Effect of Pharmaceuticals and Natural Products on Multidrug Resistance

AN ABSTRACT OF THE DISSERTATION OF

Ying Fan for the degree of Doctor of Philosophy in Pharmacy presented on

December 7, 2007.

Title: Effect of Pharmaceuticals and Natural Products on Multidrug Resistance

Mediated Transport in Caco-2 and MDCKII-MDR1 Drug Transport Models

Abstract approved:

Rosita Rodriguez Proteau

This thesis details my investigation of some pharmaceuticals and natural products on the transport of drugs which are substrates of P-glycoprotein (P-gp), such as cyclosporin A (CSA) and digoxin by using Caco-2 and MDCKII-MDR1 drug transport models. Three objectives were performed to address this goal.

The first objective was to investigate if ketoconazole (KT) can modulate P- gp, thereby, altering cellular uptake and apparent permeability (Papp) of multidrug- resistance (MDR) substrates, such as CSA and digoxin, across Caco-2, MDCKII-

MDR1, and MDCKII-wild type drug transport models. 3H-CSA/3H-digoxin

transport experiments were performed with and without co-exposure to KT. KT was found to inhibit the Papp efflux of CSA and digoxin in Caco-2 cells. In

MDCKII-MDR1 cells, KT reduced the Papp efflux of CSA and increased the Papp absorption of digoxin. The drug-diet interaction of KT with flavonoids, epigallocatechin gallate (EGCG) and xanthohumol, was evaluated as well. EGCG and xanthohumol were able to reduce the Papp efflux of KT. In the uptake studies,

EGCG and xanthohumol exhibited biphasic responses in KT uptake. Our results suggest that KT may modulate the Papp of P-gp substrates by interacting with

MDR1 protein. EGCG and xanthohumol can modulate the transport and uptake of

KT.

The second objective was to investigate if specific flavonoids such as

EGCG, epicatechin gallate (ECG), and xanthohumol can modulate the cellular uptake and the permeability of CSA and digoxin across the Caco-2 and MDCKII-

MDR1 cell transport models. 3H-CSA/3H-digoxin transport and uptake experiments were performed with and without co-exposure of 30 µM xanthohumol, EGCG and ECG. EGCG and ECG inhibited the permeability efflux of CSA in Caco-2 cells, but not in MDCKII-MDR1 cells, which suggests EGCG and ECG may modulate other efflux transporters other than MDR1. Xanthohumol was able to inhibit the uptake of CSA, but increased the uptake of digoxin; xanthohumol inhibited the efflux permeability of CSA, but not digoxin in both

Caco-2 and MDCKII-MDR1 cells. These data suggests that xanthohumol may increase the bioavailability of CSA by inhibiting MDR1-mediated efflux, whereas

digoxin may share substrate specificity with other transporters such as multidrug resistance associated protein 2 (MRP2), or interact with MDR1 at a different site than xanthohumol.

Oregon Grape Root (OGR), Mahonia aquifolia, is native to the West coast of North America. Berberine as an alkaloid found in the OGR as well as berbamine have been reported to have significant anticancer activity. The last objective of my studies was to evaluate if berberine and berbamine can modulate

P-gp, thereby, altering the Papp of P-gp substrates across Caco-2 and MDCKII-

MDR1 drug transport models. OGR extract inhibited the efflux of CSA and digoxin with significant inhibition seen at low concentration of 0.1 mg/ml.

Berberine and berbamine significantly reduced the efflux Papp of CSA, whereas berberine did not have a measurable effect on the Papp of digoxin in the Caco-2 cells. In the MDCKII-MDR1 cells, berberine and berbamine inhibited both the efflux Papp of CSA and digoxin. Our data suggests that OGR extract, berberine, and berbamine can inhibit P-gp thereby may increase the bioavailability of drugs that are substrates of P-gp.

©Copyright by Ying Fan

December 7, 2007

Effect of Pharmaceuticals and Natural Products on Multidrug Resistance Mediated Transport in Caco-2 and MDCKII-MDR1 Drug Transport Models

by Ying Fan

A DISSERTATION

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Presented December 7, 2007

Commencement June 2008

Doctor of Philosophy dissertation of Ying Fan presented on December 7, 2007.

APPROVED:

______

Major Professor, representing Pharmacy

______

Dean of the College of Pharmacy

______

Dean of the Graduate School

I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request.

______

Ying Fan, Author

ACKNOWLEDGMENTS

I would like to express my gratitude to my major advisor, Dr. Rosita

Rodriguez Proteau, for her guidance in the experimental investigation, for her encouragement and financial support. I also would like to thank my committee members, Dr. J. Mark Christensen, Dr. Zhengrong Cui, Dr. Virginia M. Lessa, and

Dr. Bruce E. Coblentz for their support and academic guidance throughout my graduate career at Oregon State University.

My appreciation also goes to Dr. Ling Zhang and Dr. Bingnan Han for their assistance on the LC/MS work, Dr. Cristobal Miranda for his helpful advise in experimental design, and Dr. Taifo Mahmud and Dr. Philip Proteau for their valuable HPLC and mass spectrometry suggestions.

I am very grateful to my current and former group of colleagues for their mentorship, insight, and most importantly, friendship. I would especially like to acknowledge Dr. John Mata, Dr. Jinlong Cheng, Ms. Jean Brown, Ms. Kelly Hall,

Dr. Chien Nyugen, Mr. Brian Sloat, Ms. Hang Le, and Ms. Uyen Le for their friendship and encouragement throughout the years.

Finally, I would like to express my gratitude to my wonderful family for their support and encouragement. I am forever indebted to them for their patience and love through these challenging years.

CONTRIBUTION OF AUTHORS

Dr. John Mata was involved with experimental design and writing of Chapter 3.

Ms. Jean Brown assisted with some experiments in Chapter 3.

TABLE OF CONTENTS

Page

1. GENERAL INTRODUCTION...... 1

1.1 Background ...... 1

1.2 P-glycoprotein (P-gp)...... 6

1.2.1 Function...... 6 1.2.2 Structure ...... 6 1.2.3 Location...... 9 1.2.3 Substrates of P-gp ...... 12 1.2.4 Inhibitors of P-gp ...... 14

1.3 Drug-drug and drug-diet interaction ...... 15

1.3.1 Mechanism of drug-drug and drug-diet interactions...... 15 1.3.2 Examples of drug-drug interaction...... 21 1.3.3 Examples of drug-diet interaction...... 33

1.4 Methods for investigating the multidrug resistance mediated drug-drug interactions and drug-diet interactions...... 48

1.4.1 In vitro...... 48 1.4.2 In vivo...... 56

2. KETOCONAZOLE MODULATES MULTIDRUG RESISTANCE- MEDIATED TRANSPORT IN CACO-2 AND MDCKII-MDR1 DRUG TRANSPORT MODELS ...... 57

2.1 Abstract ...... 58

2.2 Introduction...... 59

2.3 Materials and Methods...... 63

2.3.1 Chemicals...... 63 2.3.2 Cell culture...... 63 2.3.3 Transport studies ...... 65 2.3.4 MDR assay...... 67 2.3.5 Uptake studies ...... 68 2.3.6 Statistical analysis ...... 69

TABLE OF CONTENTS (Continued)

Page

2.4 Results...... 70

2.5 Discussion ...... 88

3. EFFECT OF DIETARY FLAVONOIDS ON TRANSPORTERS IN CACO-2 AND MDCKII-MDR1 CELL TRANSPORT MODELS...... 96

3.1 Abstract ...... 97

3.2 Introduction...... 98

3.3 Materials and methods ...... 103

3.3.1 Chemicals...... 103 3.3.2 Cell culture...... 103 3.3.3 Transport studies ...... 104 3.3.4 Uptake studies ...... 106 3.3.5 Liquid Chromatography/Mass Spectrometry (LC/MS) analysis ...... 107 3.3.6 Statistical analysis ...... 108

3.4 Results...... 109

3.5 Discussion ...... 124

3.6 Appendix...... 131

4. BERBERINE AND BERBAMINE MODULATE P-GLYCOPROTEIN...... 136

4.1 Abstract ...... 137

4.2 Introduction...... 139

4.3 Materials and methods ...... 145

4.3.1 Chemicals and reagents...... 145 4.3.2 Oregon grape root extract preparation ...... 146 4.3.3 Cell culture...... 147 4.3.4 Chemical exposure...... 148 4.3.5 Cytotoxicity assays...... 151

TABLE OF CONTENTS (Continued)

Page

4.3.6 Transport studies ...... 152 4.3.7 Real time quantitative PCR analysis of MDR1 mRNA...... 154 4.3.8 High Performance Liquid Chromatography (HPLC) analysis...... 155 4.3.9 Liquid Chromatography/Mass Spectrometry (LC/MS) analysis ...... 157 4.3.10 Statistical analysis ...... 157

4.4 Results...... 158

4.5 Discussion ...... 187

5. GENERAL CONCLUSION ...... 197

BIBLIOGRAPHY...... 200

APPENDICES ...... 243

LIST OF FIGURES

Figure Page

1.1 Schematic diagram of multidrug transporters...... 3

1.2 P-glycoprotein (P-gp) function ...... 7

1.3 Structure of P-glycoprotein...... 10

1.4 Tissue location of transport proteins...... 11

1.6 Chemical structure of digoxin...... 25

1.7 Chemical structure of ketoconazole...... 28

1.8 Chemical structure of MK-571 ...... 31

1.9 Chemical structure of verapamil ...... 32

1.10 Chemical structure of (-)-epigallocatechin-3-gallate (EGCG)...... 39

1.11 Chemical structure of xanthohumol...... 43

1.12 Chemical structure of berberine...... 45

1.13 Chemical structure of berbamine ...... 47

1.14 The Transwell® plate with inserts ...... 50

1.15 Principle of the VybrantTM Multidrug Resistance (MDR) Assay ...... 54

2.1 Effect of increasing concentrations of ketoconazole, 0.03, 0.1, 0.3, 1.0, and 3.0 µM, on 1 µCi 3H-ketoconazole across Caco-2 cells for the apical (A) to basolateral (B) and B to A transport of ketoconazole ...... 71

2.2 Effect of increasing concentrations of ketoconazole (KT), 0.03, 0.1, 0.3, 3 1.0, 3.0 and 10.0 µM, on the apparent permeability (Papp) of 1 µCi H-KT across MDCKII-MDR1 cells for the apical (A) to basolateral (B) and B to A transport of KT...... 72

2.3 Effects of ketoconazole (KT) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cells ...... 74

LIST OF FIGURES (Continued)

Figure Page

2.4 Effects of ketoconazole (KT) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII-MDR1 cells ...... 76

2.5 The effect of ketoconazole (KT) and transport inhibitors on the transport of 0.5 µM 3H-cyclosporin A (CSA) and 0.5 µM digoxin (1 µCi) across MDCKII-wild type cells ...... 79

2.6 Effects of (-)-epigallocatechin-3-gallate (EGCG) and xanthohumol (XN), on the transport of 1 µM, 3H-ketoconazole (KT, 1 µCi) from the basolateral to apical direction in Caco-2 cells...... 82

2.7 The influence of increasing concentrations of ketoconazole (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 µM) on calcein retention in Caco-2 cells (Panel A) and MDCKII-MDR1 cells (Panel B)...... 84

2.8 The effects of increasing cyclosporin A (CSA) and digoxin concentrations (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 µM) on 1.0 µM 3H- ketoconazole (0.1 µCi) uptake using Caco-2 cells (Panel A) and MDCKII- MDR1 cells (Panel B) ...... 85

2.9 The effects of increasing (-)-epigallocatechin-3-gallate (EGCG) or xanthohumol (XN) concentrations (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 µM) on 1.0 µM 3H-ketoconazole (0.1 µCi) uptake using Caco-2 cells (Panel A) and MDCKII-MDR1 cells (Panel B)...... 87

3.1 Effects of various transport inhibitors on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cell monolayers ...... 110

3.2. Effects of transport inhibitors on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII-MDR1 cells ...... 112

3.3 Effects of transport inhibitors on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII wild-type cells ...... 114

LIST OF FIGURES (Continued)

Figure Page

3.4 The effects of increasing EGCG or xanthohumol (XN) concentrations (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 µM) on (0.1 µCi, 0.5 µM) 3H-cyclosporin A (CSA, Panel A) or 3H-digoxin (Panel B) uptake using Caco-2 cells...... 116

3.5 The effects of increasing EGCG or xanthohumol (XN) concentrations (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 µM) on (0.1 µCi, 0.5 µM) 3H-cyclosporin A (CSA, Panel A) or 3H-digoxin (Panel B) uptake using MDCKII-MDR1 cells ...... 117

3.6 Effects of Menhop® extract on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cell monolayers ...... 119

3.7 Effect of xanthohumol and xanthohumol derivatives on transport of transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H- digoxin (1 µCi) across Caco-2 cell monolayers...... 120

3.8 High performance liquid chromatograph (HPLC) chromatograms and Mass Spectrometry (MS) of xanthohumol. Panel A: HPLC chromatogram of xanthohumol standard...... 122

3.9 High performance liquid chromatograph (HPLC) chromatograms and Mass Spectrometry (MS) of Menohop® extract...... 123

3.10 Cytotoxicity (3-(4, 5-demethythiazole-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay results of Menohop® extract in Caco-2 cells at 4 hours...... 132

3.11 Effects of various transport inhibitors on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across LLC- MDR1 cell monolayers ...... 134

4.1 Time course of the transport of 100 µM berberine (Panel A) or 100 µM berbamine (Panel B) from the apical to the basolateral (A to B, □) and the B to A (♦) across Caco-2 cells ...... 163

LIST OF FIGURES (Continued)

Figure Page

4.2 Time course of the transport of 100 µM berberine (Panel A) or 100 µM berbamine (Panel B) from the apical to basolateral (A to B, □) and the B to A (♦) across MDCKII-MDR1 cells...... 164

4.3 Effects of berberine and berbamine on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) in the Caco-2 cells ...... 166

4.4 Effects of berberine and berbamine on transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII-MDR1 cells ...... 167

4.5 Effects of berberine and berbamine on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII wild-type (MDCKII-WT) cells...... 168

4.6 Effect of Oregon grape root extract 1 (E1) on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cells ...... 171

4.7 Effect of Oregon grape root extract 2 (E2) on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cells ...... 172

4.8 High performance liquid chromatograph (HPLC) chromatograms of berberine (Panel A), berbamine (Panel B), Oregon grape root extract 1 (E1) (Panel C) and Oregon grape root extract 2 (E2) (Panel D)...... 175

4.9 Mass Spectrometry (MS) of berberine, berbamine, E1 and E2 ...... 176

4.10 Effects of Oregon grape root extract 1 aqueous portion (EA1) and organic portion (EO1) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) across Caco-2 cells ...... 181

4.11 Effects of Oregon grape root extract 1 aqueous portion (EA1) and organic portion (EO1) on the transport of 0.5 µM 3H-digoxin (digoxin, 1 µCi) across Caco-2 cells ...... 182

LIST OF FIGURES (Continued)

Figure Page

4.12 Effects of Oregon grape root extract 2 aqueous portion (EA2) and organic portion (EO2) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) across Caco-2 cells ...... 183

4.13 Effects of Oregon grape root extract 1 aqueous portion (EA2) and organic portion (EO2) on the transport of 0.5 µM 3H-digoxin (digoxin, 1 µCi) across Caco-2 cells ...... 184

4.14 Oregon grape root extract 1 (E1) and Oregon grape root extract 2 (E2) up-regulation of mRNA level of human MDR1 (ABCB1) in Caco-2 cells (Panel A) and LS-180 cells (Panel B)...... 186

LIST OF TABLES

Table Page

1.1 Typical substrates and inhibitors of P-glycoprotein (P-gp) ...... 8

1.2 Examples of herbs or drugs inhibit cytochrome P450 enzymes ...... 18

1.3 Composition of green tea extract capsules...... 38

2.1 Comparison of efflux ratios of [3H] cyclosporin A (CSA) and [3H] digoxin with various concentrations of ketoconazole (KT) in Caco-2 and MDCKII-MDR1 cells ...... 77

3 2.2 Effect of ketoconazole (KT) on the apparent permeability (Papp) of [ H] cyclosporin A (CSA) and [3H] digoxin in Caco-2, MDCKII-MDR1, and MDCKII-wild type (MDCKII-WT) cells from the basolateral to apical direction ...... 80

4.1 Typical substrates of P-glycoprotein (P-gp) ...... 142

4.2 Dose-response cytotoxicity data of berberine and berbamine from the 3-(4, 5-demethythiazole-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays in Caco-2, MDCKII-MDR1 and MDCKII wild-type (MDCKII-WT) cells after 4 hours exposure...... 159

4.3 Dose-response cytotoxicity data of Oregon grape root extract (E1) and Oregon grape root extract (E2) from the 3-(4, 5-demethythiazole-2yl)-2, 5- diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays in Caco-2 and LS-180 cells...... 161

3 4.4 Effect of berberine and berbamine on apparent permeability (Papp) of [ H] cyclosporine A (CSA) and [3H] digoxin in Caco-2, MDCKII-MDR1 and MDCKII wild-type (MDCKII-WT) cells from basolateral to apical direction ...... 169

3 4.5 Comparison of apparent permeability (Papp), efflux ratio and IC 50 of [ H] cyclosporin A (CSA) and [3H] digoxin with various concentrations of Oregon grape root extract 1 (E1) and Oregon grape root extract 2 (E2) in Caco-2 cells...... 173

LIST OF APPENDICES

Protocol Page

Protocol A: Real time Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) ...... 244

Protocol B: Ketoconaozle Multidrug Resistance (MDR) assay...... 255

Protocol C: Cytotoxicity Studies...... 257

Protocol D: Menohop® Liquid Chromatography/Mass Spectrometry LC/MS... 260

Protocol E: Oregon Grape Root Extract Liquid Chromatography/Mass Spectrometry (LC/MS)...... 262

Protocol F: Oregon Grape Root Extract Purification...... 265

Protocol G: Oregon Grape Root Extract Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC/MS/MS) Analysis...... 268

Protocol H: Protein Separation...... 271

Protocol I: Protein Determination ...... 274

EFFECT OF PHARMACEUTICALS AND NATURAL PRODUCTS ON MULTIDRUG RESISTANCE MEDIATED TRANSPORT IN CACO-2 AND MDCKII-MDR1 DRUG TRANSPORT MODELS

1. GENERAL INTRODUCTION

1.1 Background

Drug transport across bio-membranes occurs by active and facilitated transport processes and simple passive diffusion. It depends on the physicochemical properties of the compound. It is believed that physicochemical properties, such as size, lipophilicity and metabolic processes are the main determinants of the bioavailability of most of the drugs. However, there are many drugs that do not follow a passive diffusion process, but are actively transported through intestinal enterocytes into portal blood vessels or back to the intestinal lumen. Therefore, the differences of plasma concentrations of the drug are due to different expression of levels of both metabolic enzymes and drug transporters in organ barriers or excretion organs (Fromm, 2004; Gerloff, 2004).

