). Figure 3-25. Alanine amino transferase (ALT)le vels after 28 days during the final study, mean SEM n=13 (**=P<0.01).

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86 ** 0 GF J RS G FJ D S GFJ 0 G FJ RS GFJ DS GF J 0 GFJ RS GFJ DS GF J 0 100 200 300 3500 3600 3700 3800 3900 40000 SV 20 SV 80 SV AST [U/L] 0 GFJ RS GFJ DS GF J 0 GFJ R S GFJ DS GF J 0 GFJ R S GFJ DS G FJ 0 100 200 300 3500 3600 3700 3800 3900 40000 SV 20 SV 80 SV AST [U/L] *** *** *** ** *** *** *** Cont rol SV53 S V200 0 1000 2000 3000 4000*AST [U/L] Figure 3-26. Aspartate amino transferase (AST) le vels after 12 days dur ing the pilot study mean SEM Control n=7, SV53 n=8, SV200 n=4 (*=P<0.05). Figure 3-27. Aspartate amino transferase (AST) le vels after 28 days during the final study, mean SEM n=13 (**=P<0.01).

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87 before after 0 200 400 600 800 1000 10000 15000 20000 25000 30000 CPK [U/L] Figure 3-28. Creatine kinase (CPK) leve ls before and after decapitation n=9. 0 G FJ R S G FJ D S GFJ 0 G FJ R S GFJ DS GFJ 0 200 400 600 80020 SV 80 SV *** ***CPK [U/L] Figure 3-29. Creatine kinase (C PK) levels after 28 days during the final study, mean SEM, n=13 (**=P<0.01).

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88 0 GFJ RS GFJ DS GFJ 0 GFJ RS GFJ DS GFJ 0 GFJ RS GFJ DS GFJ 0 5 10 150 SV 20 SV 80 SV **Absolute Liver Weight [g]A 0 GFJ R S GFJ D S GFJ 0 GFJ R S GFJ D S GFJ 0 G FJ R S GFJ D S GFJ 0 5 10 150 SV 20 SV 80 SV *** *** *Absolute Liver Weight [g]B 0 GFJ RS GFJ DS GF J 0 GFJ RS GF J D S G FJ 0 GFJ RS GF J D S G FJ 0 200 400 600 800 10000 SV 20 SV 80 SV Absolute Spleen Weight [mg]C 0 G FJ RS GFJ DS GFJ 0 GFJ R S GFJ DS GFJ 0 GFJ RS GFJ DS GFJ 0.0 0.5 1.0 1.50 SV 20 SV 80 SV *Absolute Heart Weight [g]D 0 GF J RS GFJ DS GFJ 0 GFJ RS GFJ DS GFJ 0 GF J RS G F J DS GFJ 0 1 2 3 40 SV 20 SV 80 SV Absolute Testis Weight [g]E 0 GFJ RS GF J DS GF J 0 GF J RS GFJ DS GFJ 0 GF J RS GFJ DS GFJ 0 20 40 600 SV 20 SV 80 SV Absolute Adrenal Gland Weight [mg]F 0 GFJ RS GFJ DS GFJ 0 GFJ RS GFJ DS GF J 0 G FJ R S GFJ DS GF J 0.0 0.5 1.0 1.5 2.0 2.50 SV 20 SV 80 SV Absolute Kidney Weight [g]G Figure 3-30. Absolute organ weights after 28 da ys during the final study, mean SEM, n=13 A) liver B) liver c) Spleen D) heart E) testis F) adrenal glands G) kidney

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89 0 GFJ R S GFJ D S GFJ 0 GFJ R S G FJ D S G FJ 0 GFJ RS GFJ DS GFJ 0.00 0.01 0.02 0.03 0.040 SV 20 SV 80 SV ARelative Liver Weight [g/g] 0 GFJ R S G FJ D S G FJ 0 G FJ RS GFJ DS GFJ 0 GFJ R S GFJ D S GFJ 0.00 1.00 2.00 3.000 SV 20 SV 80 SV Relative Spleen Weight [mg/g]B 0 GFJ RS GFJ DS GFJ 0 GFJ R S GFJ D S G FJ 0 G FJ RS GFJ DS GFJ 0.000 0.001 0.002 0.003 0.0040 SV 20 SV 80 SV Relative Heart Weigth [g/g]C 0 GFJ RS GFJ DS GFJ 0 GFJ R S GFJ D S GFJ 0 GFJ RS GFJ DS GFJ 0.000 0.005 0.010 0.0150 SV 20 SV 80 SV Relative Testis Weight [g/g]D 0 GFJ RS G FJ DS G FJ 0 G FJ RS G FJ DS G FJ 0 G FJ RS G FJ D S G FJ 0.00 0.05 0.10 0.15 0.200 SV 20 SV 80 SV Relative Adrenal Gland Weight [mg/g]E 0 G FJ RS G FJ DS GFJ 0 GFJ R S G FJ D S G FJ 0 G FJ RS G FJ DS G FJ 0.000 0.002 0.004 0.006 0.008Legend Legend Legend ** ***Relative Kidney weight [g/g]F 0 G FJ RS G FJ DS G FJ 0 G FJ RS GFJ D S G FJ 0 G FJ R S G FJ D S G FJ 0.000 0.002 0.004 0.006 0.008Legend Legend Legend ** ** ***Relative Kidney weight [g/g]G Figure 3-31. Organ indices after 28 days during the final study, m ean SEM, n=13 A) liver B) spleen C) heart D) testis E) adre nal glands F) kidney G) kidney

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90 Table 3-1. Preparation procedure for simvastatin suspensions with respective dosing volume. Study Dose Amount simvastatin Amount CMC Vehicle (30mL) Dosing volume SV 53 160mg 240mg Water 0.1mL/10g Pilot SV200 600mg 240mg Water 0.1mL/10g SV20 120mg 240mg Water 0.05mL/10g SV20RS 120mg 240mg RS 0.05mL/10g SV20DS 120mg 240mg DS 0.05mL/10g SV80 480mg 240mg Water 0.05mL/10g SV80RS 480mg 240mg RS 0.05mL/10g Final SV80DS 480mg 240mg DS 0.05mL/10g Table 3-2. Naringin (NAR) / nari ngenin (NAG) standard curve NAR(0.058g) Dissolve in 2mL DMSO (50,000 M) NAG(0.03g) Dissolve in 2mL DMSO (55,094 M) take) 1 mL take) 0.1 mL Concentration Add both and fill to 5mL with 50:50 methanol:water NAR(M) NAG(M) Take Fill to Solvent 450 49.6 0.45mL 10mL 50:50 methanol:water 350 38.6 0.350mL 10mL 50:50 methanol:water 250 27.5 0.250mL 10mL 50:50 methanol:water 150 16.5 0.150mL 10mL 50:50 methanol:water 50 5.5 0.050mL 10mL 50:50 methanol:water