Drug transporters consist of uptake and efflux transporters. Uptake carriers facilitate the cellular influx of nutrients and vitamins and to reabsorb endogenous compounds, such as glucose, amino acids, small peptides or nucleosides. They use electrochemical gradients or ions as a driving force enabling transport even against 2 a concentration gradient. The main uptake carriers are organic anion transporting polypeptide (OATP), organic anion transporter (OAT), organic cation transporters

(OCT), concentrative nucleoside transporters (CNT), dipeptide transporters

(PEPT) and monocarboxylate carriers (MCT) (Ho and Kim, 2005).

The structures of the efflux transporters or multidrug resistance (MDR) transporters can be defined based on four distinct transporter superfamilies (Figure

1.1): Major facilitator superfamily (MFS family), resistance-nodulation-division

(RND) family, small multidrug resistance (SMR) family and ATP-binding cassette

(ABC) family (Higgins, 2007). These transporters play an important role in the defending system for transporting xenobiotics and endogenous compound.

Multidrug resistance transporters are clinically important because they can render cells resistant to many chemotherapeutic agents, such as anticancer drugs and antibiotics (Borst and Elferink, 2002; Gottesman, 2002; Poelarends et al., 2002).

Furthermore, some mammalian multidrug resistance transporters also play an important role in the absorption, distribution, and elimination of drugs and xenobiotics, and are probably responsible for clinical drug-drug interactions

(Lown et al., 1997b; Schinkel, 1998; Goh et al., 2002). Multidrug resistance transporters are present in all living organisms from prokaryotes to mammals. In humans, there are three major types of MDR proteins: P-glycoprotein (P-gp,

ABCB1/MDR1), multidrug resistance associated protein (ABCC/MRP), and breast cancer resistance protein (ABCG/BCRP). These efflux transporters all belong to the ABC superfamily of membrane proteins. 3

Outer membrane Transmembrane domain (TMD) Periplasmic Cell domain exterior

Cytoplasma

Nucleotide-binding Transmembrane N-teminus domain (NBD) domain (TMD)

SMR MFS ABC transporters RND transporters transporters transporters

Figure 1.1 Schematic diagram of multidrug transporters

ABC, ATP-binding cassette; RND, resistance-nodulation-division; SMR, small multidrug resistance; MFS, major facilitator superfamily

P-gp is one of the intensely studied efflux transport proteins in respect to its structure and function (Ambudkar et al., 1999; Loo and Clarke, 1999a; Hrycyna,

2001; Ambudkar et al., 2003). It transports many structurally and pharmacologically unrelated neutral and positively charged hydrophobic compounds (Mizuno et al., 2003; Chan et al., 2004). It also prevents the uptake of toxic compounds from the gut into the body and protects important organs, such as the brain, against toxins. It was first found as a surface phosphoglycoprotein overexpressed in cultured cells selected for MDR (Juliano and Ling, 1976) and was subsequently cloned from mouse and human cells based on amplification of the MDR locus (Chen et al., 1986; Gros et al., 1986).

MRPs confer resistance to a large variety of drugs and contribute to cellular efflux of endogenous anionic glutathione, glucuronate conjugates and cyclic nucleotides (Evers et al., 2000). Ten members of the MRP family are known, and at least seven of them (MRP1, 2, 3, 4, 5, 6 and 7) have been shown to confer resistance to one or more drugs used to treat cancer (Kool et al., 1999; Borst et al., 2000; Struk et al., 2000; Wijnholds et al., 2000b; Chen et al., 2001; Chen et al., 2003). However, only MRP1 has been shown to be likely have significance in clinical drug resistance. MRP1 (ABCC1) was isolated from an MDR lung cancer cell line where MDR1 was not expressed (Cole et al., 1992). It has similar transport specificity to MDR1, but drugs are often conjugated with glutathione and other anions, or are co-transported with glutathione (Borst et al., 1999). MRP2, also known as canalicular multispecific organic anion transporter (cMOAT), 5 transports relatively hydrophilic compounds, including glucuronide, glutathione, and sulfate conjugates of compounds (Suzuki and Sugiyama, 2002; Chan et al.,

2004). MRP2 has been shown to be involved in resistance to vincristine, cisplatin, and etoposide (Cui et al., 1999; Kawabe et al., 1999). Therefore, MRP2 has a potential to have significance in clinical drug resistance.

BCRP is a novel protein in the human ABC transporter proteins. It is also known as mitoxantrone-resistance protein (MXR) or placenta-specific ABC protein (ABCP). It transports relatively hydrophilic anticancer agents (Doyle and

Ross, 2003). Unlike P-gp and MRPs, which are arranged in two repeated halves,

BCRP is a half-transporter consisting of only one nucleotide binding domain followed by one membrane-spanning domain. At present, four human members of

BCRP, namely ABCG1, ABCG2, ABCG5 and ABCG8, have been identified. The expression of BCRP has been detected in hematological malignancies and solid tumors. In addition, BCRP is also highly expressed in the apical membrane of the epithelium in the small intestine, in the liver canalicular membrane, and at the luminal surface of human brain microvessels (Ito et al., 2005; Meyer zu

Schwabedissen et al., 2006). Therefore in addition to conferring resistance against chemotherapeutic agents, BCRP plays an important role in drug absorption (small intestine), distribution (blood brain barrier) and elimination (liver).

Approximately 50% of marketed drugs have been identified to be P-gp substrates and/or inhibitors (Keogh and Kunta, 2006), which indicates a major role 6 of P-gp in the multidrug resistance system (Germann, 1996). Therefore, this thesis was focused mainly on P-gp.

1.2 P-glycoprotein (P-gp)

1.2.1 Function

P-gp is a 170-KD protein, which consists of 1280 amino acids with 12 trans-membrane segments and two ATP binding sites (Gottesman and Pastan,

1993). It utilizes ATP hydrolysis as an energy source, exporting the compounds from the intracellular to the extracellular domain or interacting with drug molecules trapped within the cell membrane lipid bilayer (Figure 1.2). Therefore,

P-gp protects cells from toxins and exports a large amount of its substrates which attempt to pass into the cell membrane. In humans, only MDR1 gene encodes for the MDR-related P-gp efflux pump (Ueda et al., 1987). However, in mice, there are two isoforms of the genes which encodes P-gp, mdr1 a and mdr1 b (Croop et al., 1989). They have similar functions (Borst et al., 1999) and coding sequences

(Devault and Gros, 1990) as the single human MDR1 gene. P-gp is responsible for the transport of different hydrophobic substrates such as vinblastine, cyclosporin

A, dexamethasone and digoxin (Table 1.1).

1.2.2 Structure

P-gp is a transmembrane glycoprotein. It contains two homologous halves, each consisting of one hydrophobic domain with six transmembrane segments 7

Pharmaceuticals or natural products

Lipid bilayer

Figure 1.2 P-glycoprotein (P-gp) function

P-Gp, P-glycoprotein; ATP, adenosine triphosphate; ADP, adenosine diphosphate; Pi, inorganic phosphate. Modified from Marzolini et al., 2004. 8

Table 1.1 Typical substrates and inhibitors of P-glycoprotein (P-gp)

Substrates Inhibitors

Anticancer drugs Antihypertensive agents Actinomycin D, Daunonibicin, Carvedilol, Nicardipine, Daunorubicin, Docetacel, Reserpine Doxorubicin, Etoposide, Imatinib, Paclitaxel, Tamoxifen, Teniposide, Antiarrhythmics Vinblastine, Vincristine Amiodarone, Propafenone

Antibiotics Antibiotics Cefazolin, Erythromycin, Ofloxacin Clarithromycin, Erythromycin,

Antihistamines Antidepressants Fexofenadine, Terfenadine Fluoxetine, Paroxetine, Sertraline Ca2+-channel blockers Diltiazem, Verapamil Ca2+-channel blockers Verapamil Cardiac drugs Digoxin, Digitoxin, Quinidine Cardiac drugs Quinidine Fluorencent dyes Neuroleptics Rhodamine 123 Chloropromazine Flupenthixol HIV protease inhibitors Ritonavir, Inidnavir, Saquinavir Opioids Methadone, Pentazocine Immunosuppressants Cyclosporin A, Sirolimus, Tacrolimus Immunosuppressants Valspodar Cyclosporin A, Sirolimus, Tacrolimus, Valspodar Steroids Dexamethasone, Methylprednisolon

Miscellaneous drugs Amitryptiline, Colchicine, Itraconazole, Lansoprazole, Loperamide, Losartan, Morphine, Phenytoin, Rifampicin

References: (Ambudkar et al., 1999; Kerb et al., 2001; Litman et al., 2001; Dietrich et al., 2003; Ho and Kim, 2005) 9

and one hydrophilic nucleotide-binding domain (Figure 1.3). These two halves are connected by a short flexible linked polypeptide (Hyde et al., 1990). P-gp is synthesized in the endoplasmic reticulum and the carbohydrate moeity being subsequently modified in the Golgi apparatus prior to export to the cell surface

(Loo and Clarke, 1999b). Although P-gp is glycosylated on its first extracelluar loop, the role of glycosylation is still not clear. An experiment which used P-gp mutant cells lacking the N-terminal glycosylation sites showed that substrate transport was not affected (Schinkel et al., 1993). Therefore, glycosylation may alter the trafficking and the stability of P-gp within the plasma membrane. The secondary and tertiary structures of P-gp have not been fully elucidated.

1.2.3 Location

P-gp was first discovered in cancer cells, but later it was also found in normal tissues (Figure 1.4), such as the apical membrane of intestinal epithelial cells (Mukhopadhyay et al., 1988), the biliary canalicular membrane of liver, the luminal membrane of proximal tubular epithelial cells in kidney (Demeule et al.,

2001) and the luminal membrane of the endothelial cells forming the blood-brain barrier (BBB), blood-cerebro spinal fluid barrier (BCSFB) and blood-testis barrier

(Cordon-Cardo et al., 1989; Fromm, 2000; Wijnholds et al., 2000a).

Extracelluar

1 2 3 4 5 6 7 8 9 10 11 12

NH2

NBD NBD

COOH Intracellular

Figure 1.3 Structure of P-glycoprotein

NBD, nucleotide-binding domain. Modified from Pal and Mitra, 2006.

Intestinal efflux Biliary excretion Hepatic uptake P-gp, OCT1, P-gp, MRP2 OATP2, OCT1, MRP2, MPR1, OATP8, OATP2, OATP3 OATP3

Renal secretion OAT1, OAT3, OCT1, OCT2, Brain transport OATP P-gp, OAT3, MRP1, MRP5, OATP1

Renal secretion P-gp, MRP1

Figure 1.4 Tissue location of transport proteins

P-gp, P-glycoprotein; OCT, organic cation transporters; MRP, multidrug resistance associated protein; OATP, organic anion transporting polypeptide; OAT, organic anion transporter. Modified from Ayrton and Morgan, 2001. 12

In mice, the mdr1a P-gp is mainly on the apical surface of intestinal enterocytes and on the luminal side of capillary endothelial cells of the brain. The expression of mdr1b is predominant in the adrenal gland and the secretory epithelial cells of the ovaries (Croop et al., 1989; Schinkel et al., 1995a).

The surfaces of intestine and kidney are lined by a single layer of epithelial cells. Epithelial cells function as a barrier to select nutrients and toxic xenobiotics by the uptake and efflux transport systems. Up to now, ABC transporters and solute carriers (SLCs), such as H+/peptide transporters and organic ion transporters have been found in the intestine and kidney.

P-gp was also found in the apical membrane of endothelial cells in BBB and BCSFB. Besides P-gp, other ABC transporters (such as MRPs and BCRP), organic anion transporters and large amino-acid transporters have been found in

BBB and BCSFB (Schinkel and Jonker, 2003; Begley, 2004). Existing of these efflux transporters helps the BBB prevent the influx of most compounds from the blood to the brain, but also becomes a major obstacle for the delivery of therapeutics to the brain.

1.2.3 Substrates of P-gp

Substrates of P-gp are expected to freely diffuse into the cells, and can be recognized by P-gp in the context of the plasma membrane (Pastan and Gottesman,

1991). The structure-activity relationship for P-gp substrates has not been clearly defined. The lipophilicity and the number of hydrogen bonds appear to be the 13 relevant parameters, as both have been proportionally correlated to the affinity of compounds for P-gp (Ecker et al., 1999). The range of substrates transported by P- gp is broad and includes a variety of pharmacologically distinct agents used in cancer chemotherapy, hypertension, allergy, infections, immunosuppression, neurology, and inflammation. Theoretical model and in vitro experiments indicated that P-gp can recognize its substrates before they reach the cytoplasma

(Stein, 1997; Litman et al., 2003). Lipinski et al. reported a ‘rule-of -5’ method which can be used to predict the intestinal absorption based on physicochemical properties. It indicated that the compounds which have poor intestinal absorption could possibly satisfy any two of the characteristics: molecular weight > 500; number of hydrogen bond donors > 5 (O-H or N-H group); number of hydrogen bond acceptors > 10 (O or N); calculated log P > 5 (Lipinski et al., 2001). There is also some evidence showing that many drugs which are substrates of P-gp are also substrates of drug-metabolizing enzymes, such as cytochrome P450 (CYP) 3A4.

The overlap between CYP3A4 and P-gp substrates may result in part from the coordinated regulation and tissue expression of CYP3A4 and MDR1 in organs such as the liver and intestine. Interestingly, both genes were found to locate on the same chromosome in close proximity, 7q22.1 and 7q21.1 for CYP3A4 and

MDR1, respectively (Kim et al., 2000). However, the extent of substrate overlap for them is not complete, because there are some substrates of CYP3A4 that are not transported by P-gp, such as midazolam (Kim et al., 1999). Conversely, there are also some compounds that are P-gp substrates, but they do not interact to a 14 significant extent with CYP enzymes such as digoxin, fexofenadine, celiprolol, and talinolol (Milne and Buckley, 1991; Trausch et al., 1995; Marzolini et al.,

2004).

1.2.4 Inhibitors of P-gp

Like P-gp substrates, the chemical structures and pharmacological actions of P-gp inhibitors are not related to their inhibitory actions. It has been reported that there is a good correlation between P-gp inhibition and physicochemical parameters. It is suggested that a highly effective P-gp modulator candidate should possesses a log P value of 2.92 or higher, 18 atom-long or longer molecular axis, at least one tertiary basic nitrogen atom, and high energy in the highest occupied orbit (Wang et al., 2003).

Inhibition of P-gp could potentially result from the blockage of specific recognition of the substrate, binding of ATP, ATP hydrolysis, or coupling of ATP hydrolysis to translocation of the substrate. Most reversing agents block P-gp by acting as competitive or non-competitive inhibitors (Garrigos et al., 1997) and by binding either to drug interaction sites (Dey et al., 1997) or to other modulator binding sites, leading to allosteric changes. Modulators such as verapamil are substrates of P-gp and inhibit the transport function in a competitive manner without interrupting the catalytic cycle of P-gp (Ford, 1996). Cyclosporin A, as one of the reversing agents, inhibits P-gp function by interfering with both substrate recognition (Tamai and Safa, 1991) and ATP hydrolysis (Tamai and 15

Safa, 1991). Because ATP hydrolysis is required for transport (Azzaria et al.,

1989; Loo and Clarke, 1995), modulators that inhibit ATPase activity are unlikely to be transported by P-gp.

1.3 Drug-drug and drug-diet interaction

1.3.1 Mechanism of drug-drug and drug-diet interactions

It is known that clinically significant drug-drug interactions occur when the efficacy or toxicity of a drug is altered by the administration of another drug.

Traditionally, the cause of many clinically important drug interactions has been considered as altering the metabolic clearance of a drug, particularly through cytochrome P450 metabolism (von Moltke et al., 1998; Dresser et al., 2000).

However, more and more evidence suggested that some drug transporter proteins, such as P-gp (MDR1), MRPs, and BCRP also cause changes in the bioavailability of the drug (Johne et al., 2002; Giessmann et al., 2004; Sparreboom et al., 2005).

Primary active efflux transporter, such as MDR1, limits the uptake of its substrates, digoxin and cyclosporin A (Gottesman et al., 2002). In addition, BCRP,

MRP2, and MRP4 are expressed in the apical membranes of intestinal enterocytes and MRP1, 3, 5, 6 are expressed in the basolateral membranes of intestinal enterocytes, which also limit the absorption of xenobiotics in the intestine (Chan et al., 2004). The expression of these efflux transporter proteins were also found in 16

Caco-2 cells and correlates with the expression in normal human jejunum (r2 =

0.90) (Taipalensuu et al., 2001).

Drug-diet interaction also involves the intestinal and hepatic metabolism by

CYPs and induction or inhibition of drug efflux proteins such as P-gp and MRPs

(Wilkinson, 1997; Evans, 2000; Ioannides, 2002). Therefore, consumption of diet supplements or herbs that are able to modulate efflux proteins or CYPs may cause clinically relevant drug-diet interactions and alter drug bioavailability (Fugh-

Berman, 2000; Fugh-Berman and Ernst, 2001; Izzo and Ernst, 2001).

Pharmacokinetic drug-drug or drug-diet interactions

Pharmacokinetic drug-drug or drug-diet interactions are caused by altering absorption, metabolism, distribution and excretion of drugs. One mechanism for this is the induction or inhibition of hepatic and intestinal Cytochrome P450 and P- gp (Hebert et al., 1992; Lin and Lu, 1998; Evans, 2000; Zhou et al., 2003; Keogh and Kunta, 2006). The CYP system oxidizes a broad spectrum of drugs by a number of metabolic processes that can be enhanced or reduced by various compounds known as inducers or inhibitors. The clinical importance of any drug- drug or drug-diet interactions depends on factors that are drug-, patient- and route of administration-related (Dresser et al., 2000). Rifampicin, a potent inducer of

CYP2C9, decreased the mean area under the concentration-time curve (AUC) of glibenclamide by 39% (p < 0.01), decreased the peak plasma concentration (Cmax) by 22% (p = 0.01), and shortened the mean half life (t½) of glibenclamide from 2.0 17 hours to 1.7 hours (p < 0.05), when rifampicin was coadminstered with glibenclamide in healthy volunteers (Niemi et al., 2001). A well known CYP3A4 inhibitor, ketoconazole, increased the Cmax and AUC of dexloxiglumide by 32% and 36%, respectively, when dexloxiglumide was coadminstered with ketoconazole at steady state (200 mg once daily for 5 days) (Jakate et al., 2005).

Coadministration of garlic (Allium sativum, CYP3A4 inhibitor) extract (10 mg) with ritonavir 400 mg single dose for 4 days in healthy human volunteers decreased the AUC of ritonavir by 17% compared with administration of ritonavir alone (Gallicano et al., 2003).