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91 Table 3-3. Bergamottin and dihydroxybergamottin standard curve BG( 0.0169 g) Dissolve in 1mL DMSO (50,000 M) DHB( 0.0186g) Dissolve in 1mL DMSO (50,000 M) take)25microl take) 10microl Concentration Add both and fill to 5mL with 50:50 methanol:water (=BG 250microM DHB 100microM) BG(M) DHB(M) Take Fill to Solvent 125 50 1mL 2mL 50:50 methanol:water 62.5 25 0.5mL 2mL 50:50 methanol:water 25 10 0.2mL 2mL 50:50 methanol:water 6.25 2.5 0.05mL 2mL 50:50 methanol:water 2.5 1 0.01mL 2mL 50:50 methanol:water

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92 Table 3-4. Dosing scheme of the final study Group Treatment N per group 1 Water control 13 2 Grapefruit juice (regular strength) control (RS) 13 3 Grapefruit juice (double strength) control (DS) 13 4 Simvastatin (low dose) control (SV20) 13 5 Simvastatin (high dose) control (SV80) 13 6 SVlow dose + GFJRS (SV20RS) 13 7 SVlow dose + GFJDS (SV20DS) 13 8 SVhigh dose + GFJRS (SV80RS) 13 9 SVhigh dose + GFJDS (SV80DS) 13 SVlowdose = 20 mg/kg Simvastatin, SVhighdose = 80 mg/kg Simvastatin, GFJRS = Grapefruit juice concentrate dilute w ith the same volume of water, GFJDS = Grapefruit juice concentrate dilute with half the volume of water

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93 Table 3-5. Muscle damage incidence and severity. Study Dose (mg/kg x day-1) IncidenceSeverity Control 0/7 SV53 0/8 Pilot SV2001 1/4 ** Control 0/12 RS 1/13 DS 0/13 SV20 0/11 SV20RS 0/10 SV20DS 1/12 SV80 0/11 SV80RS 0/12 Final SV80DS 0/13 1 four animals died in this group due to toxicity.*=mild muscle damage, **=moderate muscle damage, ***=severe muscle damage

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94 CHAPTER 4 DETERMINATION OF ACUTE AND CHRONIC EFFECT OF GRAPEFRUIT JUICE ON SIMVASTATIN PLASMA CONC ENTRATIONS IN RATS Specific Aim Assess the magnitude to which GFJ changes the simvastatin (SV) plasma levels after long term use in the rat model and assess th e possibility of adaptive processes. Material and Methods Chemicals Simvastatin (SV), 99.4% pure, wa s a kind gift from Merck Sharp Dome (MSD, West Point PA., USA. Simvastatin hydroxy aci d (98% pure) for calibration a nd quality control standards was purchased from Toronto Research chemicals, North York, ON, Canada. D6-simvastatin and D6-simvastatin hydroxy acid as internal standard s were also purchased from Toronto Research Chemicals. Isotopic purity for both compounds was >99%. Methanol and Acetonitrile (Optima Grade) and Formic and acetic acid were purchas ed from Fisher Scientific. Methylamine was purchased from Sigma-Aldrich (Saint Louis, MO ). Solid Phase Extraction columns (Versaplate C8 100mg) were purchased from Varian Inc. Heparinized, mixed gender pooled rat plasma from Sprague DAwley rats was purchased from Biocemed (Winchester, VA). Stock and Work Solutions Stock solutions for Simvasta tin acid were prepared by dissolving 1000g simvastatin hydroxy acid in 1 mL ACN/H2O 60:40. Stock solutions for simvastatin lactone were prepared by dissolving 1000g simvastatin lactone in 1 mL ACN. 100g and 0.5 g stock solutions were prepared as follows: Simvastatin acid: 100 g/mL: 0.2 mL (1000 g/mL SVacid Stock)/2mL 60:40 ACN:H2O 0.5 g/mL: 0.010 mL (100 g/mL SVacid Stock)/2mL 60:40 ACN:H2O

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95 Simvastatin lactone: 100 g/mL: 0.2 mL (1000 g/mL SV-acid Stock)/2mL ACN 0.5 g/mL: 0.010 mL (100 g/mL SV-acid Stock)/2mL ACN Calibration Quality Control Standards Calibration and quality control standards were prepared according to the spiking scheme in table 4-1. Analytical System and Ch romatographic Conditions The LC/MS/MS system consisted of a Ther mo Finnigan Surveyor HPLC autosampler, Thermo Finnigan Surveyor MS quaternary pump and a Thermo Finnigan TSQ Quantum Discovery triple quadrupole mass spectrometer. The TSQ Quantum mass spectrometer was equipped with an electrospray (E SI) ion source and operated in th e negative mode from 0 to 2.9 minutes and in positive mode from 2.9 to 4.5 minutes. The ESI source spray was set orthogonal to the ion transfer capillary tube. The mass sp ectrometer was calibrated with a solution of polytyrosine-1,3,6 according to th e manufacturer. The MS/MS c onditions were optimized by infusing simvastatin and simvastatin hydroxy acid in the mobile phase. For quantification, the TSQ Quantum was ope rated in single reaction monitoring mode (SRM). The acquisition parameters were: spray voltage 3kV for simvastatin hydroxy acid and 5 kV for simvastatin lactone, source CID 5 V, and heated capillary temperature at 325oC. Nitrogen was used as the sheath and auxiliary gas and set to 45 and 15 (arbitrary units), respectively. The argon collision gas pressure was set to 1.5 mTorr. The collision energy was 23 eV for Simvastatin hydroxy acid and simvastatin hydroxy aci d-D6 (internal standard) and. 19 eV for Simvastatin lactone and simvastatin lactone-D6 (inter nal standard).

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96 The selected reaction monitoring scheme follo wed transitions of the [M-H]precursor to selected product ions with the following values: m/z 435.3 319.1 for simvastatin acid and 441.3 319.1 for simvastatin acid-D6 and [M+CH3NH3]+ precursor to selected product ions m/z 450.3 285.1 for simvastatin lactone and 456.3 285.1 for simvastatin lactone-D6. The instrument was operated in normal resoluti on with peak width (F WHM) set to 0.7 Th at Q1 and to 0.7 Th at Q3. The scan time was 300 ms for each transition. SRM data were acquired and processed using ThermoFinnigan XCa libur software version 1.4, service release 1 (Thermo Electron Corporation, San Jose, CA, USA). Separation of the analytes was achieved usi ng a Phenomenex (Phenomenex, Torrance, CA) Luna PhenylHexyl, 100 2.0 mm, 3 analytical column. The mobile phases used for the analysis were ACN (mobile phase A) and 1m M methylammonium acetat e in deionized water (mobile phase B). The mobile phases were degassed and filtered through a 0.22 Nylon 66 membrane before use. The analyses were performed under isocratic conditions using 70% mobile phase A and 30% mobile phase B. The flow rate was 0.2 mL/min. Experiment Design Non-fasted male Sprague-Dawley rats wei ghing 239-346 g were used. The animals were handled daily a couple of days before the study star ted in order to reduce stress and adapt them to the researcher. Body weight was checked daily as well. The male Sprague-Dawley rats had free access to food and water during the experiment except for the first two hours after drug administration. The animals were di vided in the groups shown in table 3-3. On the first day of the experiment the rats were dosed w ith their respective simvastatin dose (SVlowdose = 20 mg/kgxday-1 (SV20) Simvastatin, SVhighdose = 80 mg/kgxday-1 (SV80) Simvastatin) and their grapefruit juice permutations (s imvastatin and regular strength grapefruit juice; SV20RS and SV80RS, and simvastatin and double strength grapefruit jucie; SV 20DS and SV80DS). To