Herbs or drugs may inhibit CYPs by three mechanisms: competitive inhibition, non-competitive inhibition, and mechanism-based inhibition (Shou et al., 2001; Zhou et al., 2003). Competitive inhibition may occur between drug or herbal constituent and drug, which are often metabolized by the same CYP enzyme. Non-competitive inhibition is caused by the binding of drug or herbal components containing electrophilic groups, such as imidazole or hydrazine group, to the haem portion of the CYP molecule. The mechanism-based inhibition of

CYP is caused by the formation of a complex between drug or herbal metabolite and CYP. The examples for these inhibitions were summarized in the Table 1.2. 18

Table 1.2 Examples of herbs or drugs inhibit cytochrome P450 enzymes

Inhibition Type Inhibitor Enzyme References

Competitive inhibition diallyl sufide CYP2E1 (Teyssier et al., 1999) (from garlic)

dextromethorphan CYP2D6 (Ciccone et al., 2006)

Non-competitive piperine CYP2E1 (Dalvi and Dalvi, 1991) inhibition (from peper) (Kang et al., 1994)

hyperforin CYP2D6 (Obach, 2000) (from St John’s wort)

Mechanism-based diallyl sulfone CYP2E1 (Jin and Baillie, 1997) inhibition 19

Among these, competitive and noncompetitive inhibition are the most common mechanisms responsible for drug interactions (Kunze et al., 1996; Yao et al.,

2001).

Herbs or drugs may also modulate the expression and activity of transport proteins, such as P-gp and OATP. For example, St John’s wort has been found to induce intestinal P-gp in vitro and in vivo (Durr et al., 2000; Perloff et al., 2001;

Hennessy et al., 2002). Furanocoumarins and bioflavonoids found in fruit juices are inhibitors of OATP. When they are co-administered together with fexofenadine

(substrate of OATP), they decreased the fexofenadine AUC, Cmax and urinary excretion values to 30-40%, whereas no change in the tmax, t½, renal clearance, or urine volume in humans (Dresser et al., 2002). Grapefruit juice has been reported as an inhibitor of OATP, whereas its inhibitory effect of P-gp has been determined to be modest (Mizuno et al., 2003).

Quinidine, an inhibitor of P-gp, when co-administrated with digoxin (a substrate of P-gp) 1mg intravenous dose, reduced digoxin total body clearance by

26% (from 2.98 to 2.22 ml/min/kg), and lengthened digoxin elimination t½ from

34.2 to 51.8 hr (Wandell et al., 1980). Many other drugs, such as cyclosporin A

(Dorian et al., 1988), verapamil (Pedersen et al., 1983), and itraconazole (Partanen et al., 1996) have the similar effect on digoxin, indicating they may have interaction with P-gp as well.

Pharmacodynamic drug-drug or drug-diet interactions

Combined use of herbs may alter the effects of drugs. Synergistic or additive therapeutic effects may cause toxic effects or decrease drug efficacy resulting in therapeutic failure. These effects often result from the competitive or complementary effect of the drug and the combined herbal constituents or drug on the same drug target.

An interaction between kava and alprazolam in a 54-year old man

(Almeida and Grimsley, 1996) indicated that combination using of kavalactones and alprazolam can cause a lethargic and disoriented state in humans, because they both acted on the same γ-aminobutyric acid (GABA) receptors in central nervous system (Jussofie et al., 1994; Yuan et al., 2002). In another study, an 80-year old woman with Alzheimer’s disease fell into coma when taking a low dose of the atypical antidepressant, trazodone and ginkgo (Galluzzi et al., 2000), which indicated that it may be associated with enhanced GABA-related neuronal activity in the brain by ginkgo.

Aspirin and ibuprofen co-administration has been reported to be associated with an increased risk of cardiovascular mortality (Kurth et al., 2003; MacDonald and Wei, 2003; Kimmel et al., 2004). The mechanism suggested was that ibuprofen inhibited the access of aspirin to the cyclooxigenase (COX)-1 acetylation site in platelets resulting in antagonizing irreversible platelet inhibition.

When aspirin was given 2 hours before a daily dose of ibuprofen, it successfully inhibited platelet aggregation (Catella-Lawson et al., 2001). However, when 21 ibuprofen was administered three times daily, it competitively prevented aspirin from accessing its target serine and inhibiting platelet aggregation (Catella-Lawson et al., 2001)

1.3.2 Examples of drug-drug interaction

Cyclosporin A

Cyclosporin A (Figure 1.5) is a potent immunosuppressive drug in the prevention of allograft rejection. It is an antibiotic produced by the fungus

Tolypocladium inflatum Gams which binds to the immuno-philin cyclophilin A

(Friedman and Weissman, 1991; Schreiber, 1991) and inhibits the calcium- dependent serine/threonine phosphatase calcineurin, abrogating transcription of lymphokines, such as interleukin-2 (IL-2) (Friedman and Weissman, 1991; Liu et al., 1991). Cyclosporin A is metabolized by CYP3A4 to form hydroxylated and N- demethylated derivatives in both liver and intestine (Kronbach et al., 1988;

Combalbert et al., 1989; Kolars et al., 1991). As an immunosuppressive agent, the effective concentration of cyclosporin A is 0.083 to 0.208 µM (100 to 250 ng/ml)

(Rogers et al., 1984; Kahan, 1985; Heidecke et al., 1987), whereas the clinical therapeutic range for cyclosporin A is 0.083 to 0.665 µM (100 to 800 ng/ml)

(Hamilton et al., 1987; McMillan, 1989; List et al., 1993).

Many studies have shown that P-gp plays an important role in the oral bioavailability of cyclosporin A. Kaplan et al. evaluated the bioavailability of 22

H3C H3C H C 3 H3C N OH H3C CH3 N CH3 CH H N 3 O O O N O CH3 N CH3 H3C O H C H3C 3 H C O N CH3 3 HN H3C O CH O N 3 O CH3 H H3C O N N H3C N H CH3 CH O 3 H3C CH3

Figure 1.5 Chemical structure of cyclosporin A 23 cyclosporin A in an intestinal transplant recipient who required a very large dose

(1200 mg/dose, 3 times daily) of cyclosporin A to achieve acceptable blood concentration. They found that the oral bioavailability of cyclosporin A was 6% and P-gp was highly expressed at allograft ileum (Kaplan et al., 1999). Masuda et al. reported that a large oral dose of cyclosporin A (703.9 ± 385.4 mg/day, for 13 days) needs to be used in a recipient of living donor liver transplantation, whose intestinal mRNA level of P-gp was increased markedly (Masuda et al., 2003).

Cyclosporin A also has been used as one of the first generation multi-drug resistance modulators to reverse MDR and improve chemotherapy (Tan et al.,

2000). In addition, cyclosporin A also has been reported to be an inhibitor of P-gp, and involved in many drug-drug interactions (Sparreboom and Nooter, 2000), such as increasing the plasma concentration and decreasing the clearance of digoxin and etoposide (Carcel-Trullols et al., 2004; Englund et al., 2004). Cyclosporin A also increased the brain penetration of P-gp substrates nimodipine and verapamil (Liu et al., 2003; Sasongko et al., 2005). Shitara et al. reported that the transporter- mediated uptake of cerivastatin, a potent HMG-CoA reductase inhibitor (statin), is inhibited by cyclosporin A at a low concentration (Ki was 0.3 to 0.7 µM), whereas the in vitro metabolism of cerivastatin is inhibited with Ki 0.2 µM. They suggested that this drug-drug interaction was caused by a transporter-mediated process such as OATP2 mediated uptake rather than a metabolic process (Shitara et al., 2003).

Besides having interaction with P-gp, cyclosporin A has been found to modulate drug transport by MRP1 and BCRP by using cytometry in cells overexpressing 24

MRP1 and BCRP (Pawarode et al., 2007). In this thesis, cyclosporin A is used as a substrate for P-gp.

Digoxin

Digoxin (Figure 1.6) is an important cardiotonic drug having a very narrow therapeutic window of 0.64 to 2.56 nM (0.5 to 2.0 µg/L) (Mooradian, 1988). It is cleared by both the kidney and liver, and due to the large volume of distribution

(i.e. 4 to 7 L/kg) (Hanratty et al., 2000), digoxin exhibits a long half-life (30 to 40 hours) (Kramer et al., 1974; Kramer et al., 1979; Rodin and Johnson, 1988;

Westphal et al., 2000). The bioavailability of digoxin is approximately 90% from capsules and 70 to 80% from tablets (Smith, 1985). It has been reported that digoxin is predominantly excreted in the urine as unchanged form (Sumner and

Russell, 1976) and P-gp plays an important role in the excretion (Schinkel et al.,

1995b). However, the transport of digoxin into hepatocytes is by both passive diffusion and OATP 8 in human (Kullak-Ublick et al., 2001).

Digoxin-drug interactions have been reported (Okudaira et al., 1988; Rodin and Johnson, 1988; Fromm et al., 1999; Lau et al., 2004; Igel et al., 2007). For example, rifampin, an OATP2 inhibitor, has been reported to increase the AUC

(from 0 to 60 min) of digoxin from 3880 ± 210 nM · min to 5200 ± 240 nM · min in the recirculating liver preparation (Lau et al., 2004). Quinidine, an inhibitor of

P-gp, significantly increased AUC and Cmax of digoxin by 3-fold (p < 0.05) and 25

Figure 1.6 Chemical structure of digoxin

3.8-fold (p < 0.001), respectively in healthy human volunteers (Igel et al., 2007).

In Fromm et al. study, quinidine inhibited digoxin transport by 57% in Caco-2 cells, also increased plasma digoxin concentrations by 73% in wild type mice, compared with 19.5% in mdr1 knock out mice (Fromm et al., 1999). Oral administration of a cardioselective β-blocker talinolol, which is known as being secreted by P-gp into the lumen of the gastrointestinal tract, significantly increased the AUC of digoxin to 23% and significantly increased the maximum serum levels by 45% in humans (Westphal et al., 2000). Durr et al. reported that the AUC of digoxin was decreased by 18%, when St. John’s wort extract was given for 14 days in healthy human volunteers (Durr et al., 2000). It also has been reported that

P-gp significantly modified digoxin plasma concentration and the inhibition of P- gp can increase the toxicity of digoxin, such as nausea, disequilibrium, and vision alteration, in systemic exposure in humans (Hooymans and Merkus, 1985; Fromm et al., 1999; Westphal et al., 2000). In addition, digoxin is used as a standard substrate for P-gp to evaluate if other drugs or natural products can modulate P-gp.

For example, Gurley et al. used digoxin to determine whether supplementation with goldenseal or kava kava modified P-gp in twenty healthy volunteers.

Goldenseal or kava kava did not affect digoxin pharmacokinetics, which indicated that these supplements are not potent modulators of P-gp (Gurley et al., 2007). In an in vitro study, digoxin was used as the probe to investigate the inhibition profiles of new drug candidates (Rautio et al., 2006). Recently, digoxin was used as a model drug to estimate the human intestinal P-gp activity in relation to age, 27 gender and medical treatment (rifampicin) in 32 healthy people (Larsen et al.,

2007). Their results suggested that variations in P-gp activity attributable to age and gender are of minor importance compared to the intra-individual variation.

The results also indicated that rifampicin increases P-gp activity by increasing both

MDR1 mRNA and P-gp levels (Larsen et al., 2007). Because digoxin is a well established substrate of P-gp, digoxin is used as a substrate of P-gp in this thesis.

Ketoconazole

Ketoconazole (Figure 1.7) is an imidazole derivative which is effective against a wide range of fungal pathogens (Dixon et al., 1978; Odds et al., 1980) and for treating androgen-dependent diseases such as advanced prostate cancer

(Pont et al., 1984; Jubelirer and Hogan, 1989). It is almost insoluble in water, except at a pH lower than 3. Although the spectrum of activity of ketoconazole is similar to other imidazole derivatives, such as miconazole, econazole and clotrimazole, ketoconazole is effective when administered orally (Huang et al.,

1986). It is recommended that an adult take 200 mg or 400 mg ketoconazole daily.

The peak concentrations of ketoconazole for human was ranged from 8 µM to 19

µM for 200 mg/day dose (Huang et al., 1986) and 13 µM to 26 µM for 400 mg/day dose (Baxter et al., 1986). Ketoconazole is a known inhibitor of CYP3A4

(Ghosal et al., 1996) and it is used clinically to enhance drug absorption of 28

N Cl N

H2C Cl

O O O

O N N C CH3 C H2

Figure 1.7 Chemical structure of ketoconazole

CYP3A4 substrates such as the immunosuppressant cyclosporin A (First et al.,

1993; Odocha et al., 1996), antimalarial mefloquine (Ridtitid et al., 2005), and antipsychotic quetiapine (Grimm et al., 2006). Regarding the mechanism of modulating CYP3A4, it is shown that ketoconazole is able to block the pregane X receptor (PXR) activation, thereby repressing the coordinated activation of genes involved in ketoconazole metabolism (Wang et al., 2007).

Ketoconaozle has also been reported to inhibit the multi-drug resistance P- gp in various in vitro (Siegsmund et al., 1994; Yumoto et al., 1999; Wang et al.,

2002a) and in vivo models (Ward et al., 2004; Kageyama et al., 2005; Machavaram et al., 2006). For example, ketoconazole treatment (200 mg orally, once daily for 5 days) increased the Cmax, AUC, and t½ of ranitidine (a substrate for P-gp) by 78%,

74%, and 56%, respectively in human volunteers (Machavaram et al., 2006).

Ketoconazole was also used as a known P-gp inhibitor to assess the impact of P-gp efflux on the intestinal extraction of verapamil (Johnson et al., 2003). Because P- gp requires ATP to be functional, ketoconazole can directly inhibit complex I of the respiratory chain in the mitochondria thereby inhibiting ATP synthesis

(Rodriguez and Acosta, 1996).

Because ketoconazole is commonly used in combination with cyclosporin

A to reduce the overall cost of drug therapy by decreasing the dose of cyclosporin

A by inhibiting CYP3A4 and P-gp (Saad et al., 2006), and it is effective against a wide range of fungal pathogens and used to treat androgen-dependent diseases, it 30 was chosen as one of the interested pharmaceuticals to be evaluated on multidrug resistance mediated transport in this thesis.

Other drugs

MK-571

MK-571 (Figure 1.8) is a specific leukotriene D4 (LTD4) receptor antagonist, an anionic quinoline. It specifically inhibits at least MRP1 and MRP2, and has been shown to sensitize MRP1- and MRP2-expressing cell lines (Gekeler et al., 1995; van Asperen et al., 1997; Chen et al., 1999). In this thesis, MK-571 was used to inhibit MRPs.

Verapamil

Verapamil (Figure 1.9) is a calcium channel blocker. It has been used for many years for the treatment of cardiovascular diseases (Weiner et al., 1983;

Echizen et al., 1985; Reicher-Reiss and Barasch, 1991). Verapamil inhibits the function of P-gp, which makes malignant cells more susceptible to cytotoxic drugs

(Cass et al., 1989; Solary et al., 1991). Besides the functional inhibitory effect on

P-gp, Muller et al. demonstrated that verapamil can down-regulate the mdr1 gene in leukemic cell lines (Muller et al., 1994; Muller et al., 1995). In this thesis, verapamil was used to inhibit MDR1. 31

CH3 Cl N S N

CH3 S

O O Na

Figure 1.8 Chemical structure of MK-571

H3CO CN OCH3 CH3

H3CO C CH2CH2CH2 N CH CH OCH 2 2 3 H3C CH3

Figure 1.9 Chemical structure of verapamil

1.3.3 Examples of drug-diet interaction

Herbal medicines received a lot of attention by the scientific and clinical communities. In the European market, herbal medicines represent an important pharmaceutical market with annual sale of $7 billion and in the United States, the sale of herbal medicine increased from $200 million in 1988 to more than $3.3 billion in 1997 (Mahady, 2001). The opportunity for a diet-drug interaction is an everyday occurrence, especially when total drug absorption is altered. Potential interaction of herbal medicines with drugs is becoming a major safety concern, especially for drugs with narrow therapeutic window, such as warfarin and digoxin, because it may lead to severe adverse reactions that are sometimes life- threatening. Therefore, it is extremely important to characterize, analyze, and predict these diet-drug interactions.

Grapefruit juice

Grapefruit is a widely consumed fruit in the United States. It is popular for taste and health potential. Many reports of grapefruit juice-drug interactions have been published. For example, Bailey et al. reported that grapefruit juice increased the oral bioavailability of felodipine (a calcium channel antagonist) to higher than

2.5-fold compared with that seen with water (Bailey et al., 1989; Bailey et al.,

1991). The mean AUC of atorvastatin acid (metabolized by CYP) were found to 34 be increased by grapefruit juice by 83% in healthy subjects (Ando et al., 2005).

Furanocoumarin, a derivative identified from grapefruit juice, strongly inhibited the catalytic activity of CYP3A4 and caused the decrease of the first-pass metabolism of orally administered therapeutic drugs catalyzed by CYP3A4

(Fukuda et al., 1997; Guo et al., 2000; Tassaneeyakul et al., 2000). It also has been found that most drugs which are investigated for an interaction with grapefruit juice are substrates for CYP3A4, which include felodipine (Bailey et al., 2000;

Takanaga et al., 2000), cyclosporin A (Hollander et al., 1995; Mangano et al.,

2001), nisodipine (Bailey et al., 1993), nitrendipine (Soons et al., 1991), nifedipine

(Bailey et al., 1991; Rashid et al., 1995) and amlodipine (Josefsson et al., 1996).

Regarding its target, Lown et al. demonstrated that the target of grapefruit juice was specific to the small intestine compared to the liver, and the ingestion of grapefruit juice resulted in loss of enteric CYP3A4 protein, but no mRNA (Lown et al., 1997a). However, grapefruit juice produces less CYP3A inhibition as compared with highly potent inhibitors such as ketoconazole and ritonavir

(Greenblatt et al., 2000; Venkatakrishnan et al., 2000).

Besides producing mechanism-based inhibition of intestinal drug metabolism, grapefruit juice also inhibited intestinal P-gp-mediated efflux transport of drugs to increase its oral bioavailability, such as cyclosporin A.

However, it does not enhance the absorption of digoxin, which is also a known substrate of P-gp. This probably attribute to the inherent bioavailability of digoxin

(Dresser and Bailey, 2003). In Soldner et al. report, grapefruit juice was able to 35 increase the efflux ratio of CYP3A4/P-gp substrates, fexofenadine and losartan, whereas there is no effect on the efflux ratio of substrates of CYP3A4, such as felodipine and nifedipine, in MDCKII-MDR1 cells (Soldner et al., 1999). In addition, it has been shown that grapefruit juice can interact with other drug transporters as well. For instance, grapefruit juice decreased OATP 1A2-mediated drug-uptake transport in vitro by more than 50% (Dresser et al., 2002). However, grapefruit juice did not affect drug efflux transport by P-gp at concentrations 10- fold higher, which indicated that grapefruit juice may produce potent in vitro impairment of OATP1A2 relative to P-gp carrier-mediated transport. In human, grapefruit juice at commonly consumed quantity (300 ml) reduced the absorption of fexofenadine, a drug transported by OATP1A2 and P-gp, to 58% of that with water (Dresser et al., 2005), which suggested that grapefruit juice may interact with OATP1A2 and P-gp in human.