PAGE 97

97 establish a pharmacokinetic baseline profile, blood samples (500 l) were taken from the sublingual vein at 0, 1, 2, 4, 6, 8, 12 hours after drug administration (three blood collections per day; e.g. time points 1, 4, 8; then two week s washout, then the remaining time points 0, 2, 6, 12 in the same animals). After the acute phase two blood samples 1000 l was taken each week for 4 weeks right before dosing the drug (trough level) and 2 hours af ter dosing (peak level) figure 3-4. After each blood sample, approximately 1000 l of isotonic saline were replaced by i.p. injection in order to maintain the blood fluid. The blood samples were analyzed for Simvastatin, Simvastatin acid. The animals were fed grapefruit juice (GFJ) (5 mL/kg) at different c oncentrations (double and regular strength), or water (5mL/kg) and S imvastatin through an oral feeding needle daily. At the end of the study the rats were sacrif iced, blood was collected for determination of simvastatin plasma levels. Sample Preparation and Extraction Samples were collected into heparinized tube s and stored on ice unt il further processing. Samples were centrifuged for 15 minutes at r oom temperature at 2800 rpm. The plasma was collected, aliquoted and stored at -80oC until analysis. The plasma was thawed at room temperature and 100 microL were transferred in to a 2 mL deep well plate. 400 microL of DDwater and 10 L of internal standard were added to each well. The plate was sealed and vortexed for 1 min. The C8 columns were preconditioned with 2x 1mL Optima grade methanol and equilibrated 2x1mL DDwater. The previously prepared samples were then loaded onto the columns and low vacuum <7in Hg was applied. The SPE cartridges were then washed with 1x1mL DDwater, 1x1mL 3.5% formic acid and 1x1 mL DDwater. Low vacuum (<7in Hg) was applied for each step. After the last washing st ep, high vacuum (20 in Hg) was applied for 3 min to dry the cartridges. The elution was then pe rformed using 1x1mL of optima grade methanol

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98 and 1x1mL of 2% formic acid in methanol. The co lumns were dried for 1 min. after each elution step. The samples were then dried in a centrivap overnight at 37oC and reconstituted in 200microL 70:30 Acetonitrile:Methylammoniumacet ate buffer (1 mM pH4.5). 20 L of the reconstitute was injected onto the HPLC column. Pharmacokinetic Analysis Pharmacokinetic analysis was performe d using WinNonlin 5.2 Professional Build 200701231637 (Pharsight Corp., Mountain View, CA). The Noncompartmental analysis algorithm was used and the parameters AUClast, AUCinf, T1/2, Tmax, Cmax, Vz/F and CL/F were evaluated. Statistical Analysis Final study: Samples were an alyzed using SAS version 9 (Cary, NC, USA). All samples were analyzed using a Two Way ANOVA with a B onferroni multiple comparison post hoc test to compare treatments to each other. Differences were considered statistically significant at P<0.05. As set a priori, the following comparisons were evaluated, SV20 compared with SV20Rs and SV20DS. We also compar ed SV80 with SV80RS and SV80DS. Results The interand intraday variability during va lidation was less than 20%. The calibration range was linear from 0.5ng/mL to 750 ng/mL. Grapefruit juice did not have a significant effect on the pharmacokinetic parameter of either Simvastatin lactone or its hydroxy acid form when administered as 20 mg/kg over 28 days. There were no significant differenc es in either regular strength or double strength grapefruit juice group. RS and DS GFJ significantly increased th e exposure of a 80 mg/kg simvastatin lactone during the first administration by 1.3 fold. The main active metabolite exposure increase by 1.3

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99 fold. During the chronic treatment with RS GFJ the Cmax of SV on day 7 and day 28 increased by around 1.8 fold and 1.7 fold for the hydroxy acid. Cmax of SV increased maximally by 2fold on day 28 and 2.1 fold on day 14 for SVA. Acute Treatment Simvastatin lactone SV20: The Area Under the Curve from 0 to 12 hours (AUC0-12) of Simvastatin lactone in the 20 mg/kg group increased from 127.710.1 (h*ng/mL) when give with water to 228.3.1 (h*ng/mL) and 187.2.9 (h*ng/mL) when given with RS GFJ and DS GFJ respectively. These changes however were not statistically significant. The AUC from 0 to infinity (AUCinf) increased from 137.1.9 (h*ng/mL) when dosed with water to 241.8.4 (h*ng/mL) and 365.0.4 (h*ng/mL) when given with RS GFJ and DS GFJ respectively. Similar to AUC0-12 these changes were not statisti cally significant. (Figure 4-1) Cmax, Tmax and half life remained unchanged. The vol ume of distribution (Vz/F) decreased in the SV20DS group compared to the wate r control from 690.7.8 (L/kg) to 360.3.4 (L/kg) (P<0.05). The clearance of simvasta tin decreased from 156.7.4 (L/h/kg) when the drug was dosed with water to 95.3.4 (L/h/kg) and 99.6.3 (L/h/kg) when dosed with RS and DS GFJ respectively (Table 4-2). SV80: The Area Under the Curve from 0 to 12 hours (AUC0-12) of simvastatin lactone in the 80 mg/kg group increased significantly from 651.4.6 (h*ng/mL) when administrated with water to 869.6.5 (h*ng/mL) and 889.5.6 (h*ng/mL) wh en dosed with RS (P<0.001) and DS (P<0.001) GFJ respectively. The AUC from 0 to infinity (AUCinf) increased from 807.5.3

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100 (h*ng/mL) in the control group to 9 63.0.0 (h*ng/mL) and 1104.5.4 (h*ng/mL) when dosed with RS (P<0.001) and DS (P<0.001) GFJ respectively. The Cmax in the SV80RS group increased from 226.2.0 (ng/mL) to 312.2.6 (ng/mL) when comparing to the control group (P< 0.05). The increase in the SV80DS group was not statistically significan t. In addition the Tmax increased from 0.8.1 (h ) to 1.2.1 (h) comparing the control group to the RS (P<0.001) GFJ group. The increase to 1.0.0 (h) in the DS GFJ group was not statistically significant. The half life, volume of distribut ion and Clearance (Vz/F and CL/F) remained unchanged (Table4-3) Simvastatin hydroxy acid SV20: The Area Under the Curve from 0 to 12 hours (AUC0-12) of simvastatin lactone in the 20 mg/kg group increased from 92.7.9 (h*ng/mL) wh en coadministered with water to 117.4.2 (h*ng/mL) and 135.5.2 (h*ng/mL) when administer ed with RS and DS GFJ. However these changes were not statistically significan t. The AUC from 0 to infinity (AUCinf) increased from 99.8.8 (h*ng/mL) in the control group to 128.5.4 (h*ng/mL) and 142.6.5 (h*ng/mL) when dosed with RS (P<0.001) and DS (P<0.001) GFJ respectively. Similar to AUC0-12 these changes were not statistically significant. (Figure 4-3) Cmax, Tmax, T1/2 remained unchanged. The Vz/F decreased from 871.3.9 (L/kg) in the water control group to 425.1.4 (L/kg) when administered concomitantly with DS GFJ (<0.05). The Cl/F decreased from 212.1.8 (L/ kg) in the water c ontrol group to 148.6.8 (L/kg) when administered concomitantly with DS GFJ (<0.05) (Table 4-4). SV80: The Area Under the Curve from 0 to 12 hours (AUC0-12) of simvastatin lactone in the 80 mg/kg group increased from 546.937.5 (h*ng/mL) when coadministered with water to

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101 659.3.9 (h*ng/mL) and 716.3.5 (h*ng/mL) when administered with RS and DS GFJ. The increase with DS GFJ was statistically signi ficant (P<0.001), however the increase in plasma concentration when the drug was administered with RS GFJ did not reach significance. The AUC from 0 to infinity (AUCinf) increased from 648.6.7 (h*ng/mL) in the control group to 735.8.7 (h*ng/mL) and 857.1.1 (h*ng/mL) when dosed with RS (P<0.001) and DS (P<0.001) GFJ respectively. Of these changes only the increase with coadmi nistration of DS GFJ showed to be statistically si gnificant (P<0.01) (Figure 4-4) Cmax, Tmax, T1/2, Vz/F and Cl/F rema ined unchanged. (Table 4-5) Chronic Treatment Simvastatin lactone SV20: The Cmin as well as the Cmax during the 28 days of study remained unchanged when comparing the two juice groups with the control group (Figure 4-5) SV80: There was no difference in the trough concentr ation of simvastatin lactone in the RS GFJ group. The peak concentrations in the RS GFJ group were increased only on day 7 and 28 (168.5.5 ng/mL compared to 96.8.8 ng/ mL and 111.4.3 ng/mL compared to 64.0.5 ng/mL respectively). We did find a significan t increase in the trough concentration of Simvastatin lactone in the DS GFJ group on day 7 (137.8.2 ng/mL compared to 13.8.3 ng/mL). Furthermore the peak concentrati on was increase on day 14 (186.0.0 ng/mL to 109.4.9 ng/mL), and 28 (130.4.3 ng/mL to 64.0.5 ng/mL), but did not reach statistical significance on day 7 and day 21 (Figure 4-6, Table 4-6).

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102 Simvastatin hydroxy acid SV20: The Cmin as well as the Cmax during the 28 days of study remained unchanged when comparing the two juice groups with the control group (Figure 4-7) SV80: There was no difference in the trough concentr ation of simvastatin lactone in the RS GFJ group. The peak concentrations in the RS GFJ group were increased only on day 7 and 28 (111.0.5 ng/mL compared to 68.6.8 ng/ mL and 98.5.8 ng/mL compared to 57.4.8 ng/mL respectively). We found a significant increas e in the trough concentr ation of Simvastatin lactone in the DS GFJ group on day 7 (41.0 10.0 ng/mL compared to 9.1.0 ng/mL). Furthermore the peak concentration was increase on day 14 (140.9.6 ng/mL to 65.9.2 ng/mL), 21 (151.1.8 ng/mL to 90.9.1 ng/mL ) and 28 (96.6.6 ng/mL to 57.4.8 ng/mL ), but did not reach statistical signif icance on day 7 (Figure 4-8, Table4-7). Conclusions As hypothesized, Grapefruit juice in either c oncentration (DS or RS) administered daily did not seem to have a relevant effect on the pharmacokinetics on a low acute or chronic dose of simvastatin. When administered with a high dose of simvastatin however, concentrations were elevated up to the last day of the study. Concentr ations of the main active metabolite were also elevated up to the end of the study. It seems that the interaction persist ove r the full range of the study.

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103 0 5 10 15 0 50 100 150 SV20 SV20RS SV20DS Time [h]Concentration [ng/ml] Figure 4-1. Plasma concentrations of simvastatin lactone in rat plasma after a 20 mg/kg oral dose. Data is shown as meanSEM (n=13) 0 5 10 15 0 100 200 300 400 SV80 SV80RS SV80DS Time [h]Concentration [ng/ml] Figure 4-2. Plasma concentrations of simvastatin lactone in rat plasma after a 80 mg/kg oral dose. Data is shown as meanSEM (n=13)

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104 0 5 10 15 0 20 40 60 SVA20 SVA20RS SVA20DS Time [h]Concentration [ng/ml] Figure 4-3. Plasma concentrations of simvasta tin hydroxy acid in rat plasma after a 20 mg/kg oral dose. Data is shown as meanSEM (n=13) 0 5 10 15 0 100 200 300 SVA80 SVA80RS SVA80DA Time [h]Concentration [ng/ml] Figure 4-4. Plasma concentrations of simvasta tin hydroxy acid in rat plasma after a 80 mg/kg oral dose. Data is shown as meanSEM (n=13)

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105 0 10 20 30 0 10 20 30 40 SV20 SV20RS SV20DS Time [days]Concentration [ng/ml] Figure 4-5. Plasma concentrations of simvastatin lactone in rat plasma after a 20 mg/kg oral dose from day 7 to day 28. Data is shown as meanSEM (n=13) 0 10 20 30 0 50 100 150 200 250 SV80 SV80RS SV80DS Time [days]Concentration [ng/ml] Figure 4-6. Plasma concentrations of simvastatin lactone in rat plasma after a 80 mg/kg oral dose from day 7 to day 28. Data is shown as meanSEM (n=13)

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106 0 10 20 30 0 5 10 15 20 25 SVA20 SVA20RS SVA20DS Time [days]Concentration [ng/ml] Figure 4-7. Plasma concentrations of simvasta tin hydroxy acid in rat plasma after a 20 mg/kg oral dose from day 7 to day 28. Data is shown as mean SEM (n=13) 0 10 20 30 0 50 100 150 200 SVA80 SVA80RS SVA80DDS Time [days]Concentration [ng/ml] Figure 4-8. Plasma concentrations of simvasta tin hydroxy acid in rat plasma after a 80 mg/kg oral dose from day 7 to day 28. Data is shown as mean SEM (n=13)

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107 Table 4-1 Spiking scheme for calibra tion and quality control standards Standard Concentration (ng/mL) Total Plasma Amount (mL) Stock Solution (g/mL) ACID Stock Solution (g/mL) LACTONE Spiking Amount (L) 0 10 0 0 0 0.5 10 0.5 0.5 10 1 10 0.5 0.5 20 10 10 0.5 0.5 200 100 10 100 100 10 250 10 100 100 25 500 10 100 100 50 750 10 100 100 75 2.0 LC 10 0.5 0.5 40 150.0 MC 10 100 100 15 400.0 HC 10 100 100 40 2.0 LC 5 0.5 0.5 20 150.0 MC 5 100 100 7.5 400.0 HC 5 100 100 20