St. John’s Wort (Hypericum perforatum)

St. John’s Wort extract has been reported as a potent inducer of CYP2B6 and CYP3A4 in vitro, and the main effective component is hyperforin (Moore et al., 2000; Goodwin et al., 2001). Hyperforin is a potent ligand for the orphan nuclear receptor, pregnane X receptor (PXR) which can mediate activation of

PXR, up-regulating both CYP3A4 and ABCB1 (the gene for CYP3A4 and P-gp, respectively) and leading to reduced bioavailability of many drugs, such as midazolam (by 56%) and digoxin (by 18%) (Durr et al., 2000; Dresser et al., 36

2003). Some in vivo studies in mice, rats and humans indicated that St John’s Wort extract is able to modulate various CYP enzymes, such as CYP3A and CYP3E1

(Durr et al., 2000; Roby et al., 2000; Bray et al., 2002). For instance, administration of St. John’s Wort extract at 140 or 280 mg/kg/day in mice for 3 weeks resulted in a 2-fold increase in both the CYP3A and CYP3E1 activities

(Bray et al., 2002). St. John’s Wort may also be able to induce intestinal P-gp in vitro or in vivo (Durr et al., 2000; Perloff et al., 2001; Hennessy et al., 2002). St.

John’s Wort at 300 µg/ml and hypericin at 3 µM caused a 4- and 7- fold increase, respectively, in the expression of P-gp in LS-180 intestinal carcinoma cells and decreased accumulation of rhodamine 123 (P-gp substrate) after chronic exposure

(Perloff et al., 2001). Also, the administration of St. John’s Wort extract to rats

(1000 mg/kg) and to healthy human volunteers (300 mg/dose, 3 times/day) for 14 days resulted in a 3.8-fold, and 1.4-fold increase of intestinal P-gp expression, for rats and healthy human volunteers, respectively (Durr et al., 2000).

Because St. John’s Wort induces P-gp, it may reduce the efficacy of drugs which are substrates of P-gp. Therefore, self-medication with St. John’s Wort may cause treatment failure due to subtherapeutic plasma concentrations of many pharmaceuticals. For example, two renal transplant recipients with self- medication of St. John’s Wort consequently had sub-therapeutic concentrations of cyclosporin A. After discontinuation of St. John’s Wort, the patients’ cyclosporin

A concentrations returned to therapeutic levels (Barone et al., 2001). This 37 indicated that St. John’s Wort may cause significant reduction in plasma concentration of cyclosporin A.

Green tea (EGCG)

The consumption of tea is a very ancient habit. Legends from China and

India indicate that it was initiated about five thousand years ago. Historically, tea has been lauded for various beneficial health effects (Yang and Landau, 2000;

Riemersma et al., 2001; Rietveld and Wiseman, 2003; Siddiqui et al., 2004). Many people are interested in the oxidant/antioxidant effects for diseases such as cancer, coronary heart disease, and diabetes. The possible cancer-preventive potential of tea has received much attention because of the demonstrated anticancer activities of tea preparation in laboratory studies (Dreosti et al., 1997; Conney et al., 1999).

These studies indicate that polyphenols have a high antioxidant power which can protect cells against the adverse effects of reactive oxygen species. The compositions of green tea are listed in Table 1.3 (Pisters et al., 2001). Catechins are characterized by di- or tri-hydroxyl group substitution of four major catechins,

(-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC). Among these components, EGCG (Figure

1.10) is the major catechin in tea, which account for more than 50% of the total catechin in tea (McKay and Blumberg, 2002; Konishi et al., 2003). 38

Table 1.3 Composition of green tea extract capsules

Strength of capsule, mg 110 200 270 Composition of green tea extract, % Catechins, total 26.9 EGCG 13.2 EGC 8.3 ECG 3.3 EC 2.2 Caffeine 6.8 Protein 19.8 Lipid 0.1 Amino acids 4.5 Ash 10.8 Other (carbohydrate, flavonoid, etc.) 31.1

EGCG, (-)-epigallocatechin-3-gallate; EGC, (-)-epigallocatechin; ECG, (-)- epicatechin gallate; EC, (-)-epicatechin. Modified from Pisters et al., 2001.

HO O OH

OH OH O

Figure 1.10 Chemical structure of (-)-epigallocatechin-3-gallate (EGCG).

To determine the bioavailability of tea catechins in humans, 18 individuals were given various amounts of green tea, and the time-dependent plasma concentration and urinary excretion of tea catechins were evaluated (Yang et al.,

1998). After taking 1.5, 3.0, and 4.5 g of decaffeinated green tea solids (dissolved in 500 ml water), the maximum plasma concentration (Cmax) of EGCG was 0.71

µM (3.0 g), the Cmax of EC was 0.66 µM (4.5 g), and the Cmax of EGC was 1.80

µM (4.5 g). These Cmax values were observed at 1.4 – 2.4 hours after ingestion of the tea preparation. The half-life of EGCG was 5.0 to 5.5 hours and the half-life of

EGC or EC is 2.5 to 3.4 hours. EGC and EC, but not EGCG, were excreted in the urine.

EGCG and tea have been reported to have anti-tumor activity as well

(Kono et al., 1988; Xu et al., 1996; Pisters et al., 2001; Dashwood et al., 2002; Ju et al., 2005; Siddiqui et al., 2006). For example, EGCG at the dose of 0.08% or

0.16% in drinking fluid has been found to decrease the small intestinal tumor formation in Apc min/+ mice (a mouse model for human intestinal cancer) (Ju et al.,

2005). Green tea (2%, w/v) and black tea (1%, w/v) were able to inhibit colon carcinogenesis in rats exposed to the cooked meat heterocyclic amine 2-amino-3- methyl-imidazo[4, 5-f]quinoline (IQ) (Xu et al., 1996).

In addition, EGCG, the most abundant flavonoid in green tea, as well as

ECG, have been reported to modulate the transport of substrates for MDR1.

EGCG and ECG have been reported to reversibly inhibit the MDR1-mediated 41 transport of vinblastine (Jodoin et al., 2002). Authors also indicated that green tea polyphenols and their principal catechins inhibited the photolabelled P-gp and increased the accumulation of rhodamine-123 in multidrug resistant cells CHRC5.

In the study of Kitagawa et al., EGCG and ECG were found to increase the accumulation of rhodamine-123 and daunorubicin in P-gp over-expressing KB-C2 cells (Kitagawa et al., 2004). Mei et al. found that EGCG at 10 µg/ml enhanced doxorubicin cytotoxicity in the drug-resistant KB-A-1 cells by 2.5 times and they indicated that EGCG has reversal effects on the MDR phenotype in vitro (Mei et al., 2004). In this thesis, the Caco-2 and MDCKII-MDR1 cell drug transport models were used to investigate the effect of EGCG on drug efflux and also to characterize the relative potency of its interaction with MDR1 substrates, cyclosporin A and digoxin.

Hop (xanthohumol, Menohop®)

Hop (Humulus lupulus) has been used as an alternative treatment to the conventional method, such as hormone replacement therapy, for menopausal symptoms (Huntley, 2004; Heyerick et al., 2006). Hops has been studied for more than 50 years as a possible source of estrogenically active compounds (Koch and

Heim, 1953) with 8-prenylnaringenin being identified as a potent phytoestrogen

(Milligan et al., 1999).

Xanthohumol (Figure 1.11) is the most abundant flavonoid in Humulus lupulus and has antioxidative (Miranda et al., 2000a), anti-mutagenic (Miranda et al., 2000b), and chemopreventive activities (Miranda et al., 1999). It is present in hop flower extracts and in beer (Stevens et al., 1999; Stevens and Page, 2004); inhibits nitric oxide production in mouse macrophage RAW 264.7 cells (Casaschi et al., 2004); and inhibits triglyceride and apolipoprotein B secretion in HepG2 cells (Zhao et al., 2003). Direct binding assays of purified cytosolic nucleotide- binding domain of P-gp suggested that prenylated xanthones was able to modulate

MDR1-mediated transport (Bois et al., 1998; Bois et al., 1999; Tchamo et al.,

2000). In one of our previous papers, xanthohumol has been shown to increase the uptake of 3H-digoxin and to decrease the uptake of 3H-CSA in Caco-2 cells

(Rodriguez-Proteau et al., 2006). In addition, xanthohumol significantly inhibited the efflux permeability of CSA but not in digoxin transport, which indicated that xanthohumol affects the transport of CSA in a manner that is distinct from the digoxin efflux pathway (Rodriguez-Proteau et al., 2006). Recently, the transport and accumulation of xanthohumol were studied using Caco-2 cells and it has been found that the mechanism of its low bioavailability is due to the specific binding of xanthohumol to cytosolic proteins, such as Keap 1, in intestinal epithelial cells

(Pang et al., 2007). In this thesis, the modulation of the P-gp-mediated transport by xanthohumol was investigated using Caco-2 and MDCKII-MDR1 drug transport models. 43

OH OH HO O

Figure 1.11 Chemical structure of xanthohumol 44

Menohop®, a new supplement invented in Belgium, is produced from hop by a patented process. Menohop® is used to ease the menopausal transition period and inhibits the proliferation of breast and uterine cancer cells (Heyerick et al.,

2006). It contains isoxanthohumol, 8-prenylnaringenin, 6-prenylnaringenin and xanthohumol. Because Menohop® contains xanthohumol as its primary active component, we hypothesize that Menohop® might have the similar effect as xanthohumol in modulating P-gp mediated transport.

Oregon grape root (isoquinoline alkaloids, berberine, berbamine)

Oregon grape root (Mahonia aquifolia), which is also known by the names of barberry, mountain grape and holly-leaved barberry, has a direct action on the skin, such as treating psoriasis and eczema (Succar, 1999; Bernstein et al., 2006;

Donsky and Clarke, 2007). In folk medicine, it is used for chronic eruptions, rashes associated with pustules, and rashes associated with eating fatty foods

(Dattner, 2003). It also has been reported to be used for gallbladder disease (Moga,

2003).

Berberine (Figure 1.12) is one of the alkaloids found in the Oregon grape root and has been used for many years in traditional Eastern medicine as an effective medication for the treatment of gastro-enteritis and secretory diarrhea.

Other pharmacological effects reported for berberine and its related proberine

N MeO

OMe

Figure 1.12 Chemical structure of berberine 46 alkaloids include antimicrobial (Kaneda et al., 1991), antiarrhythmic (Sanchez-

Chapula, 1996), anticancer (Iizuka et al., 2000), anti-inflammatory (Ckless et al.,

1995; Kuo et al., 2004) and antiproliferative effects (Jantova et al., 2007).

Berbamine (Figure 1.13), another bisbenzylisoquinoline alkaloids, is widely used in traditional Chinese medicine as a source of leukogenic, anti-arrhythmic, antihypertensive, and anticancer activity (Liu et al., 1983; Shiraishi et al., 1987; Li et al., 1989).

Many studies have shown the interaction of berberine and berbamine with

P-gp (Tian and Pan, 1997; Lin et al., 1999a; Stermitz et al., 2000; He and Liu,

2002). For example, it has been reported that 24 hour berberine treatment up- regulated P-gp expression in human and murine hepatoma cells (Lin et al., 1999a) as well as being able to up-regulate P-gp expression in cultured bovine brain capillary endothelial cells (He and Liu, 2002). Berbamine has been found to down regulate the expression of MDR1 mRNA and P-gp after 72 hour treatment in human erythroleukemic cells by using reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry (Han et al., 2003) as well as having similar activity to verapamil in reversing MDR in breast cancer cell line (Tian and Pan,

1997). However, there are few publications showing the P-gp mediated drug-drug interaction with berberine (He and Liu, 2002; Maeng et al., 2002; Pan et al., 2002), whereas there are no publications discussing P-gp mediated drug-drug interaction with berbamine or the crude extract of their original source Oregon grape root.

OMe

N Me OMe

OMe O

Figure 1.13 Chemical structure of berbamine

In this thesis, we hypothesized that berberine, berbamine and Oregon grape root extract can modulate P-gp, thereby altering the apparent permeability of drugs that are substrates of P-gp, like cyclosporine A (CSA) and digoxin.

1.4 Methods for investigating the multidrug resistance mediated drug-drug interactions and drug-diet interactions

1.4.1 In vitro

Because possible toxicity may arise from the P-gp mediated drug-drug interactions and drug-diet interactions, it is important and necessary to develop effective in vitro screenings that identify interactions at initial stages in the drug development process as well as predict their effects in vivo. The following are the methods used commonly in cell-based models.

Caco-2 cell transport model

Caco-2 cells were originally used to model human intestinal absorption in the late 1980s (Hidalgo et al., 1989; Artursson et al., 2001). Later, this model has become the standard method for predicting human intestinal drug absorption and for mechanistic drug transport determination (Artursson, 1990). Caco-2 cells were originally isolated from a human colon adenocarcinoma that undergoes spontaneous enterocytic differentiation to mimic the epithelial cells of the human small intestine (Hilgers et al., 1990). Normally, Caco-2 cells reach confluency within 3-6 days and reach the stationary growth phase after 10 days in culture 49

(Braun et al., 2000). The differentiation is usually completed within 20 days. Fully differentiated Caco-2 cells form an epithelial membrane with a barrier function similar to the human colon (Artursson et al., 1993), but express carrier proteins similar to the small intestine (Hidalgo et al., 1989; Baker and Baker, 1992). The active transport systems for bile acids, amino acids, and sugars as well as P-gp, are functionally expressed (Audus et al., 1990; Gutmann et al., 1999; Walle et al.,

1999; Hirohashi et al., 2000; Taipalensuu et al., 2001). The apparent permeability coefficients (Papp) can be determined for the specific compounds which have a good correlation with in vivo absorption (Artursson and Karlsson, 1991; Gres et al., 1998). As a result of these properties, the Caco-2 cell model has become a reliable and predictive tool to infer possible adverse effects in vivo and allows for the prediction of oral drug availability (Gres et al., 1998; Artursson et al., 2001).

For transport experiments, Caco-2 cells normally are cultured on a semipermeable polycarbonate surface on the inserts that establishing apical and basolateral chambers (Figure 1.14). The apical and basolateral chambers represent the luminal side and blood supply of the gastrointestinal tract, respectively. In order to evaluate the integrity of the cell monolayers, the transepithelial electrical resistance (TEER) is measured before and after an experiment to evaluate the integrity of the cellular junctions (Yee, 1997). Studies are typically performed with low passage numbers (21-30), because as the passage number of Caco-2 cells increases, there is a reduction in the functional expression of a brush border 50

Apical side (A): Intestinal lumen Transwell® Insert

A Culture Medium B Basolateral side (B): Blood supply

= Caco-2/MDCKII Cells

Figure 1.14 The Transwell® plate with inserts

51 enzyme and several transport proteins (Yu et al., 1997; Anderle et al., 2003;

Behrens et al., 2004; Mizuma et al., 2004). The high passages of Caco-2 cells will have less morphological heterogeneity, higher trans-epithelial electrical resistance and lower carrier-mediated transport (Yu et al., 1997).

Typically, the method for measuring the apparent permeability coefficient in Caco-2 cells is with radiolabelled compounds (Gres et al., 1998). However, radiolabelled compounds are expensive to synthesize and the disposal of radioactive waste is expensive. Now, high performance liquid chromatography

(HPLC) coupled with ultraviolet (UV) or fluorescence detection is used for quantification (Ucpinar and Stavchansky, 2003), but this approach requires that the compounds have a strong UV or fluorescent chromophore for adequate sensitivity

(Wang et al., 2000; Kerns and Di, 2006; Marzo and Bo, 2007).

The quantification of compound transported in the Caco-2 cells has been improved with the application of liquid chromatography-mass spectrometry (LC-

MS) (Caldwell et al., 1998) and LC-MS-MS (Stevenson et al., 1999; Wang et al.,

2000). Compared with the radiolabelling approach and HPLC methods, LC-MS and LC-MS-MS are faster and more sensitive. For instance, chromatographic separations have been developed for LC-MS-MS analysis of samples from Caco-2 cell that have a throughput of 2 min/sample (Romanyshyn et al., 2000). Another advantage of these mass spectrometry based methods is that it is able to distinguish the test compounds from their metabolites. In addition, MS-MS also facilitates the 52 identification of these metabolites, so that one can add another dimension to the information that obtained using Caco-2 cells (Li et al., 2003).

MDCK and LLC cell transport models

Recombinant cellular in vitro drug transport models have become important tools for drug development to predict drug position in human. Such cellular models are facilitated the determination of substrate specificities and the kinetics of hepatic and renal drug transporters. Models have been established using polarized epithelial cell lines such as the porcine kidney-derived LLC-PK1 and the canine kidney-derived Madin Darby canine kidney (MDCK) cells. These enable directional drug transport studies and simulate hepatic and renal drug transport.

Examples where these cells have been used include the expression of human

MDR1 in LLC-PK1 (Schinkel et al., 1995a) and MDCK cells (Pastan et al., 1988); expression of human MRP2 in LLC-PK1 (Chen et al., 1999), MDCK (Cui et al.,

1999), and MDCKII cells (Jacquemin et al., 1994); and the expression of human

OATP8 in MDCKII cells (Konig et al., 2000).

The MDCK cells are one of the most common epithelial cell lines for studying cell growth regulation and metabolism (Rabito, 1986; Mendoza, 1988).

Since the late 1990s, these cells have been used for studying transport mechanisms in renal epithelia (Hunter et al., 1993a; Irvine et al., 1999). When cultured on semipermeable supports, MDCK cells will differentiate into columnar epithelial cells with well-formed tight junctions (Irvine et al., 1999). In the parental MDCK cells, 53 there is no active transport of large neutral amino acids and bile acids, whereas, transport of monocarboxylic acids and small peptides was active (Putnam et al.,

2002a; Putnam et al., 2002b). MDCK cell model has been reported as a good in vitro model for drug transport and interaction studies with drugs (Rothen-

Rutishauser et al., 1998). The MDCK II cells are the subclone of MDCK cells and have been well characterized to overexpress human MDR1, MRP1, MRP2 (Evers et al., 1998; Tang et al., 2002) and are useful models for drug transport for the comparison with Caco-2 and MDCKII wild-type cells (Tang et al., 2002).

MDCKII-MDR1 cells have been used for transfection with the MDR1 gene, which codes for P-gp. It has been shown to be a good in vitro model for determining whether compounds interact with P-gp (Polli et al., 2001; Tang et al.,

2002; Taub et al., 2005). The main advantage of using MDCKII cells is the minimal culturing time of 7 days before using for experimentation, whereas Caco-

2 cells require 21 days. Because there are several transporters involved in Caco-2 cells and in order to investigate if P-gp is involved in drug transport, MDCKII-

MDR1 cells were used in this thesis.

Multidrug Resistance (MDR) Assay

The VybrantTM Multidrug resistance assay (Figure 1.15), is based on the fluorescence micro-plate method developed by Tiberghien and Loor (Tiberghien and Loor, 1996). It provides a rapid and simple method for large-scale screening of

MDR inhibitors. This assay utilizes the fluorogenic dye calcein acetoxymethyl 54

Cell membrane Normal cell

Esterase Calcein AM Calcein

MDR protein

Esterase Calcein AM Calcein

MDR cell

Figure 1.15 Principle of the VybrantTM Multidrug Resistance (MDR) Assay

ester (calcein AM) as a substrate for the efflux protein. Calcein AM is non- fluorescent, and it can rapidly penetrate the plasma membrane of normal cells.