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108 Table 4-2. Pharmacokinetic parameters of simvastatin lactone after a 20 mg/kg oral dose in rats. Data is shown as mean SEM (n=13) (*=p<0.05, **=P<0.01, ***=P<0.001) AUClast (h*ng/mL) AUCinf (h*ng/mL) Cmax (ng/mL) Tmax (h) T1/2 (h) Vz/F (L/kg) Cl/F (L/h/kg) 0 GFJ 127.7.1 137.1.9 61.0.8 0.9.1 2.9.2 690.7.8 156.8.5 RS GFJ 228.3.1 241.8.4 90.8.2 1.1.2 2.7.4 429.6.4 95.4.4*** DS GFJ 187.2.9 365.0. 4 77.1.4 1.6.5 5.3.1 360.3.9* 99.6.4** Table 4-3. Pharmacokinetic parameters of simvastatin lactone after a 80 mg/kg oral dose in rats. Data is shown as mean SEM (n= 13) (*=p<0.05, **=P<0.01, ***=P<0.001) AUClast (h*ng/mL) AUCinf (h*ng/mL) Cmax (ng/mL) Tmax (h) T1/2 (h) Vz/F (L/kg) Cl/F (L/h/kg) 0 GFJ 651.4.6 807.5.3 226.2.0 0.8.1 3.6.6 504.7.4 111.4.6 RS GFJ 869.6.5***963.0.0 312.2.6* 1.2.1** 3.4.3 417.2.5 85.0.9 DS GFJ 889.5.6***1104.5.4 291.7.0 1.0.0 3.8.5 405.2.6 79.1.4

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109 Table 4-4. Pharmacokinetic parameters of simvastatin hydroxy acid after a 20 mg/kg oral dose in ra ts. Data is shown as meanSEM (n=13) (*=p<0.05) AUClast (h*ng/mL) AUCinf (h*ng/mL) Cmax (ng/mL) Tmax (h) T1/2 (h) Vz/F (L/kg) Cl/F (L/h/kg) 0 GFJ 92.7.9 99.8.8 37.9.6 1.1.3 2.8.2 871.3.9212.1.8 RS GFJ 117.4.2 128.5.4 41.9.7 1.2.4 3.2.6 812.0.8 166.4.7 DS GFJ 135.5.2 142.6.5 41.9.7 2.2.6 2.0.1 425.1.4*148.6.8* Table 4-5. Pharmacokinetic parameters of simvastatin hydroxy acid after a 80 mg/kg oral dose in ra ts. Data is shown as meanSEM (n=13) (**=P<0.01, ***=P<0.001) AUClast (h*ng/mL) AUCinf (h*ng/mL) Cmax (ng/mL) Tmax (h) T1/2 (h) Vz/F (L/kg) Cl/F (L/h/kg) 0 GFJ 546.9.5 648.6.7 199.2.8 0.9.3 3.3.4 594.6.8 135.8.4 RS GFJ 659.3.9 735.8.7 234.6.3 1.0.0 3.4.3 549.3.7 111.9.9 DS GFJ 716.3.5*** 857.1.1** 244.6.0 1.0.0 4.1.7 534.8.0 100.6.0

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110 Table 4-6. Trough(Cmin) and peak(Cmax) concentrations of simvastatin lactone after a 20 mg/kg and 80 mg /kg oral dose in rats. Data is shown as meanSEM (n=13) (*=p<0.05, **=P<0.01, ***=P<0.001) Cmin7 (ng/mL) Cmax7 (ng/mL) Cmin14 (ng/mL) Cmax14 (ng/mL) Cmin21 (ng/mL) Cmax21 (ng/mL) Cmin28 (ng/mL) Cmax28 (ng/mL) 20 mg/kg 0 GFJ 1.9.6 24.1.4 1.7.4 26.2.2 0.5.1 18.6.7 5.3.7 15.4.0 RS GFJ 5.0.0 18.0.2 3.0.3 26.5.4 1.5.2 21.0.7 0.2.1 17.7.9 DS GFJ 2.1.7 26.1.0 2.2.7 18.5.4 1.3.2 24.6.5 5.1.4 24.8.3 80 mg/kg 0 GFJ 13.8.3 96.8.8 16.8.3109.4.9 16.6.9 121.0.28.1.1 64.0.5 RS GFJ 49.8.9 168.5.5** 4.8.9 109.9.0 18.5.4 111.0.060.5.0111.4.3* DS GFJ 137.8.2*** 155.9.6 14.7.2186.0.0**9.9.6 189.7.04.1.0 130.4.3***

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111 Table 4-7. Trough(Cmin) and peak(Cmax) concentrations of simvastatin hydroxy acid after a 20 mg/kg a nd 80 mg/kg oral dose in rats. Data is shown as meanSEM (n=13) (*=p<0.05, **=P<0.01, ***=P<0.001) Cmin7 (ng/mL) Cmax7 (ng/mL) Cmin14 (ng/mL) Cmax14 (ng/mL) Cmin21 (ng/mL) Cmax21 (ng/mL) Cmin28 (ng/mL) Cmax28 (ng/mL) 20 mg/kg 0 GFJ 0.9.3 20.4.8 1.7.4 16.3.3 0.4.1 13.2.8 3.5.0 11.4.8 RS GFJ 2.6.6 13.9.2 3.0.3 15.8.8 1.0.2 17.8.0 0.1.1 15.1.6 DS GFJ 1.4.5 19.3.3 2.2.7 18.4.1 1.0.2 18.7.5 3.4.9 17.4.4 80 mg/kg 0 GFJ 9.1.0 68.6.8 16.8.365.9.2 12.6.890.9.1 6.3.8 57.4.8 RS GFJ 8.7.4 111.0.5**4.8.9 89.4.5 12.2.278.1.7 39.2.298.5.8* DS GFJ 41.0.0*** 103.2.0 14.7.2140.9.6***7.3.0 151.1.8*3.1.8 96.6.6*

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112 CHAPTER 5 DETERMINATION OF CHANGES IN S TEADY STATE CONCENTRATIONS OF ATORVASTATIN AFTER LONG TERM GRAPEFRUIT JUICE CONSUMPTION Background Current research demonstrates the increases in plasma concentration of certain statins when taken concomitantly with gr apefruit juice. Even though a number of clinical trials have been published about this interact ion, it remains unclear whether th is interaction is clinically relevant and results in a higher in cidence in muscle damage than a regular high dose of statins. In 2004 a case report about a 59 year old man from th e north of the country was published in the scientific literature and soon afterwards was picked up by the mass media [87]. This patient suffered from hypercholesterolaemia and his initia l dose of 10 mg/day ator vastatin was steadily increased to 60 mg/day, which is on the higher and of the therapeutic dosing range. Additionally to changes in his diet and exerci se, he also started to consume gr apefruit juice that he squeezed himself. This patient was diagnosed with rha bdomyolysis [87]. A similar case was reported with another patient that was diagnosed with rhabdom yolysis after having tolerated simvastatin well for 2 years and after tolerating the increase in dose from 40-80mg for 6 month. She stared consuming grapefruit juice 4 days before the ap pearance of the first symptoms [88]. These case reports demonstrate the necessity to evaluate the long term eff ects of grapefruit on the exposure of atorvastatin. Specific Aim Assess the magnitude by which GFJ changes the steady state atorvastatin plasma levels after long term concomitant use. The magnitude of the interaction was assessed in a time dependent manner over a period of 90 days.