Once inside the cell, it can be cleaved by endogenous esterases, and transform into calcein, which is fluorescent. In MDR cells, there are high levels of multidrug resistance efflux proteins and they can extrude non-fluorescent calcein AM from the plasma membrane, reducing accumulation of fluorescent calcein in the cytosol

(Figure 1.15). By measuring the increase in intracellular calcein fluorescence, the degree of inhibition of the activity of multidrug resistance protein can be quantitated. This assay is commonly used to screen the interaction of compounds with P-gp. For instance, the inhibitory effect of kava-kava (a perennial shrub native to the South Pacific Islands and traditionally used for calming and relaxing effects) on P-gp activity has been investigated using calcein uptake assay to see the effect of kava-kava on the uptake of calcein-AM in murine monocytic leukemia

P388 cells (Weiss et al., 2005). Calcein inhibition assay was also used to investigate the potential interaction of drug candidates with P-gp (Rautio et al.,

2006). Furthermore, calcein-AM based assay was used to screen compounds for P- gp interactions in blood-brain barrier (Bubik et al., 2006). In this thesis, this assay was used as one of the methods to investigate the interaction of ketoconazole with

P-gp as well.

Quantitative real time RT-PCR

Real time reverse transcriptase polymerase chain reaction (RT-PCR) quantitates the differences in mRNA expression. This method is particularly valuable when amounts of RNA are low, because it requires amplification.

One of the mechanisms of modulating P-gp is through activating pregane X receptor (PXR). In this thesis, real time RT-PCR was used to look at the effect of

Oregon grape root extract on alteration of mRNA level of P-gp in different cells, then investigate whether P-gp is modulated through activating PXR pathway or not.

1.4.2 In vivo

In addition to the in vitro methods, the in vivo methods are usually used afterwards to investigate the biological effect to correlate with in vitro mechanistic studies to fully understand the overall interactions. Generally, the in vivo models include the in situ/ex vivo models (isolated perfused organs) (Dagenais et al., 2000;

Cisternino et al., 2001; Lau et al., 2004), and in vivo knockout mice models

(Schinkel et al., 1997; Jonker et al., 2001; Sweet et al., 2002).

2. KETOCONAZOLE MODULATES MULTIDRUG RESISTANCE- MEDIATED TRANSPORT IN CACO-2 AND MDCKII-MDR1 DRUG TRANSPORT MODELS

Ying Fan & Rosita Rodriguez-Proteau

Xenobiotica, 2007, in press

2.1 Abstract

1. The hypothesis tested was that ketoconazole can modulate P-glycoprotein, thereby, altering cellular uptake and apparent permeability (Papp) of multidrug- resistant substrates, such as cyclosporin A (CSA) and digoxin, across Caco-2,

MDCKII-MDR1, and MDCKII-wild type cell transport models.

2. 3H-CSA/3H-digoxin transport experiments were performed with and without co- exposure to ketoconazole and 3H-ketoconzole transport experiments were performed with and without co-exposure to dietary flavonoids, epigallocatechin-3- gallate and xanthohumol. Ketoconazole (3 µM) reduced the Papp efflux of CSA and digoxin from 5.07 × 10-6 to 2.91 × 10-6 cm/sec and from 2.60 × 10-6 to 1.41 ×

10-6 cm/sec, respectively, in Caco-2 cells. In the MDCKII-MDR1 cells, ketoconazole reduced the Papp efflux of CSA and increased the Papp absorption of digoxin. Cellular uptake of ketoconazole in the Caco-2 cells was significantly inhibited by CSA and digoxin whereas epigallocatechin-3-gallate and xanthohumol exhibited biphasic responses.

3. In conclusion, ketoconazole modulates the Papp of P-glycoprotein substrates by interacting with MDR1 protein. Epigallocatechin-3-gallate and xanthohumol modulate the transport and uptake of ketoconazole. 59

2.2 Introduction

Ketoconazole (KT) is a prominent broad-spectrum oral antifungal agent used in the treatment of systemic mycoses (Ringel, 1990) as well as for the treatment of androgen-dependent diseases such as advanced prostate cancer (Pont et al., 1984; Jubelirer and Hogan, 1989). KT has been well established as an inhibitor of cytochrome P450 3A4 (CYP3A4) (Ghosal et al., 1996) which is taken advantage of by clinicians to enhance drug absorption of CYP3A4 substrates such as the immunosuppressant cyclosporin A (CSA) (First et al., 1993; Odocha et al.,

1996). KT has also been reported to inhibit the multi-drug resistance (MDR) P- glycoprotein (P-gp) in various in vitro models (Siegsmund et al., 1994; Yumoto et al., 1999; Wang et al., 2002a) as well as in rats (Kageyama et al., 2005), monkeys

(Ward et al., 2004), and humans (Machavaram et al., 2006). Because P-gp requires

ATP to be functional, KT can indirectly inhibit P-gp function by directly inhibiting complex I of the respiratory chain in the mitochondria thereby inhibiting ATP synthesis (Rodriguez and Acosta, 1996). As for KT being a substrate of P-gp, this is still questionable. A study using mdr1a deficient mice suggests that KT is not a

P-gp substrate (Kim et al., 1999) whereas another study using immobilized P-gp from a MDA435/LCC6MDR1 cell line on an open tubular column shows KT to be a moderate-low affinity P-gp substrate (Moaddel et al., 2004).

The study of P-gp and multi-drug resistant proteins (MRPs) is of great importance because these efflux transporters are present in the intestinal tissue that 60 is directly exposed to orally administered drugs and the dietary milieu. P-gp is known for its importance in the body's cellular defense mechanism against environmental carcinogens and toxins within our diet by pumping them out of the intestinal cell back into the gut lumen before it is completely absorbed (Hunter and

Hirst, 1997; Sun et al., 2004). MRPs confer resistance to a large variety of drugs and contribute to cellular efflux of endogenous anionic glutathione, glucuronide conjugates and cyclic nucleotides (Evers et al., 2000). Among various forms of these transporters, P-gp plays a major role in the disposition of many drugs

(Germann, 1996) and it has been shown to be important in reducing gastrointestinal absorption of drugs that are P-gp substrates such as CSA (Schinkel et al., 1995b) and digoxin, a cardiac glycoside (de Lannoy and Silverman, 1992;

Schinkel et al., 1995b).

Drug-drug interactions of KT with CSA has been evaluated in the current study because KT is commonly used concomitantly with CSA to reduce the overall cost of drug therapy by decreasing the dose of CSA administered by inhibiting CYP3A4 as well as P-gp (Saad et al., 2006). We also evaluated the interaction of KT and digoxin because digoxin has a narrow therapeutic index, is a substrate of P-gp, is commonly used in the assessment of P-gp-mediated transport; and, is poorly metabolized by the intestine (de Lannoy and Silverman, 1992).

Thus, inhibition of intestinal P-gp by KT can increase oral drug absorption of CSA and digoxin, thereby enhancing therapeutic efficacy or eliciting adverse effects, respectively. 61

The drug-diet interaction of KT with epigallocatechin-3-gallate (EGCG) was evaluated because EGCG is the most abundant flavonoid in green tea and one of the most common ingredients of several dietary supplements and vitamins

(Nagle et al., 2006; Seeram et al., 2006). Similarly, MenoHop®, which contains xanthohumol (XN) the most abundant prenylated flavonoid in Humulus lupulus and an ingredient that imparts flavor and bitterness to beer, was evaluated because it is the first “hop” food supplement available on the market. MenoHop® eases the menopause transition period in women and inhibits the proliferation of breast and uterine cancer cells (Heyerick et al., 2006). Moreover, our previous work has shown XN to be a potent inhibitor of the MDR1-mediated efflux of CSA but not digoxin in Caco-2 and MDCKII-MDR1 cells (Rodriguez-Proteau et al., 2006).

Caco-2 cells have been shown to be a predictive tool to infer possible adverse effects in vivo and allows for direct examination of the dynamics of drug absorption and efflux across polarized epithelial cells for prediction of oral bioavailability (Gres et al., 1998; Artursson et al., 2001). mRNA expression levels of efflux proteins (i.e., MDR1-3, MRP1-6) in the jejunum and Caco-2 correlate well to each other (Taipalensuu et al., 2001) as well as being functional (Hunter et al., 1993b; Gutmann et al., 1999). Madin-Darby Canine Kidney (MDCK) cells that overexpress human MRP1-3, MRP5, and MDR1 have been well characterized and are useful models for the maintenance of specialized cell surfaces (Louvard, 1980), as well as for drug transport for comparison to the Caco-2 and MDCKII-wild type cells (Tang et al., 2002). 62

Herein, we have conducted a study: i) to evaluate the uptake and transport of KT using the Caco-2 and MDCKII-MDR1 drug transport models; ii) to characterize relative potency of the KT-drug interaction with P-gp substrates, CSA and digoxin; and iii) to evaluate the potential KT-flavonoid interaction with EGCG and

XN because many humans consume either green tea-, green tea extracts-, or XN- containing products on a daily basis.

2.3 Materials and Methods

2.3.1 Chemicals

Radiolabeled 3H-ketoconazole (KT, 5.0 Ci/mmol, 99% purity) was obtained from American Radiolabeled Chemicals Inc (St. Louis, MO, USA). 3H-

Cyclosporin A (CSA, 8.0 Ci/mmol, 98.8% purity) and 3H-digoxin (37 Ci/mmol, >

97% purity) were obtained from Amersham, Inc. (Piscataway, NJ, USA). KT was obtained from Janssen Biotech N.V. (Titusville, NJ, USA). Xanthohumol (XN, >

98% purity) that was isolated, purified, and characterized by Dr. Fred Stevens was used for these studies (Stevens et al., 1999). MK-571 was obtained from Cayman

Chemical Co. (Ann Arbor, MI, USA). Epigallocatechin-3-gallate (EGCG), verapamil, CSA, and digoxin were obtained from Sigma, Inc. (St. Louis, MO,

USA). Foetal bovine serum (FBS) was purchased from Hyclone (Logan, UT,

USA). Dulbecco’s Modified Eagle Medium (DMEM) and non-essential amino acids were obtained from GibcoBRL (Rockville, MD, USA). All other reagents were analytical grade.

2.3.2 Cell culture

The Caco-2 cell line was obtained from American Type Culture Collection

(Rockville, MD, USA). The MDCKII-MDR1 and MDCKII-wild type cell lines were generous gifts from Dr. Piet Borst (The Netherlands Cancer Research

Institute). Caco-2 and MDCKII cells were maintained in a humidified chamber at 64

37 °C in air with 5% CO2, and 95% relative humidity with DMEM, 10% FBS, 0.1 mM non-essential amino acids, and 0.01% penicillin/streptomycin (Evers et al.,

1996). Cells were seeded, 300,000 cells/well, onto Transwell® inserts (Corning

Costar, Cambridge, MA, USA) and maintained until use on days 21 to 25 (Caco-2, passages 23 to 30) and 6 to 7 days (MDCKII-MDR1 and MDCKII wild-type).

The integrity of the monolayers was assessed by measuring transepithelial electrical resistance (TEER) using a World Precision Instrument, EVOM,

(Sarasota, FL) and evaluating 3H-mannitol transport. The average TEER readings were 962.0 ± 113.9 Ωcm2 (Caco-2), 833.3 ± 70.9 Ω cm2 (MDCKII-MDR1), and

623.2 ± 49.4 Ω cm2 (MDCKII wild-type). Also, permeability studies with 3H- mannitol were performed with the various treatment conditions and the transport

-6 - rate was less than 1% per hour and the Papp was 2.95 ± 0.98 ×10 , 3.24 ± 0.22 ×10

6, and 2.96 ± 0.48 ×10-6 cm/sec for the Caco-2, MDCKII-MDR1, and MDCKII- wild type cells, respectively, throughout the entire experiment. These data indicate that the cell monolayers were not comprised during the experiment. Lastly, dose- and time-response cytotoxicity studies, MTT and lactate dehydrogenase assays, were performed with all compounds used in this study in the Caco-2 and MDCKII cell lines. There was no cytotoxicity seen with any of the drug concentrations used for these transport and uptake studies in the Caco-2 and MDCKII cell lines (data not shown). 65

2.3.3 Transport studies

Transport of 3H labeled compounds (KT, CSA, and digoxin) were studied using a transport buffer consisting of Hanks’ buffered salt solution (HBSS) with

10 mM HEPES and 25 mM D-glucose at a pH of 6.8 for the apical (A) compartment and 7.4 for the basolateral (B) compartment. During experiments, the cells were maintained at 37 °C in air, 5% CO2, and 95% relative humidity. The cells were washed 3 times with transport buffer and allowed to equilibrate for 30 min before adding the test compounds. The concentrations of the MDR1 substrates were 0.5 µM for both CSA and digoxin. For the KT studies, the concentrations of

KT (1.0 µCi) used were 0.03 µM, 0.1 µM, 0.3 µM, 1.0 µM and 3.0 µM. For the flavonoid studies, 1.0 µM (1.0 µCi) KT was used with 0.3 µM, 1.0 µM, 3.0 µM,

10 µM and 30 µM for both EGCG and XN. Stock concentrations of EGCG and

XN were prepared fresh the day of the experiment and kept away from direct light.

The transporter inhibitors used were 100 µM verapamil and 50 µM MK-571. KT,

EGCG, XN and the transporter inhibitors were prepared in transport buffer at the appropriate pH (pH 6.8 for the A compartment and 7.4 for the B compartment) and allowed to incubate at 37 °C prior to the start of the experiment for 30 min. KT and the inhibitors were present in both the A and B chambers during the transport of the P-gp substrates (CSA and digoxin). A dose response analysis for KT (1.0

µCi) was performed to determine if KT could inhibit its own transport. KT 66 concentrations tested were 0.03, 0.1, 0.3, 1.0 and 3.0 µM. Ethanol was used as the solvent vehicle (0.1% v/v) for CSA and XN and DMSO (0.1%v/v) was used as the solvent vehicle for digoxin. Each experiment was performed in duplicate and repeated twice. For “non-sink” conditions, 100 µl aliquots were taken from the donor and receiver chambers at time “zero” and were taken from the receiver chamber at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 hr. An equal volume of 100 µl was replaced in the chamber at each time point. Non-sink conditions are used when ≤

10% of compound is transported across the cells during the transport experiment such that there is always a concentration gradient present. All experiments using

3H-CSA from the A to B direction were performed in non-sink conditions. For

“sink” condition experiments, the entire receiver volume was replaced at each time point and a 100 µl aliquot was taken from the receiver chamber for scintillation counting. A 100 µl sample was taken from the donor chamber at the last time point in order to calculate the mass balance of radioactivity to determine if adsorption to the cell culture transport apparatus had occurred. All transport experiments with 3H-digoxin and 3H-CSA from the B to A direction were performed under sink conditions. Each sample was placed in 0.9 ml of scintillation fluor (Cytoscint ES, ICN, Cosa Mesa, CA, USA) and read on a

Beckman LS 6500 (Palo Alto, CA, USA) scintillation counter for 3H activity.

The apparent permeability (Papp) was calculated using the following equation (Taub et al., 2005):

Papp= dQ/dt × 1/(A × C0) 67

2 Where A is the surface area of the monolayer (4.71 cm ) and C0 is the initial concentration of radiolabeled probe substrate in the donor compartment. dQ/dt is the slope of the steady-state rate constant.

Efflux ratio was determined by dividing the Papp in the B to A direction by the Papp in the A to B direction (Crivori et al., 2006):

Papp (B to A) Efflux ratio =

Papp (A to B)

2.3.4 MDR assay

The VybrantTM Multidrug Resistance (MDR) Assay (Molecular Probes,

Eugene, OR, USA) uses the fluorogenic dye, calcein-acetoxymethylester (calcein-

AM) which gets hydrolyzed to calcein, a fluorescent substrate of P-gp and MRP

(Essodaigui et al., 1998). This assay is based on the fluorescence microplate method in which calcein-AM is a highly lipid soluble dye that penetrates the plasma membrane of normal cells (Tiberghien and Loor, 1996). Once calcein-AM is inside the cell, the ester bond is cleaved resulting in the hydrophilic and fluorescent calcein (Feller et al., 1995; Essodaigui et al., 1998). We have used the protocol suggested by the manufacturer with minor modifications. Briefly, 50 µl of

KT was added to a flat bottom 96-well microplate (Falcon, Franklin Lakes, NJ,

USA) at final concentrations of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 30, and 100 µM. Phosphate buffered saline (PBS) was included as a control. Caco-2 68 cells were grown in T-162 flasks until confluent. The cells were washed two times with PBS and scraped from the flasks with a disposable cell lifter. A dilution of 5

×105 cells per 100 µl was made and added to each well. The microplate was mixed and incubated at 37 oC for 15 min. Calcein-AM (Molecular Probes, Eugene

OR, USA), 1.0 mM, was diluted to 1.0 µM in PBS and 50 µl was added to each well of the microplate to achieve a final concentration of 0.25 µM. The reagents were mixed by gentle shaking and incubated at 37 °C for 15 min. Then, the microplate was centrifuged at 200 × g for 5 min. The cells were washed by removing the supernatant and adding 200 µl of 4 oC tissue culture media. This latter step was repeated a total of three times with the last wash containing the resuspended cells in 200 µl. The calcein retention was measured by fluorescence at 494 nm excitation and 517 nm emission using a Molecular Devices fluorescence microplate reader (Spectromax Gemini Sunnyvale, CA, USA).

2.3.5 Uptake studies

The fluorescence microplate method above was adapted to use 3H-KT as the P-gp substrate rather than calcein-AM (Rodriguez-Proteau et al., 2006).

Briefly, 50 µl of EGCG or XN or P-gp substrates (CSA or digoxin) were added to a round bottom 96-well microplate (Falcon, Franklin Lakes, NJ, USA) to achieve final concentrations of 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 30, and100 µM.

PBS was included as a control. Approximately 5 × 105 cells (Caco-2 or MDCKII- 69

MDR1) were added in 100 µl volumes of cell culture media to each well, mixed, and incubated at 37 °C for 15 min. 3H-KT (0.1 µCi, 1.0 µM) was added to each well of the microplate. The reagents were mixed by gentle shaking and incubated at 37 °C for 15 min. The microplate was then centrifuged for 5 min at 200 × g and the supernatant was removed. The cells were washed 3 times with 100 µl 4 ˚C tissue culture media with the last wash containing the cells resuspended in 200 µl media. The 200 µl resuspension of cells was placed in 1.8 ml of scintillation fluor

(Cytoscint ES, ICN, Cosa Mesa, CA, USA) and read on a Beckman LS 6500 (Palo

Alto, CA, USA) scintillation counter for 3H activity.

2.3.6 Statistical analysis

All values are presented as a mean ± standard deviation (SD). The percent of drug transported during the experiment for the various treatments and the Papp values within treatment groups were compared using one-way ANOVA followed by a Dunnett’s post-hoc test. Differences between A and B transport were compared using an unpaired t-test. Tukey’s multiple comparison was performed for the comparison of various concentrations of KT with 0.03 µM KT. A probability of difference less than 0.05 (p < 0.05) was considered to be statistically significant.

2.4 Results

Because KT has been shown to be a modest inhibitor of MDR1-mediated transport (Takano et al., 1998; Ayrton and Morgan, 2001), we wanted to determine if carrier-mediated transport is involved in the transport of KT across the Caco-2 cells. The cumulative amount of 3H-KT was evaluated as a function of time

(Figure 2.1A). The transport of 1 µM 3H-KT was observed to be faster in the B Æ

A direction compared with the A Æ B direction. Figure 2.1B evaluates the effect of increasing concentrations of KT (0.03, 0.1, 0.3, 1.0 and 3.0 µM) on its own transport. The apparent permeability (Papp) of KT in the B Æ A direction was significantly greater, p < 0.01, compared with the A Æ B direction which indicates that KT was actively effluxed from the cells. There was also an increase in the efflux of KT with increasing concentrations of KT which suggests that KT can significantly enhance its own efflux at concentrations ≥ 3 µM when compared to

0.03 µM KT, p < 0.05. Next, the MDCKII-MDR1 cells were used to evaluate

MDR1-mediated transport of KT. As seen with the Caco-2 cells, KT significantly enhances its own efflux but at lower concentrations, 0.3 µM KT, while having no effect at higher concentrations (Figure 2.2). However, there was significant absorption at 0.3 and 10.0 µM KT when compared to 0.03 µM KT. As for the controls, 100 µM verapamil, P-gp inhibitor, significantly inhibited the efflux of 1

µM KT by 66.3% and 40.4% in the Caco-2 and MDCKII-MDR1 cells, 71

A 300 B to A A to B

200

100

0 0 50 100 150 200 Time (min) B

30 B to A A to B *

0 10 0 10 1 10 2 10 3 10 4 Ketoconazole (nM)

Figure 2.1 Effect of increasing concentrations of ketoconazole, 0.03, 0.1, 0.3, 1.0, and 3.0 µM, on 1 µCi 3H-ketoconazole across Caco-2 cells for the apical (A) to basolateral (B) and B to A transport of ketoconazole

Panel A: Cumulative amount of 1 µM 3H- ketoconazole transport for both transport directions. Panel B: Apparent permeability (Papp) of 0.03, 0.1, 0.3, 1.0 and 3.0 µM 3H- ketoconazole for both transport directions. Data represents mean ± standard deviation, n = 4. Statistical significance from 0.03 µM ketoconazole is shown with asterisks: *, p < 0.05. 72

MDCKII-MDR1

3 A to B B to A *

* **

Figure 2.2 Effect of increasing concentrations of ketoconazole (KT), 0.03, 0.1, 0.3, 3 1.0, 3.0 and 10.0 µM, on the apparent permeability (Papp) of 1 µCi H-KT across MDCKII-MDR1 cells for the apical (A) to basolateral (B) and B to A transport of KT

Data represents mean ± standard deviation, n = 4. Statistical significance from 0.03 µM KT is shown with asterisks: *, p < 0.05, and **, p < 0.01. 73 respectively (data not shown). Also, 50 µM MK-571, an inhibitor of MRP, inhibited the efflux of 1 µM KT by 54.8% (p < 0.05) and 19.3% in the Caco-2 and

MDCKII-MDR1 cells, respectively (data not shown). Thus, KT appears to be a substrate for both the MDR1- and MRP-mediated transport.

To evaluate the effect of KT on MDR1-mediated transport, CSA and digoxin were chosen in this study because they are well-known substrates for the intestinal efflux transporters in the Caco-2 drug transport model and in vivo

(Augustijns, 1996). Figure 2.3 shows the effect of KT on the transport of 3H-CSA and 3H-digoxin in Caco-2 cells. Figure 2.3A shows that increasing concentrations of KT (0.03, 0.1, 0.3, 1.0 and 3.0 µM) significantly decreased the Papp of CSA from the B Æ A direction (p < 0.05) with 1.0 and 3.0 µM KT having the greatest effect (p < 0.01) when compared to control conditions. As shown in Figure 2.3B,

3 µM KT significantly decreased the Papp of digoxin (p < 0.01) from the B Æ A direction when compared with control conditions. These observations suggest that

KT can effectively inhibit the efflux transporters involved in the transport of CSA and digoxin with KT having a higher affinity to transporter(s) utilized by CSA.

Because there are many transporters expressed in Caco-2 cells, such as acid-base transporters (Osypiw et al., 1994), amino acid and peptide transporters

(Thwaites et al., 1994), and efflux pumps like P-gp, MRP2, breast cancer resistance protein (Hilgendorf et al., 2000; Ebert et al., 2005; Balimane et al.,

2006) among many others, we wanted to evaluate if KT can specifically modulate

MDR1-mediated transport using the MDR1-overexpressing MDCKII-MDR1 cells. 74

CACO-2

A. Cyclosporin A

B to A 5.5 5.0 * * * 4.5 ** 4.0 3.5 ** 3.0 2.5 2.0 1.5 1.0 0.5 0.0

B. Digoxin

3 B to A

2 *** **

Figure 2.3 Effects of ketoconazole (KT) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cells. Panel A: Effects of 0.03, 0.1, 0.3, 1.0, and 3.0 µM KT on the transport of CSA from the basolateral (B) to the apical (A) direction. Panel B: The effect of 0.03, 0.1, 0.3, 1.0, and 3.0 µM KT on the transport of digoxin from B to A direction. All data represents mean ± standard deviation, n = 4. Statistical significance from 0.5 µM CSA or 0.5 µM digoxin controls are shown with asterisks, **, p < 0.01, and *, p < 0.05. 75

We employed 1 µCi of 0.5 µM 3H-CSA or 0.5 µM 3H-digoxin alone or in combination with KT (0.03, 0.3 and 3 µM), 100 µM verapamil (inhibitor of

MDR1) and 50 µM MK-571 (inhibitor of MRP2). As shown in Figure 2.4A, 100

µM verapamil and 50 µM MK-571 significantly reduced the Papp of CSA from the

B Æ A direction (p < 0.05) in the MDCKII-MDR1 cells. KT was able to significantly decrease the Papp of CSA from the B Æ A direction at concentrations as low as 0.03 µM KT (p < 0.01). In contrast, KT did not have any significant effect on the efflux of 0.5 µM digoxin at all concentrations evaluated (Figure

2.4B); however, the Papp of digoxin from A Æ B direction was significantly increased at ≥ 0.3 µM KT, p < 0.05, (data not shown).

To compare the effect of KT modulating the efflux of CSA and digoxin, efflux ratios of the Papp of the B to A and the A to B transport across Caco-2 and

MDCKII-MDR1 cell monolayers were determined (Table 2.1). KT significantly decreased the efflux ratios of CSA and digoxin in both the Caco-2 and MDCKII-

MDR1 cells (p < 0.01). The effect was seen as low as 0.03 µM KT for CSA in both Caco-2 (6.99 to 4.4) and MDCKII-MDR1 cells (6.67 to 3.09). For the digoxin, significance was seen at 3 µM for the Caco-2 cells (3.64 to 1.44) and 0.3

µM for the MDCKII-MDR1 cells (7.83 to 3.77). This suggests that KT has a greater effect in inhibiting the efflux of digoxin in MDCKII-MDR1 cells than in the Caco-2 cells.

As a control for the MDR1-overexpressing MDCKII-MDR1 cell line, experiments using the MDCKII-wild type cells under the same experimental 76

MDCKII-MDR1

A. Cyclosporin A

20 )

-6 B to A 15 *

10 ** ** ** ** (cm/sec 10 x

app 5 P

T T T SA K K C M M M µ µ µ 3 5 0 0.3 3.0 µM K 0. + + 0. + 50 µM MK-571 + + 100 µM verapamil B. Digoxin 3 B to A ) -6

2 *

1 (cm/sec x 10 (cm/sec app P

0 1 T in 7 K KT ox K-5 M KT M µ µM dig .3 µM verapamil .03 0 3.0 0 + + µM M 0 µM + 5 0.5 + + 100 µ Figure 2.4 Effects of ketoconazole (KT) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII-MDR1 cells. Panel A: The effect of 100 µM verapamil, 50 µM MK-571, and KT (0.03, 0.3, and 3.0 µM) on the transport of CSA from the basolateral, B, to the apical, A, direction. Panel B: The effect of 100 µM verapamil, 50 µM MK-571, and KT (0.03, 0.3, and 3.0 µM) on the transport of digoxin from the B to A direction. D: digoxin. Data are represented as mean ± standard deviation, n = 4. Statistical significance from 0.5 µM CSA or 0.5 µM digoxin controls are shown with asterisks, **, p < 0.01, *, p < 0.05. 77

Table 2.1 Comparison of efflux ratios of [3H] cyclosporin A (CSA) and [3H] digoxin with various concentrations of ketoconazole (KT) in Caco-2 and MDCKII-MDR1 cells

Data are represented as mean ± standard deviation, n = 4. Statistical significance from 0.5 µM [3H]-CSA or 0.5 µM [3H]-digoxin controls are shown with asterisks (**) p < 0.01.

Caco-2 Efflux Ratio MDCKII-MDR1 Efflux Ratio

KT (µM) 0.5 µM 3H-CSA 0.5 µM 3H-Digoxin 0.5 µM 3H-CSA 0.5 µM 3H-Digoxin

0.0 6.99 ± 1.45 3.64 ± 0.49 6.67 ± 1.70 7.83 ± 1.90 0.03 4.40 ± 1.64** 2.74 ± 0.40 3.09 ± 1.08** 5.59 ± 2.85 0.3 3.62 ± 1.28** 2.97 ± 0.61 2.85 ± 1.06** 3.77 ± 1.28** 3.0 2.18 ± 0.81** 1.44 ± 0.43** 1.90 ± 0.22** 2.80 ± 0.78**

conditions were also performed. In Figure 2.5A, KT significantly inhibited the efflux of CSA (p < 0.01) in the MDCKII-wild type cells at concentrations ≥ 0.03

µM KT. In contrast, Figure 2.5B shows that the efflux of digoxin was significantly inhibited by 3.0 µM KT (p < 0.05). These results are similar to the Caco-2 data shown in Figure 2.2.

Table 2.2 is a summary of the effect of KT on the Papp of CSA and digoxin in Caco-2, MDCKII-MDR1 and MDCKII-wild type cells from the B Æ A direction. KT significantly decreased the Papp of CSA in the B Æ A direction at concentrations as low as 0.03 µM in the Caco-2 (5.07 × 10-6 to 4.24 × 10-6 cm/sec, p < 0.05), MDCKII-MDR1 (17.80 × 10-6 to 8.70 × 10-6 cm/sec, p < 0.01) and

MDCKII-wild type cells (21.66 × 10-6 to 15.73 × 10-6 cm/sec, p < 0.01). At higher concentrations (0.3 and 3.0 µM KT), the inhibitory effect of KT on CSA efflux reached a plateau. For digoxin, the significant inhibitory effect of KT on the efflux of digoxin was seen at 3 µM KT in the Caco-2 (2.60 ×10-6 to 1.41 × 10-6 cm/sec, p

< 0.01) and MDCKII-wild type cells (7.71 × 10-6 to 6.18 ×10-6 cm/sec, p < 0.05).

In the MDCKII-wild type cells, there was a significant increase, p < 0.01, in the efflux of digoxin at 0.03 µM KT. This suggests that KT has a biphasic response that is concentration-dependent in modulating P-gp. Also, the Papp of CSA and digoxin was significantly higher in the MDCKII-wild type cells than the MDCKII-

MDR1 cells. This may be due to other efflux transporters present in the MDCKII- wild type cells that CSA and digoxin are utilizing. 79

MDCKII-WILD TYPE

A. Cyclosporin A 30

) B to A -6

20 ** ** ** ** **

(cm/sec x 10 10 app P

0 T mil 571 K KT KT a - M M p K µM ra M 3 µ .0 µ M 0. 3 0.5 µM CSA µ 0.03 + + 0 + µM ve 5 0 + + 10 B. Digoxin 11 ** B to A 10 )

-6 9 8 7 * 6 ** 5 4 (cm/sec 10 x 3

app 2 P 1 0

n il T oxi am -571 g K M verap M µ 3.0 µM K M + 0.3 µM KT + 5 µM di µ + 0.03 µM KT 0. 50 + 100 +

Figure 2.5 The effect of ketoconazole (KT) and transport inhibitors on the transport of 0.5 µM 3H-cyclosporin A (CSA) and 0.5 µM digoxin (1 µCi) across MDCKII-wild type cells. Panel A: The effect of 100 µM verapamil, 50 µM MK- 571, and KT (0.03, 0.3, and 3.0 µM) on the transport of CSA from the basolateral (B) to the apical (A) direction. Panel B: The effect of 100 µM verapamil, 50 µM MK-571, and KT (0.03, 0.3, and 3.0 µM) on the transport of digoxin from the B to A direction. Data are represented as mean ± standard deviation, n = 4. Statistical significance from 0.5 µM CSA or 0.5 µM digoxin controls are shown with asterisks, **, p < 0.01, *, p < 0.05. 80

3 Table 2.2 Effect of ketoconazole (KT) on the apparent permeability (Papp) of [ H] cyclosporin A (CSA) and [3H] digoxin in Caco-2, MDCKII-MDR1, and MDCKII- wild type (MDCKII-WT) cells from the basolateral to apical direction

Data represents mean ± standard deviation. Statistical significance from 0.5 µM [3H]-CSA or 0.5 µM [3H]-digoxin controls are shown with asterisks (* p < 0.05, ** p < 0.01) n = 4. Comparison between the MDCKII-MDR1 and MDCKII-wild type controls (0.5 µM [3H]-CSA or 0.5 µM [3H]-digoxin) were performed († p < 0.05, †† p < 0.01) n = 4.

3 -6 3 -6 [ H] CSA Papp ( × 10 ) cm/sec [ H] Digoxin Papp ( × 10 ) cm/sec

KT (µM) Caco-2 MDCKII-MDR1 MDCKII-WT Caco-2 MDCKII-MDR1 MDCKII-WT

† 0.0 5.07 ± 0.24 17.80 ± 2.42 21.66 ± 0.41 2.60 ± 0.52 2.22 ± 0.44 7.71 ± 0.08†† 0.03 4.24 ± 0.47* 8.70 ± 1.90** 15.73 ± 1.27** 2.43 ± 0.27 2.00 ± 0.42 10.57 ± 0.26** 0.3 4.19 ± 0.43* 7.69 ± 1.52** 14.41 ± 1.87** 2.56 ± 0.39 1.87 ± 0.53 7.53 ± 0.45 3.0 2.91 ± 0.29** 8.13 ± 0.39** 15.21 ± 0.13** 1.41 ± 0.25** 1.68 ± 0.48 6.18 ± 0.49*

The ubiquitous presence of flavonoids in tea, beer, and many dietary supplements may be particularly important classes of modulators, because of their known interactions with monocarboxylate and multidrug-associated resistant proteins (MRP1 and MRP2) (Vaidyanathan and Walle, 2003), organic anion- transporting polypeptides and P-gp (Dresser et al., 2002), especially if these sub- stances are consumed on a daily basis and in sufficient quantities. Thus, we evaluated the drug-diet interaction of KT with EGCG and XN, the latter of two being present in tea and beer, respectively. As shown in Figures 2.6A-D, XN (0.3 to 10 µM) appeared to have a slight inhibitory effect on the B Æ A transport of KT in the Caco-2 cells when compared to control; however, only 30 µM XN, Figure

2.6E, significantly inhibited the efflux of KT when compared to control (p < 0.01).

The accumulative amount of 1.0 µM KT was a significantly decreased at 3 hr,

51.24 ± 9.73 to 27.99 ± 5.16 nmol/cm2, when co-exposed with 30 µM XN. Also,

30 µM XN significantly decreased the Papp of 1.0 µM KT in B Æ A direction from

19.31 ± 2.47 x 10-6 to 12.37 ± 2.41 x 10-6 cm/sec (p < 0.01, data not shown). In comparison, 3, 10 and 30 µM EGCG significantly inhibited the B Æ A transport of KT compared with control (Figures 2.6C-E). The accumulative amount of KT decreased at 3 hr from 51.24 ± 9.73 nmol/cm2 to: 18.60 ± 1.05, 27.54 ± 6.13 and

26.20 ± 12.24 nmol/cm2, for 3, 10 and 30 µM EGCG, respectively. Moreover,

EGCG significantly inhibited the Papp of 1.0 µM KT in B Æ A direction at 3.0 µM

(19.31 ± 2.47 x 10-6 to 12.01 ± 1.27 x 10-6 cm/sec, p < 0.05, data not shown).

A B

0.3 µM EGCG and XN 1.0 µM EGCG and XN ) 80 EGCG ) 80 2 EGCG 2 70 XN 70 XN 60 60 CONTROL CONTROL 50 50 40 40 30 30 20 20 10 10 H] KT (nmol/cm KT H] H] KT (nmol/cm KT H] 3 3 [ 0 [ 0 0 50 100 150 200 0 50 100 150 200 Time (min) Time (min)

C D

3.0 µM EGCG and XN 10.0 µM EGCG and XN ) ) 80 80 EGCG 2 2 EGCG 70 70 XN XN 60 60 CONTROL 50 CONTROL 50 40 40 30 30 20 20 10 10 H] KT (nmol/cm KT H] H] KT (nmol/cm 3 3 [ [ 0 0 0 50 100 150 200 0 50 100 150 200 Time (min) Time (min)

30.0 µM EGCG and XN

) 80 EGCG 2 70 XN 60 CONTROL 50 40 30 20 10 H] KT (nmol/cm KT H] 3

[ 0 0 50 100 150 200 Time (min)

Figure 2.6 Effects of (-)-epigallocatechin-3-gallate (EGCG) and xanthohumol (XN), on the transport of 1 µM, 3H-ketoconazole (KT, 1 µCi) from the basolateral to apical direction in Caco-2 cells

The concentrations of EGCG and XN were: 0.3, 1.0, 3.0, 10.0 and 30.0 µM, Figures 6A, 6B, 6C, 6D, and 6E, respectively. Data represents mean ± standard deviation, n = 4. 83

We then utilized a functional assay to specifically evaluate the interaction of KT with MDR1 in the Caco-2 and MDCKII-MDR1 cells. The VybrantTM assay uses calcein-AM, which is hydrolyzed to calcein, a fluorescent compound that is a substrate of P-gp. Figure 2.7A shows the effect of increasing concentrations of KT

(0.001 µM through 100 µM) on the retention of calcein in the Caco-2 cells. There was a dose-dependent inhibition of KT on the retention of calcein that appears to be biphasic. At 100 µM KT, there was approximately a 6-fold increase in the level of calcein retention when compared to control. In the MDCKII-MDR1 cells,

Figure 2.7B shows the effect of increasing concentration of KT (0.001 µM through

100 µM) on the retention of calcein. There was about a 50% increase in the level of calcein retained when compared to the control followed by saturation at concentrations of KT higher than 0.01 µM. These data suggest that KT can modulate MDR1-mediated transport.