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113 Material and Methods Chemicals Atorvastatin (compound # 0134298-0038A batch # 62) and D5-Atorvastain (compound # 0134298-0038A batch # 53) reference standard were provided as a kind gift by Pfizer, USA. Human Plasma was provided by Ci vitan Regional Blood System. Grapefruit juice was provided by the Florida Department of Citrus. The juice was 100% Florida juice as found on the domestic market meeting USDA grade A standards. Grapefruit juice was from one homogeneous lot, packaged in a shelf stable form. Stock and Work Solutions Atorvastatin: Stock solution 1 (1mg/mL) was prepared by dissolving 0.0250 g of Atorvastatin in methanol up to a volume of 25 mL in a graduated flask. Stock solution 2 (100 g/mL) was prepared by transfe rring a 0.1 mL aliquote of Stock solution 1 and adding a volume of 0.9 mL 50/50 wa ter/acetonitrile in a micr o centrifuge tube. Stock solution 3 (10 g/mL) was prepared by transferring a 0.1 mL aliquot of standard solution 2 and adding a volume of 0.9 mL plasma in a micro centrifuge tube. Stock solution 4 (1 g/mL) was prepared by transferring a 0.2 mL aliquot of standard solution 3 and adding a volume of 1.8 mL plasma in a micro centrifuge tube. Stock solution 5 (100 ng/mL) was prepared by transferring a 0.2 mL aliquot of standard solution 4 and adding a volume of 1.8 mL plas ma in a micro centrifuge tube. Next, these solutions were used to spike the calibration and quality control plasma samples. Where calibration curve samples were made in concentrations from 25-0.25 ng/mL and quality control samples were made in concentrations 18-0.5 ng/mL.

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114 D5-Atorvastatin: 0.010 g was dissolved in 10 mL methanol to obtain a stock solution of 1 mg/mL. Calibration Standards Atorvastatin calibration standard s were prepared in human plas ma from standard solutions 3, 4 and 5, according to the following pipetting scheme (Tbl. 5-1). Calibration Curve total volume: 40 mL a set of 40 was prepared. 1mL was transferred from each tube to obtain 40 complete calibration sets with a to tal volume of 1mL/standard sample. Atorvastatin Quality Control Standards Atorvastatin quality control standards were prepared in human plasma from standard solutions 2, 3 and 4, according to the following pi petting scheme. (Tbl. 5-2) Quality controls total volume: 1mL a set of 2 was prepared. 1mL was transferred from each tube to obtain 40 complete calibration sets with a to tal volume of 1mL/standard sample. Analytical System and Mobile Phase The HPLC system consists of a LDC CM 4000 pump, Perkin Elmer ISS-200 autoinjector, an adequate precolumn, a YMC Jsphere H 80 S-4 2.0 x 15 mm column, part #JH085041502WT followed by a Quattro LC-Z MS/MS system. The mobile phase consisted of acetonitrile : 0.1 % formic acid (70:30). The mobile phase was filtered and degassed prior to use. The flow rate was 0.2 mL/min, injection volume was 40L. Analytical Method The daughter ions of Atorvastatin (paren t ion 559 m/z) 440 m/z and D5-Atorvastatin (parent ion m/z 564) daughter i on m/z 445 were detected after electrospray ionization MS/MS analysis. The capillary voltage was set to 2.80 kVo lt, the cone voltage to 35 Volt, the extractor voltage and RF Lens voltage to 10 Volt and 0.20 Volt respectively. The source block was heated

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115 to 140 C and the desolvation temperature was 350 C. The setting for the collision energy was 25.0 eV. Experiment Design The clinical trial was performed at the Wats on Clinic in Lakeland, Fl under the supervision of Patrick Reddy. Samples were kindly provi ded for analysis to the Department of Pharmaceutics, University of Florida, Gainesville, FL. Patients were on atorvastatin for a 90 day run in phase. Patients with steady state levels of atorvastatin were eligible for this study. Pl asma was taken before commencement of the study. After the 0 time point was taken 10 ounces of grap efruit juice daily were added to the diet and monthly blood samples were taken. Blood samp ling time points were 30, 60 and 90 days after enrollment. Sample Preparation and Extraction Plasma samples were thawed at 37C. They were then vortexed and processed. 1.0 mL plasma, 50L internal standard and 0.5 mL 1.0 N NaOH were adde d in that order to a 16 x 125 mm screw cap culture flask and vortexed for 60 sec. 5 mL diethyl ether was added to each tube. The tubes were then capped and shaken horiz ontally for 15 min. The solutions were then clarified by centrifugatio n for 10 min at 2600 X g. The bottom aqueous layer was frozen in an isopropanol/dry ice bath for 10 min and the top organic layer was discarded. The aqueous layer was then thawed at 37C for 5 min. 2.0 mL 1.0 N H3PO4 was added to each sample tube and the tubes were vortexed for 60 s. After adding 10 mL of diethyl ether each sample vial was capped, shaken and centrifuged as before. The bottom aque ous layer was frozen and the ether layer was decanted into a clean tissue culture flask. The ether layer was evaporated in a water bath at 37C. The residue was reconstituted in 0.2 mL ammonium acetate (20 mM,

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116 pH4):acetonitrile:is opropanol (60:40:1), vortexed for 60 s and centrifuged at 2600 x g for 10 min. The supernatants were placed in 0.2 mL injection vials. Statistical Analysis Repeated measure one way ANOVA with Dunne tts multiple comparison post hoc test was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com. Differences we re considered statistically significant at P<0.05. Results The atorvastatin calibration wa s linear in the range form 0.25-25 ng/mL. The coefficient of determination of the calibration curve was 0.998228 (Figure 5-1). Interday variation was < 15%. Subjects that had no sample for the initial pre grapefruit juice time point were not considered in the analysis. After elimination of these subjects, a total of 121 datasets was analyzed. After the statistical analysis, no significant differences could be observed. Concentrations at timepoint 30 days were not significantly different fr om concentrations at timepoint 0. Concentrations after 60 days were not different from the beginning. Additionally, atorvastatin concentrations after 90 da ys were not different from their ini tial values (Figure 5-2). Conclusions This study could demonstrate that the chroni c concomitant administration of grapefruit juice in a realistic amount of 10 ounces per day does not lead to significantly higher plasma concentrations of the active form of atorvastatin. Simultaneous ch ronic administration to existing steady state concentrations did not lead to their augmentation either after 30, 60 or 90 days.

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117 Figure 5-1.Calibration curve for ator vastatin acid 0.25ng/ml to 25 ng/ml 1 3 0 6 0 9 0 0 10 20 30 40 50 Time [days]Concentration [ng/ml] Figure 5-2. Concentrations of at orvastatin from day 1 to day 90. Data shown as mean SEM (n=121)

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118 Table 5-1. Atorvastatin calibrati on standards for plasma samples Nr. Concentratio n (ng/mL) Volume to add ( L) Standard Solution concentration Plasma Volume (mL) Blank 0 ------40 1 25 0.1 10 (g/mL) 39.9 2 20 0.08 10 (g/mL) 39.92 3 10 0.04 10 (g/mL) 39.96 4 5 0.2 1 (g/mL) 39.8 5 2.5 0.1 1 (g/mL) 39.9 6 1.0 0.4 100 (ng/mL) 39.6 7 0.5 0.2 100 (ng/mL) 39.8 8 0.25 0.1 100 (ng/mL) 39.9 Table 5-2. Atorvastatin quality cont rol standards for plasma samples Nr Concentration Volume to add Standard Solution Plasma Volume (ng/mL) ( L) concentration ( L) I 18 0.144 10 ( g/mL) 79.856 II 5 0.4 1 ( g/mL) 79.6 III 0.5 0.4 100 (ng/mL) 79.6

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119 CHAPTER 6 CONCLUSIONS Discussion Grapefruit juice has been shown to interact with several orally administered drugs. This is especially concerning, since most patients who ha ve an existing condition might also want to benefit from the presumed beneficial properties of grapefruit juice. In most cases the expected clinical effect will be minor a nd of little clinical relevance. Conceptually, drugs which shows a high degree of interaction with gr apefruit juice can be s ubstituted by a drug not interacting, if the patient wants to continue his grapefruit juice consum ption. This should however be limited to therapeutic equivalence and consid ered only after a careful risk be nefit analysis. From the online database access statistics we can also deduct that there is a great interest in the general population and among health care professionals fo r comprehensive scientific information. The great number of almost 9 million hits shows the enor mous interest in scientific information. This number is however to be considered carefully. A hit is generated whenever a user sends a request to the website. This is also true for sites that dont exist and return an Error 404file not found error page. Files might be a better estimator, since this is a count for actual hits resulting in a file being sent back. Site is the number of IP addr esses sending the request. Ca re should be taken in the interpretation of this number ; many users can appear to be co ming from the same server IP. Visits occur when a remote site makes a request to the server. As long as the same site keeps making the request within a certain time period, it will be considered the same visit. A good estimator for actual pages being requested and not the single items that make up the page is the page access statistic. A relatively constant es timate of returning visitors since June 2006 strengthens our assumption of a great need for a comprehensive information source about grapefruit juice interactions. This estimate can be obtained by subtracting hits from files.

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120 We can further conclude that the degree of in teraction and the potential for adverse events of toxic effects depends widely on the type, bran d and batch of grapefruit juice used. Different concentrations of grapef ruit juice most likely lead to a different degree of effect [63, 65]. De Castro et al. reported in 2006 about the high variation of flavonoid and furanocoumarin content in different brands, batches, a nd dilutions of grapefru it juice [62]. Many of the publications about grapefruit juice interactions do not measure the amount of pote ntial interaction compounds in their juice. No study has been performed so fa r where the same juice was given in different dilutions to establish a crude dose relationship curve. Mixing of diffe rent varieties of fruits, from different origin and harvest times during the juice production make it almost impossible to achieve a reproducible effect for the patient who just purchases grapefruit juice. The Horticultural Science Department of the Universi ty of Florida mentions a total of 12 different varieties of grapefruit [37]. Fu rthermore the interaction seems to be limited to drug with a low bioavailability. In case of a drug with a 95% bioa vailability, inhibitions of intestinal metabolism will less likely result in significant changes of plasma concentration than a drug that has a bioavailability of 5%. In this ca se increase in 5% bioa vailability can easily result in a 2 fold increase in plasma concentration. Additionally pa tients taking medications at the higher end of the therapeutical range might be more prone to adverse events or toxicological manifestations. Researchers have developed methods such as heat treatment or UV treatment of grapefruit juice to eliminate drug intera ctions by removing interacting compounds. The removal of bergamottin and dihydroxybergamottin however does not seems to account for the total interaction and other component s might be involved. Heat treat ment had another detrimental effect: taste, flavor and nutrie nt content were compromised [68]. Furthermore the physiological safety of UV irradiated grapefruit juice has not been investigated [211]. Paine et al. used

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121 absorption resin to remove furamocoumarins. Th e consumption of the furanocoumarin free juice resulted in lower felodipi ne exposure that regular grapefruit juice [212]. Some important questions and puzzles re main for the health care professionals. Adjustment of drug doses is common and mostly linked to clinical effe ct. How should physicians respond, if a patient had this medication adjusted while he was taking grapefruit juice? It might be common practice to recommend the cessation of grapefruit jui ce consumption. The fact that his enzyme levels return to normal and that this might result in a lower drug exposure, possibly linked to less therapeutic eff ect has to be considered. Therefore we studied the influence of chronic grapefruit juice ingesti on on the probability of myopathy development. The main concern ab out this kind of inte raction study with food remains; the reproducibility. Our analysis if th e juice shows once more, as it has been shown before by De Castro et al.[62] that the interacting compounds in grapefruit jui ce vary greatly. Dihydroxybergamottin was clearly more than two fo ld higher in the double strength juice as in the regular strength juice. Naringi n concentrations were even equa l in one of the regular strength juices compared to the double st rength juice. In retrospect it seems better to make both dilution of juice out of the concentrate in order to have a real two fold differe nce in concentration of interacting compounds. We could not repeat the results of Westwood et al. [205] who could show muscle damage in rats at a dose of 80 mg/kgxda y-1. It is noteworthy however, that the previous researchers used female rats for their experiments. It has b een demonstrated early on in the simvastatin development that female animals have a higher exposure when administered simvastatin than male animals [19]. This could be the reas on why muscle damage in our study was only perceptible at a 200 mg/kgxday-1 dose of simvastatin. However the 80 mg/kgxday-1 group

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122 remained unaffected. The survival rate of 50% ma de it difficult obtain a more suitable picture of the muscle damage, since the myopathy development in the dead animals could not be assessed. It shows however the toxi city of this high dose of simvasta tin. A toxicity that could not be observed in the SV80RS or SV80DS groups. Assumi ng the increase in exposure that has been observed in humans [63-65] is comparable in rats, that these two groups would have been exposed to plasma concentration comparable to a 240 mg/kgxday-1 to almost 1.3 g/kgxday-1 dose, which would definitively l ead to manifestation of toxici ty [204, 205] that has not been observed in our study. The 200 mg/kgxday-1 incl uded two animals with high AST and ALT values at the end of the study, which could be a sign of muscle or li ver toxicity, though no changes in absolute or relative liver weight could be demonstr ated. Measured GGT values were in the negative range and were hence not consid ered. Due to the choice of scarification, the CPK values in the pilot study were not usable. Statis tic evaluation and a furthe r study of the influence of decapitation on the CPK levels deemed those unusable. Comparison of the remaining groups however showed increased CPK levels in two ra ts in the SV20DS group but none in the SV80DS group. We were not capable to lowe r the cholesterol levels in any of the two studies. Even a dose of 200 mg/kgxday-1 did not l ead to a reduction in chol esterol. Interestingly the cholesterol levels after grapefruit juice administration were significan tly lower than in the water control. The fact that it might not be possible to lower cholesterol in rats howev er has been shown before. As mentioned earlier, It seems that rats can upr egulate the HMG-CoA reducatase and cholesterol levels appear to be normal even when competitive inhibitors are present [207, 208]. The study design did not allow us to answer the question of why RS DS grapefruit juice might lower the cholesterol levels. Further resear ch need to be conducted to inve stigate the exact mechanism of this effect. We could however st ill show muscle damage without lowering the cholesterol levels