Next, we modified the VybrantTM assay to evaluate the uptake of KT into the Caco-2 or MDCKII-MDR1 cells using 1.0 µM 3H-KT as the P-gp substrate.

The Caco-2 or MDCKII-MDR1 cells were pretreated with various concentrations of CSA and digoxin (0.001 through 30 µM); then, the cells were exposed 1.0 µM

3H-KT. There was a surprisingly dose-dependent inhibition of 3H-KT uptake in the

Caco-2 cells (Figure 2.8A). In contrast, this effect was not seen with the MDCKII-

MDR1 cells (Figure 2.8B). These results suggest that KT can modulate P-gp, but there may be other transporters involved in the transport of KT.

A. Caco-2

175

150

125

100

0 100 101 102 103 104 105 106

Ketoconazole [nM] B. MDCKII-MDR1 90

0 10 0 101 102 103 104 105 106

Ketoconazole [nM]

Figure 2.7 The influence of increasing concentrations of ketoconazole (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 µM) on calcein retention in Caco-2 cells (Panel A) and MDCKII-MDR1 cells (Panel B)

Data represents mean ± standard deviation, n = 4 - 8. The units are expressed as arbitrary units (AU).

A Caco-2 130 CSA 120 Digoxin

110

100 * 90 * ** 80 ** 70 ** 60 * * * * ** ** 50 ** ** 40 **

30 ** ** 20

0 10 0 101 102 103 104 10 5 CSA or Digoxin (nM)

B MDCKII-MDR1 200 CSA Digoxin 175

150

125

100

75 * 50 * ** 25

0 100 101 102 103 104 105 CSA or Digoxin (nM)

Figure 2.8 The effects of increasing cyclosporin A (CSA) and digoxin concentrations (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 µM) on 1.0 µM 3H-ketoconazole (0.1 µCi) uptake using Caco-2 cells (Panel A) and MDCKII- MDR1 cells (Panel B)

Data represents mean ± standard deviation, n = 6 - 8. Statistical significance from 1 µM ketoconazole control is shown with asterisks, **, p < 0.01, *, p < 0.05. 86

Lastly, the effect of the dietary flavonoids, EGCG and XN, on the uptake of KT in the Caco-2 and MDCKII-MDR1 cells was evaluated. The Caco-2 or

MDCKII-MDR1 cells were pretreated with various concentrations of EGCG or

XN (0.003 through 100 µM); then, the cells were exposed to 1.0 µM 3H-KT.

Figure 2.9A clearly shows that there is a dose-dependent effect of EGCG and XN on the uptake of KT in the Caco-2 cells. At low concentrations of EGCG (≤ 0.1

µM) and XN (≤ 0.3 µM), there is ~20% and ~50% reduction in the uptake of KT for EGCG and XN, respectively; yet at concentrations greater than 0.1 µM for

EGCG and greater than 0.3 µM for XN, there is approximately a 2-fold increase in the uptake of KT in the Caco-2 cells. This suggests that EGCG and XN modulate multiple transporters in the uptake of KT. In contrast, there was not a clear dose- dependent response seen in the MDCKII-MDR1 cells (Figure 2.9B).

A Caco-2 180 XN 170 EGCG * 160 150 140 130 120 110 100 90 80 70 60 50 ** * 40 ** 30 20 10 0 100 101 102 103 104 105 106 XN or EGCG (nM)

B MDCKII-MDR1 350 XN EGCG ** 300

** 250 *

200

150 ** ** ** ** 100

50 5 0 10 0 10 1 102 103 104 105 10 6 XN or EGCG (nM)

Figure 2.9 The effects of increasing (-)-epigallocatechin-3-gallate (EGCG) or xanthohumol (XN) concentrations (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 µM) on 1.0 µM 3H-ketoconazole (0.1 µCi) uptake using Caco-2 cells (Panel A) and MDCKII-MDR1 cells (Panel B)

Data represents mean ± standard deviation, n = 8. Statistical significance from 1 µM ketoconazole control is shown with asterisks, **, p < 0.01, *, p < 0.05.

2.5 Discussion

KT is a pharmaceutical that has been on the market over two decades and is still commonly used clinically for fungal infections, prostate cancer, and concomitant administration with CSA and tacrolimus. As a research tool, KT is commonly used as an inhibitor of CYP3A in many in vitro and in vivo studies, and recently, as an inhibitor of P-gp (MDR1). Because of the multiple uses of KT as well as its well-known hepatotoxicity (Stricker et al., 1986; Lake-Bakaar et al.,

1987; Findor et al., 1998), it is important to fully characterize the transport of KT as well as potential clinical MDR1-dependent interactions of KT with pharmaceuticals and dietary supplements. The concentrations of KT used in the current transport studies (0.03 – 3 µM) are substantially lower than the peak concentrations that has been reported for humans taking 200 mg/day or 400 mg/day doses, namely 8 µM to 19 µM (Huang et al., 1986) and 13 µM to 26 µM

(Baxter et al., 1986), respectively. Long-term administration will result in auto- inhibition of metabolism of KT thereby increasing its half-life (Daneshmend et al.,

1983; Huang et al., 1986). This will be a potential problem for drug accumulation and drug- and diet-drug interactions.

Figure 2.1 demonstrates that KT is actively effluxed from the Caco-2 cells and that there was an increase in efflux with increasing concentrations of KT thereby suggesting that KT can enhance its own efflux at concentrations ≥ 3 µM

KT. This efflux was significantly greater than the influx transport thus suggesting 89 that an efflux protein such as MDR1 and/or MRP is/are involved in the transport of KT in the Caco-2 cells. In a similar study by Takano et al., there was no difference in the efflux or absorption transport of 10 µM KT (Takano et al., 1998).

The high passage numbers of their Caco-2 cells (57 through 77) and the wide passage range of 20 are the most plausible explanation for the different results compared to our study which used low passage numbers (23 – 30) and a narrow range of passages. There are numerous studies that show that higher passage numbers of Caco-2 cells possess diminished characteristics/activities of transporters and enzymes (Yu et al., 1997; Anderle et al., 2003; Behrens et al.,

2004; Mizuma et al., 2004). Thus, the Caco-2 cells in the Takano et al. (1998) study may not have had functional P-gp and/or MRP to efflux KT such that there were no differences seen between the efflux and absorption of KT. Figure 2.2 shows that KT is actively effluxed from the MDCKII-MDR1 cells with significant enhancement at a low concentration which was also seen with digoxin in the

MDCKII-wild type cells (Figure 2.5). Lastly, the efflux Papp is higher in the Caco-

2 cells than the MDCKII-MDR1 suggesting that KT is actively effluxed by P-gp and MRP of which this efflux was effectively inhibited with verapamil (66%) and

MK-571 (55%).

Because of the many transporters expressed in the Caco-2 cells (Gres et al.,

1998; Artursson et al., 2001), the drug-drug interaction study of CSA and digoxin with KT were evaluated because they are well-known substrates for the intestinal efflux transporters in the Caco-2 drug transport model and in vivo (Augustijns, 90

1996; Taipalensuu et al., 2004). The MDCKII-MDR1 cells that overexpress human MDR1 were used to identify the effect of KT on MDR1-mediated transport using CSA and digoxin. Low concentrations of KT (0.03 µM) significantly decreased the efflux Papp of CSA in Caco-2, MDCKII-MDR1, and MDCKII-wild type cells (Figures 2.3A, 2.4A, and 2.5A) while higher concentrations of KT (≥ 3

µM) were needed to produce a significant inhibitory effect on the efflux of digoxin

(Figures 2.3B, 2.4B, and 2.5B). Table 2.2 summarizes effect of KT on the efflux

Papp of CSA and digoxin in Caco-2, MDCKII-MDR1 and MDCKII-wild type cells.

Overall, these data support previous research showing that KT can inhibit the

MDR1-mediated efflux of rhodamine-123 and digoxin in the Caco-2 cells (Kim et al., 1999; Yumoto et al., 1999) as well as showing that KT can effectively inhibit the efflux of transporter(s) involved in the transport of CSA. KT appears to have a higher affinity for transporter(s) utilized by CSA than for digoxin which may be due to CSA also being a substrate of the canalicular multispecific organic anion transporter (cMOAT, MRP2) (Nies et al., 1998). Thus, it is possible that KT also inhibited the MRP2-mediated efflux of CSA as MK-571 did (Figures 2.4 and 2.5), thereby having a pronounced effect on the transport of CSA than digoxin, which is not a MRP2 substrate.

Also, digoxin might be binding to different site(s) on P-gp or has very low affinity for P-gp compared to other P-gp substrates (Eneroth et al., 2001) such that low concentrations of KT was not able to significantly modulate the transport of digoxin. Interestingly, there was a significant increase in the efflux of digoxin at 91 the lowest concentration of KT used, 0.03 µM, in the MDCKII-wild type cells. In

Taub’s et al. study, they found a similar efflux enhancement of digoxin with low concentrations of KT in the MDCKII-MDR1 cells but not the Caco-2 or the

MDCKII-wild type cells whereas higher concentrations produced an inhibitory effect (Taub et al., 2005). Because it has recently been shown that digoxin is a substrate of an organic anion transport polypeptide transporter (OATP), OATP4C1

(Mikkaichi et al., 2004; Yao and Chiou, 2006), which is possibly expressed in the

MDCKII-wild type cells (Goh et al., 2002; Ng et al., 2003), KT probably induced the OATP-mediated transport of digoxin at lower concentrations whereas higher concentrations caused saturation and the inhibition of efflux transport became dominant. This suggests that KT produces a biphasic response that is concentration-dependent in modulating MDR1-mediated transport.

The biphasic response of KT on MDR1-mediated transport was also observed using a functional assay that uses calcein-AM, which is hydrolyzed to a calcein, a fluorescent substrate of P-gp and MRP (Essodaigui et al., 1998).

Increased concentrations of KT resulted in a dose-dependent increase in the retention of calcein in Caco-2 cells that appeared to be sigmoidal (Figure 2.7A) while not having the same response with the MDCKII-MDR1 cells (Figure 2.7B).

This data suggests that KT can modulate both MDR1- and MRP-mediated transport.

The uptake studies of 3H-KT in both the Caco-2 and MDCKII-MDR1 cells proved that its uptake could be modulated by the P-gp substrates, CSA and 92 digoxin. Instead of causing competitive inhibition with MDR1 and MRP, such that there was an increased amount of 3H-KT retained in the cells, there was a surprisingly significant dose-dependent decrease in the uptake of 3H-KT at concentrations as low as 0.003 µM in the Caco-2 cells but not in the MDCKII-

MDR1 cells (Figure 2.8). These results suggest that CSA and digoxin can inhibit the transporters involved in the uptake of KT which are not expressed at the same levels in the MDCKII-MDR1 cells.

It has been reported that flavonoids are able to inhibit drug efflux by directly interacting with various sites of P-gp (Conseil et al., 1998; Zhou et al.,

2004) as well as interacting with MRP1 and MRP2 (Vaidyanathan and Walle,

2003). Flavonoid derivatives like chromones, azaifoflavones, and aurones have been shown to inhibit P-gp activity (Hadjeri et al., 2003). Thus, we evaluated the drug-diet interaction of KT with EGCG and XN, which are found in tea and beer, respectively. EGCG, the most abundant flavonoid found in green or white tea, has recently been shown to be capable of modulating its own transport in vitro and in clinical studies in humans (Yang et al., 1998; Chow et al., 2001; Vaidyanathan and

Walle, 2003) as well as reversibly inhibiting MDR1-mediated transport of 3H- vinblastine in Caco-2 cells (Jodoin et al., 2002). XN, the most abundant flavonoid in Humulus lupulus, was chosen as the representative flavonoid because studies demonstrated that it has anti-mutagenic (Miranda et al., 2000b), antioxidative

(Miranda et al., 2000a) and chemopreventive activities (Miranda et al., 1999;

Gerhauser et al., 2002). Also, direct binding assays of the purified cytosolic 93 nucleotide-binding domain of P-gp suggests that prenylated xanthones modulate

MDR1-mediated transport (Bois et al., 1998; Tchamo et al., 2000). Our data is the first to show the drug-diet interaction of EGCG and XN on the transport of

KT. The data in Figure 2.6 show XN to have a slight inhibitory effect on the efflux of KT in the Caco-2 cells when compared to control; however, 30 µM XN significantly inhibited the efflux of KT. In comparison, 3, 10 and 30 µM EGCG significantly inhibited the efflux of KT compared with control. The effect of

EGCG and XN, on the uptake of KT in the Caco-2 and MDCKII-MDR1 cells resulted in a clear dose-dependent effect on the uptake of KT in the Caco-2 cells.

At low concentrations of EGCG, there was a ~20% and ~50% reduction in the uptake of KT for EGCG and XN, respectively; yet at higher concentrations of

EGCG and XN, there is approximately a 2-fold increase in the uptake of KT in the

Caco-2 cells. This suggests that EGCG and XN modulate multiple transporters in the uptake of KT. In contrast, there was not a clear dose-dependent response seen in the MDCKII-MDR1 cells (Figure 2.9B). It is important to elucidate the mechanisms and drug specificity of this efflux inhibition in order to evaluate the possible risks of consuming XN or EGCG containing foods and/or supplements when taking KT.

In summary, KT has been shown to significantly modulate MDR1- mediated transport by interacting with MDR1 proteins. In addition, our data suggests that KT may also modulate MRP-mediated transport. This effect could potentially alter the therapeutic efficacy and safety of many pharmaceuticals that 94 are substrates for either MDR1 or MRP resulting in drug-drug interactions that should be closely monitored. Moreover, there can be significant KT-dietary interactions with beverages, food or supplements containing EGCG or XN. 95

Acknowledgements

We acknowledge Dr. Cristobal Miranda for his helpful comments and suggestions. We also thank Dr. Piet Borst from The Netherlands Cancer Research

Institute for the MDCKII-MDR1 and MDCKII-wild type cells.

Acknowledgements for financial support

This publication was made possible by NIH grants AT00853, CA090890, and

ES00210. AT00853 was from the National Center for Complementary and

Alternative Medicine (NCCAM) and the contents are solely the responsibility of the authors and do not necessarily represent the official view of NCCAM, or the

National Institutes of Health.

Endnotes a) Part of this work was presented at the Society of Toxicology’s 45th annual

meeting, San Diego, CA 2006. Abstract number #1686. Fan, Y. and

Rodriguez-Proteau, R. Effects of ketoconazole on multidrug resistant-

mediated transport in Caco-2 and MDCKII-MDR1 drug transport

models. Toxicological Sciences, The Toxicologist, Volume 80, Number 1-S,

March 2006.

3. EFFECT OF DIETARY FLAVONOIDS ON TRANSPORTERS IN CACO-2 AND MDCKII-MDR1 CELL TRANSPORT MODELS

Y. Fan, R. Rodriguez-Proteau, J. E. Mata, J. J. Brown

Note: The vast majority of this chapter is published on Xenobiotica, January 2006, 36 (1): 41-58 (Rodriguez-Proteau et al., 2006)

3.1 Abstract

Flavonoids present in plants have demonstrated activity in cancer chemoprevention and treatment paradigms, modulation of signal transduction pathways, and drug transport processes. The hypothesis tested was that specific flavonoids such as epicatechin gallate (ECG), epigallocatechin gallate (EGCG) and xanthohumol will modulate the cellular uptake and the permeability (Pe) of multidrug-resistance substrates, cyclosporin A (CSA) and digoxin, across the Caco-2 and MDCKII-MDR1 cell transport models. 3H-CSA/3H-digoxin transport and uptake experiments were performed with and without co-exposure of 30 µM xanthohumol, EGCG and ECG. Xanthohumol (30 µM) reduced the efflux Pe of 0.5

µM CSA from 3.05 × 10-6 cm/sec to 0.74 × 10-6 cm/sec and 12.48 × 10-6 cm/sec to

4.02 × 10-6 cm/sec in Caco-2 and MDCKII-MDR1 cells, respectively; whereas there is no measurable effects were seen with digoxin. In uptake study, xanthohumol significantly increased uptake of 3H-digoxin and significantly decreased the uptake of 3H-CSA in Caco-2 cells. The transport data suggested that xanthohumol affects transport of CSA in a manner that is distinct from the digoxin efflux pathway and the intestinal transport of these MDR1 substrates is more complex than previously reported.

3.2 Introduction

Flavonoids are naturally occurring polyphenolic compounds found in fruits, vegetables, teas, wine and beer (Hollman and Katan, 1998; Arts et al., 2000;

Stevens and Page, 2004) that have a variety of biological and chemical activities in vitro and in vivo (Bors et al., 1997; Tobe et al., 1997; Miranda et al., 1999;

Henderson et al., 2000; Miranda et al., 2000a; Miranda et al., 2000b; Gerhauser et al., 2002). Some flavonoids inhibit lipid peroxidation (Briviba and Sies, 1994), which has been implicated in the pathogenesis of cancer, atherosclerosis, and toxic cell injury (Kehrer and Smith, 1994). Epidemiological studies suggested a health benefit from consumption of plant polyphenols found in the diet (Hollman and

Katan, 1999). For example, green tea extract supplements in human studies have demonstrated preventative beneficial effects in breast and gastrointestinal cancer, cardiovascular disease, and carcinogen-induced DNA damage (Hertog et al., 1995;

Nakachi et al., 2000; Wiseman et al., 2001; Demeule et al., 2002). However, a lack of clear scientific evidence of green tea or its constituents from well- controlled clinical trials precludes its recommended use for the aforementioned benefits (Higdon and Frei, 2003).

In the United States from 1994 to 2004, use of dietary supplements has more than doubled and is expected to increase from 9.8 billion to 49 billion by

2010 (Office, 2000). Market-driven demand for these products has outpaced 99 pharmacological research needed to characterize effects at concentrations that are not typically found in the average diet but which are expected to be ‘reasonably safe under the conditions of use recommended’ and can thus be marketed without

FDA approval (Office, 2000). Concentrations in the intestinal lumen from dietary supplements can reach levels that are substantially higher than one would expect to achieve systemically. Presently, little is known about flavonoid’s influence on the gastrointestinal mucosa immediately after oral intake. The jejunum and duodenum have been identified as probable sites of hydrolysis and absorption of many flavonoids within intestine (Spencer et al., 2003; Turner et al., 2003). The high concentrations of flavonoids in the intestine may be capable of inhibiting metabolic and transport processes at the site of drug, nutrient, and xenobiotic absorption (Kuo et al., 1998).