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123 as has been reported before [206]. The mech anism for this remains unclear, since other experiments suggest a direct relationship between HMG-CoA re ducatse inhibition and muscle damage [205]. Assessment of grip strength seems not to be the method of choice to detect and evaluate muscle damage. This test is usually performed to assess neuromuscular damage and shows great variability. Several factors such as operator and pu lling angle can further influence the outcome [213]. The most important parameter however is the histolog ical analysis of the muscle biopsies. Moderate muscle damage could be observed on the 200 mg/kgxday-1 group, but only two cases of mild muscle damage could be associated to the RS and SV20Ds group. It is interesting that no significant increase in muscle damage could be observed in the SV80 or SV80 groups combined with grapefruit juice. As an additional possible indicator for muscle damage a test for locomotor activity was performed. The rational was that muscle necr osis would decrease the locomotor activity. Mobility of the rats in the 200 mg/kgxday1 group was decreased, though it did not reach statistical significance due to th e small number of remaining in this group. It might however prove feasible to use this test as an indicator for myopathy in la boratory animals. Interestingly the 53 mg/kgxday-1 group during the pilot study had increase locomo tor activity compared to the water control. Similar observations were made during the final study. The SV80DS group showed a significant increase in locomotor activit y compared to the SV80 control. This can be explained with the penetration of simvastatin through the blood brain ba rrier [17, 19]. Once in the brain, simvastatin seems to exhibit an effect on the neurotransmitter dopamine and the regulation of dopamine receptors. It has been re ported that simvastatin can upregulate dopamine (D1 and D2) receptors [214] in the rat prefrontal cortex and can increase the dopamine content in

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124 the stratum by 110% [215]. It has further been s hown that changes in the dopaminergic system can lead to changes in behavior and locomotor activity [215]. The results of the muscle biopsies are even more exciting when looked upon in combination with the resulting plasma concentrat ions after coadministra tion with regular and double strength grapefruit juice. Regular strength GFJ did not a lter the exposure of the low dose of simvastatin and only minimally altered the expos ure of the high dose of simvastatin. This is even more interesting consideri ng that we chose a dosing scheme that would reflect a real life situation rather than an artifici al overexposure to large quantities of grapefru it juice. The animals were dosed with 5mL/kg juice, which would be e quivalent to a regular 8oz glass of RS GFJ or two 8oz glasses when we dosed with DS GFJ. Pr evious studies have showed that predosing TID with DS[63] or RS[65] GFJ can significantly alter the plasma concentrations in humans. Clear difference however can be seen in the extent to which these plasma concentrations are altered. The fact that large quantities of grapefruit juice can alter the pharmacokinetics of simvastatin has also been recognized by the regula tory authorities and is reflecte d in the labeling of the drug. We have shown that even the increase in the maxi mum concentrations over 28 days which reached a maximum of 2-fold in the 80 mg/kg group dosed with DS GFJ did not result in increased occurrence of muscle damage or decreased su rvival. Assuming dose proportionality plasma concentration would reach into the range of hi gh doses given in the pilot study which showed clear sign of myopathy and a decreased survival ra te. The lack of a significant increase in the 20 mg/kg group brings up the question whether the rat model is right to investigate GFJ drug interactions. Researching the presently published literature we encounter previous experiments were a clear interaction has been demonstrated Mangano et al. [216] showed a significant 31% increase in Cyclosporin-A exposure after coadmi nistration with GFJ in male Sprague-Dawley

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125 rats. The dose of GFJ however was 10mL/kg and hence double the dose we chose for the previously mentioned reasons. The possibility of inhibiting intestinal CYP 3A4 was also demonstrated by Grundy et al. who report an increase in nife dipine bioavailability after coadministration of 6mL/kg GFJ concentrate. RS GFJ however had no significant effect on bioavailability in their study [217]. Our results seem to fit in this overall picture. Realistic doses of GFJ seem to be able to increase the exposure of simvastatin in the rats but seem not to lead to greater muscle damage. There is however one caveat : these results should be seen in the context of an experiment performed in laboratory animal s and might not reflect interspecies differences in metabolism. Further clinical trials are needed to definitively assess th e possibility of myopathy after coadministration of simvastatin and GFJ. It was of great interest to further investig ate the effect of chronic administration of grapefruit juice on other statins in humans. Atorvastatin has a bioavailability of 12% and is administered as the active hydroxyacid form [4 ]. Even though it is metabolized by CYP 3A4 it seems not to be as strongly affected by the grap efruit juice drug interaction as simvastatin [63]. Increases in atorvastatin acid plas ma exposure (AUC) range from 1.4 fold [81] and 1.8 fold [82] with single strength gr apefruit juice to 2.5 fold when give n with double strengt h grapefruit juice [83]. All the study performed so far however are composed of e ither multiple administration of grapefruit juice and a single dose administrati on of the drug or multiple drug administration and single dosing of grapefruit juice. This is the first study were a chronic effect of simultaneous administration of drug and grapefruit juice has been investigated. Our results indicate that there is no significant difference betw een plasma concentration with or without grapefruit juice consumption. Since atorvastatin is administered in its active fo rm, it seems unlikely that this interaction is clinically relevant.

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126 The results from the in-vivo animal experiments and the clinical trial indicate the necessity to conduct a clinical interacti on trial to confirm the lack of myopathy development after increased plasma levels of simvastatin in humans. Conclusions The results of our investigations suggest that chronic simultane ous administration of grapefruit juice and simvastatin does not lead to an increased inci dence of myopathy in laboratory animals. The coadministration with grapefruit juice does not result in the same toxicity as a high dose of simv astatin would. We can further co nclude that consumption of grapefruit juice might be capable to reduce chol esterol levels in humans as we have shown in animal experiments. It remains to be investigated whether this is due to the same mechanism as exhibited by HMG-CoA reducta se inhibitors. We can furt her conclude that chronic administration of grapefruit ju ice and atorvastatin does not result in increased plasma concentration of the active hydroxy acid form. Our research about published clinical interaction trials suggest that most of the investigated drugs show no or only a minor interaction with grapefruit juice. The great number of accesses to the online database indicates the need for comprehensive scientific information.

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144 BIOGRAPHICAL SKETCH Immo Zdrojewski was born on February 13th, 1976, in Herten, Germany. He received his license to practice as pharmacist in Germany in January 2003 from the Westfaelische Wilhelms Universitaet in Muenster, Germ any. After his graduation he work ed as pharmacist for Pharbil GmbH in Waltrop, Germany, performi ng work in process validation a nd risk analysis of generic bulk production and primary and se condary packing of liquid and semi-solid dosage forms. He joined the doctoral program under the supervis ion of Dr. Hartmut Derendorf and Dr. Veronika Butterweck at the Department of Pharmaceutics of the University of Florida in August 2003. Immo Zdrojewski received his PhD in pharmaceutical sciences in May 2008 with his work about Interactions between grapefruit juice and HMG-CoA reductase inhibitors.