Some flavonoids have been reported to modulate drug efflux in multidrug- resistant (MDR) cancer cells, such as kaempferol, quercetin, and genistein and have been classified as modulators with bifunctional interactions at vicinal ATP- binding site and steroid-interacting region within a cytosolic domain of P- glycoprotein (Pgp, MDR1) (Conseil et al., 1998). Epigallocatechin-3-gallate

(EGCG), the most abundant flavonoid in tea and one of the most common ingredients of several dietary supplements and vitamins, as well as epicatechin gallate (ECG), have been reported to inhibit reversibly MDR1-mediated transport of vinblastine (Jodoin et al., 2002). Also, evidence shows that the pharmacological and structure activity relationships of flavonoid-related 100 modulators can partly inhibit MDR1-mediated transport in K562/DOX resistant cells (Ferte et al., 1999) as well as altering MDR1-independent transporters such as Na+-K--2Cl- (Cermak et al., 1998). Xanthohumol, the most abundant prenylated flavonoid in Humulus lupulus and an ingredient that imparts flavor and bitterness to beer, has been reported to have antioxidative (Miranda et al., 2000a), anti-mutagenic (Miranda et al., 2000b), and cancer chemopreventive activities

(Miranda et al., 1999; Henderson et al., 2000; Gerhauser et al., 2002). Direct binding assay of purified cytosolic nucleotide-binding domain of P-glycoprotein suggests that prenylated xanthones modulate MDR1-mediated transport (Bois et al., 1998; Bois et al., 1999; Tchamo et al., 2000). Menohop®, which contains xanthohumol as its main component, was evaluated as well, because it is the first

“hop” food supplement available on market and it eases the menopause transition period in women and inhibits the proliferation of breast and uterine cancer cells

(Heyerick et al., 2006).

Drug transport in the intestine occurs by passive and active mechanisms, which include efflux transporters. Inhibition of efflux pathways such as P- glycoprotein can increase absorption by increasing the overall permeability of the drug; thereby enhancing therapeutic effects or eliciting adverse effects. There have been numerous reports of the effect of grapefruit juice (GFJ) altering plasma levels of CSA or digoxin via enzyme or transporter inhibition due to the interplay between absorption, metabolism, and active efflux pathways (Soldner et al., 1999;

Becquemont et al., 2001). With the exception of GFJ, there is a lack of well- 101 controlled clinical studies evaluating possible risks of adverse effects of supplements containing plant polyphenols.

The Caco-2 cell model of human intestinal transport has been shown to be a predictive tool to infer possible adverse effects in vivo and allows for direct examination of the dynamics of drug absorption and efflux across polarized epithelial cell monolayers for prediction of oral bioavailability (Gres et al., 1998;

Artursson et al., 2001). Human MDR1, MRP1, MRP2, MRP3, and MRP5 are functionally expressed in the Caco-2 cells (Hunter et al., 1993a; Gutmann et al.,

1999; Walle et al., 1999; Hirohashi et al., 2000; Taipalensuu et al., 2001). Also, the Madin-Darby Canine Kidney (MDCKII) cells overexpressing human MRP1,

MRP2, MRP3, MRP5, and MDR1 have been well characterized (Louvard, 1980;

Bakos et al., 1998; Evers et al., 1998; Tang et al., 2002) and are useful models for the maintenance of specialized cell surfaces (Louvard, 1980), as well as for drug transport for comparisons to the Caco-2 and MDCKII-wild type cells (Tang et al.,

2002).

Plant polyphenols have been shown to interact with many transporters found in the intestine including: monocarboxylate transporter and multidrug- associated resistance proteins (MRP1 and MRP2) (Vaidyanathan and Walle,

2003), and organic anion-transporting polypeptides and P-gp (MDR1) (Dresser et al., 2002). We have conducted a study to evaluate the effects of a variety of common dietary plant polyphenols, such as xanthohumol, EGCG and ECG, on drug efflux using Caco-2 and MDCKII-MDR1 cell transport models. We have 102 attempted to characterize relative potency of flavonoid/drug interaction with two

MDR1 substrates, CSA and digoxin, to provide evidence of specific binding of xanthohumol to P-glycoprotein, which acts as a potent inhibitor of cyclosporin A efflux but not digoxin in both Caco-2 and MDCKII-MDR1 cells.

103

3.3 Materials and methods

3.3.1 Chemicals

Radiolabeled 3H-cyclosporin A (CSA, 8.0 Ci/mmol, 98.8% purity) and 3H- digoxin (23.4 Ci/mmol, > 97% purity) were obtained from Amersham, Inc.

(Piscataway, NJ, USA). Xanthohumol was purified (> 98% Purity) from hops by

Stevens et al. (Stevens et al., 1999). MK-571 was obtained from Cayman

Chemical Co. (Ann Arbor, MI, USA). Epigallocatechin-3-gallate (EGCG), epicatechin gallate (ECG), CSA, and digoxin were obtained from Sigma, Inc. (St.

Louis, MO, USA). Menohop® was a generous gift from Biodynamics BVBA

(Oostende, Belgium). Fetal bovine serum (FBS) was purchased from Hyclone

(Logan, UT, USA). All other reagents were analytical grade.

3.3.2 Cell culture

The Caco-2 cell line was obtained from American Type Culture Collection

(Rockville, MD, USA). MDCKII-MDR1 and MDCKII wild-type cell lines were generous gifts from Dr. Piet Borst from the Netherlands Cancer Research Institute.

Both Caco-2 and MDCKII cells were maintained at 37 °C in air with 5% CO2, and

95% relative humidity with Dulbecco’s Modified Eagle Medium, 10 % FBS, and

0.1 mM non-essential amino acids, and 0.01% penicillin/streptomycin (Evers et al., 1996; Evers et al., 1998). The cells were seeded at density 300,000 cells/well 104 onto Transwell® cell culture inserts and maintained until use on days 21-25

(Caco-2, passages 23 to 30) and 6-7 days (MDCKII-MDR1 and MDCKII wild- type). The integrity of the monolayers was assessed by measuring transepithelial electrical resistance (TEER) using a World Precision Instrument, EVOM,

(Sarasota, FL, USA) and by evaluating mannitol transport. The average TEER reading for the Caco-2, MDCKII-MDR1 and MDCKII wild-type transport experiments were 731.61 ± 106.94, 417.9 ± 16.1 and 455.7 ± 32.6 Ωcm2, respectively. Also, permeability studies with 3H-mannitol were performed and the transport rate was less than 1% per hour throughout the entire experiment, which indicates that the cell monolayer was not compromised.

3.3.3 Transport studies

Transport of 3H labeled compounds (CSA and digoxin) were studied using a transport buffer consisting of Hanks’ buffered salt solution (HBSS) with 10 mM

HEPES and 25 mM D-glucose at a pH of 6.8 for the apical (A) compartment and

7.4 for the basolateral (B) compartment. Experiments were performed at 37 °C in

® air, 5% CO2, and 95% relative humidity. The Transwell inserts were washed 3 times with transport buffer and allowed to equilibrate for 30 min prior to the addition of test compounds. The concentrations of the P-gp substrates were 0.5 µM for both CSA and digoxin. The concentrations of the flavonoids used in these studies were: 30 µM for ECG, EGCG, and xanthohumol. All the stock 105 concentrations of the flavonoids were prepared fresh on the day of experiment and kept away from direct light. The transporter inhibitors used in these studies were:

100 µM verapamil and 50 µM MK-571. Both the flavonoids and the transport inhibitors were prepared in transport buffer at the appropriate pH (pH 6.8 for the A compartment and 7.4 for the B compartment) and allowed to incubate at 37 °C prior to the start of the experiment for 30 min. The flavonoids and inhibitors were present in both the A and B chambers during the transport of the P-gp substrates

(CSA and digoxin). Ethanol was used as the solvent vehicle (0.15%v/v) for CSA, xanthohumol and Menohop® extract. DMSO (0.15%v/v) was used as the solvent vehicle for digoxin. Each experiment was performed in duplicate and repeated for each condition tested. For experiments performed in ‘non-sink’ conditions, 100 µl aliquots were taken from the donor and receiver chambers initially and from the receiver chamber at 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 hr. Non-sink conditions are used when ≤ 10% of the compound is transported across the cells over the duration of the transport experiment such that there is always a concentration gradient present.

An equal volume, 100 µl, was replaced in the chamber at each sample time point.

All experiments using 3H-CSA from A to B direction were performed in non-sink conditions. In the experiments performed in ‘sink’ conditions, the entire receiver volume was replaced at each time interval and a 100 µl aliquot was taken from the receiver volume for scintillation counting. A 100 µl sample was taken from the donor chamber at 4 hr in order to calculate the mass balance of radioactivity to determine if adsorption to the cell culture transport apparatus had occurred. Each 106 sample was placed in 0.9 ml of scintillation fluor (Cytoscint ES, ICN, Cosa Mesa,

CA, USA) and read on a Beckman LS 6500 (Palo Alto, CA, USA) scintillation

3 counter for H activity. The effective permeability coefficients (Pe) were calculated using the following equation:

Pe = (Vd · Δ%)/(A· Δt)

Where Pe is the effective permeability coefficient measured in cm/sec, Vd is the volume in cm3 of the donor compartment, A is the surface area of the monolayer

(4.71 cm2), and the Δ% is the normal percent mass transferred in a given time interval, and Δt is the length of a given time interval in seconds.

3.3.4 Uptake studies

The VybrantTM Multidrug Resistance (MDR) Assay (Molecular Probes,

Eugene, OR, USA) fluorescence microplate method was adapted and radiolabelled substrates, 3H-CSA and 3H-digoxin, were used rather than calcein-AM. Briefly, 50

µl of EGCG or xanthohumol was added to a round bottom 96-well microplate

(Falcon, Franklin Lakes, NJ, USA) to achieve final concentrations of 0.003, 0.01,

0.03, 0.1, 0.3, 1.0, 3.0, 10, 30, and 100 µM. Phosphate-buffered saline (PBS) was included as a control. All solutions and media used were pH 7.4. Approximately 5 x 105 cells (Caco-2 or MDCKII-MDR1) were added in 100 µl volumes of cell culture media to each well, mixed, and incubated at 37 °C for 15 min. 3H-CSA or

3H-digoxin (0.1 µCi, 0.5 µM) was added to each well of the microplate. The 107 reagents were mixed by gentle shaking and incubated at 37 °C for 15 min. The microplate was then centrifuged for 5 min at 200 × g and the supernatant was removed. The cells were washed 3 times with 100 µl 4 ˚C tissue culture media with the last wash containing the cells resuspended in 200 µl media. The 200 µl resuspension of cells was placed in 1.8 ml of scintillation fluor (Cytoscint ES,

ICN, Cosa Mesa, CA, USA) and read on a Beckman LS 6500 (Palo Alto, CA,

USA) scintillation counter for 3H activity.

3.3.5 Liquid Chromatography/Mass Spectrometry (LC/MS) analysis

LS/MS analysis was performed using a Surveyor LC pump with Surveyor

PDA detector, together with Surveyor autosampler system hyphenated to a LCQ advantage mass spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped with an atmospheric-pressure chemical ionization (APCI) source. The separation was achieved using a reverse-phase C18 column 150 × 4.60 mm i.d.,

Phenomenex® Luna 5 u, with 4 mm × 3 mm i.d., Phenomenex® guard column.

The mobile phases A and B were acetonitrile and 1% formic acid/ H2O. The solvent system was followed by the gradient condition: 40% ACN to 100% ACN in 1% aqueous formic acid over 30 min, at 35 min, the % ACN was returned to

40% in 2 min, and the column was equilibrated for 15 min prior to the next injection. Mass spectra were recorded on a mass spectrometer using APCI in 108 positive mode. The flow rate was kept at 0.8 ml/min and the injection volume was

10 µl. The wavelength was used at 370 nm.

3.3.6 Statistical analysis

The percent of drug/flavonoid transported during the experiment for the various treatments and the Pe values within treatment groups were compared using one-way ANOVA followed by a Dunnett’s post-test. Differences were considered significant for p < 0.05.

109

3.4 Results

Effects of various transport inhibitors on the transport of 0.5 µM 3H-CSA and 0.5 µM 3H-digoxin in Caco-2 cells

CSA and digoxin were chosen as test compounds because they are known to be substrates for the efflux transporters expressed on the apical surface of polarized epithelial cells of the intestine found in Caco-2 monolayers and in vivo

(Augustijns, 1996). Figure 3.1 shows the effect of various transport inhibitors on the transport of CSA and digoxin in Caco-2 cells. Use of various MDR1- and

MRP-mediated transport inhibitors in Caco-2 cells and MDCKII-MDR1 cells allows for some dissection of the pathways involved in the transport of drugs that are substrates for MDR1 or MRPs. We employed 0.5 µM CSA and 100 µM verapamil as inhibitors for MDR1 and 50 µM MK-571 as an inhibitor for MRP2, alone or in combination with xanthohumol. As shown in Figure 3.1A, verapamil and MK-571 significantly reduce the Pe of CSA from B to A direction in Caco-2 cells (from 3.05 ± 0.08 ×10 -6 cm/sec to 1.14 ± 0.20 ×10 -6 cm/sec, verapamil, and to 1.76 ± 0.10 ×10 -6 cm/sec, MK-571) while xanthohumol alone was very effective at inhibiting the efflux of CSA (xanthohumol inhibited efflux Pe of CSA

-6 -6 from 3.05 ± 0.08 ×10 cm/sec to 0.74 ± 0.11 ×10 cm/sec). In contrast, the Pe of

0.5 µM digoxin from B to A direction was significantly inhibited by MK-571, verapamil, and CSA (p < 0.01) but not by xanthohumol (Figure 3.1B). However, the Pe of digoxin from the A to B direction was significantly increased by MK-

571, verapamil, and CSA (p < 0.05), but not by xanthohumol (Figure 3.1B). 110

A. Cyclosporin A

3.5 B to A 3.0

) A to B -6 2.5

2.0 **

1.5 **

(cm/sec x 10 (cm/sec 1.0

e ** P 0.5 ** ** 0.0 il N N N X X CSA M µ 0 3 Verapam + 0.5 µM M µ + MK-571/ + 50 µM MK-571 + Verapamil/X 100 +

B. Digoxin

B to A 3 A to B ) ** -6 ** 2 * ** ** ** 1 (cm/sec x 10 (cm/sec e P

0 1 l 7 xin 5 mi o a CSA ig K- p M ra M D e V µM + 30 µM XN µM 5 0 . 5 µM + 0.5 µ 0 0 + 0 1 +

Figure 3.1 Effects of various transport inhibitors on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cell monolayers. Panel A: Effects of 100 µM verapamil, 50 µM MK-571, 30 µM xanthohumol (XN), plus various combinations on CSA transport from basolateral (B) to apical (A) (sink condition) and A to B (non-sink condition) directions. Data are represented as mean ± standard deviation, n = 4 - 6. Panel B: Effects of the treatment conditions on digoxin transport from the B to A and A to B directions. Data are represented as mean ± standard deviation, n = 4. Statistical significance from 0.5 µM CSA or digoxin is shown with asterisks, **, p < 0.01, and *, p < 0.05. 111

Effects of transport inhibitors on the transport of 0.5 µM 3H-CSA and 0.5 µM 3H-digoxin in MDCKII-MDR1 cells

Figure 3.2A shows that xanthohumol, as well as verapamil, are potent inhibitors of the transport of CSA in MDCKII-MDR1 cells as shown by the significant decrease in the efflux Pe (B to A direction) of CSA (from 12.48 ± 6.68

×10 -6 cm/sec to 4.02 ± 0.40 ×10 -6 cm/sec, and to 3.34 ± 1.60 ×10 -6 cm/sec, for xanthohumol and verapamil, respectively) whereas, once again, xanthohumol had no effect on digoxin transport (Figure 3.2B). Interestingly, MK571, an inhibitor of

-6 -6 MRP2, decreased the efflux Pe of digoxin (from 2.21 ×10 cm/sec to 1.45 ×10 cm/sec). EGCG and ECG have no effect on the transport of CSA or digoxin in the

MDCKII-MDR1 cells (Figure 3.2) but they do significantly inhibit the efflux of

CSA in the Caco-2 cells (data not shown). Moreover, both xanthohumol and

EGCG inhibited the A to B transport of CSA (from 4.15 ×10 -6 cm/sec to 1.11 ×10

-6 cm/sec, EGCG, to 2.68 ×10 -6 cm/sec, ECG) in the MDCKII-MDR1 cells

(Figure 3.2A). 112

A. Cyclosporin A

B to A 20 A to B

) 15 -6 *

Pe (cm/sec x 10 Pe (cm/sec 5 ** * **

N G SA mil X er M C M EC M MK-571 30 µM 30 µ 0.5 µ 30 µM EGCG 50 µ 100 µM verap

B. Digoxin

B to A 3.5 A to B 3.0 )

-6 2.5

2.0

1.5 * ** 1.0

Pe (cm/sec x 10 Pe (cm/sec *** 0.5 **

0.0

G xin XN o M GC M ECG 30 µ M E 30 µ 30 µ 50 µM MK-571 0.5 µM Dig 100 µM verapermil

Figure 3.2. Effects of transport inhibitors on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII- MDR1 cells. Panel A: Effects of 100 µM verapamil, 50 µM MK-571, 30 µM xanthohumol (XN), 30 µM EGCG, and 30 µM ECG on CSA transport from basolateral (B) to apical (A) and A to B direction. Panel B: Effects of the various treatment conditions on digoxin transport from the B to A and A to B direction. Data are represented as mean ± SD, n = 4 - 11. Statistical significance from 0.5 µM CSA or digoxin control is shown with asterisks, **, p < 0.01, and *, p < 0.05.

113

Effects of transport inhibitors on the transport of 0.5 µM 3H-CSA and 0.5 µM 3H-digoxin in MDCKII wild-type cells

We also performed experiments using the MDCKII wild-type cells under the same experimental conditions as that in MDCKII-MDR1 cells (Figure 3.3).

There was not any significance seen in the transport of digoxin or CSA with either verapamil or MK-571. Only xanthohumol significantly decreased the efflux Pe of

CSA from 8.97 ×10 -6 cm/sec to 3.26 ×10 -6 cm/sec (p < 0.05) (Figure 3.3A).

EGCG and ECG do not have any effect on the transport of CSA and digoxin in the

MDCKII-MDR1 or MDCKII wild type cells (Figure 3.2 and 3.3), but they do significantly inhibit the efflux of CSA in the Caco-2 cells (data not shown).

Moreover, both xanthohumol and EGCG inhibited the A to B transport of CSA in the MDCKII-MDR1 cells (Figure 3.2A). 114

A. Cyclosporin A

B to A 12.5 )

-6 10.0

7.5

5.0 * (cm/sec x 10 (cm/sec

e 2.5 P 0.0 l i G am CSA GC M MK-571 µ verap M 30 µM XN 5 µ 30 µM ECG 0. M 30 µM E µ 50 100 B. Digoxin

B to A

) 8 -6 7 6 5 4 3

(cm/sec x 10 (cm/sec 2 e

P 1 0

in N amil -571 CG M X G ap K 0 µ M M 3 30 µM ECG µ 30 µM E 0.5 µM Digox 50 100 µM ver

Figure 3.3 Effects of transport inhibitors on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII wild-type cells. Panel A: Effects of 100 µM verapamil, 50 µM MK-571, 30 µM xanthohumol (XN), 30 µM EGCG, and 30 µM ECG on CSA transport from basolateral (B) to apical (A) direction. Panel B: Effect of the various treatment conditions on digoxin transport from the B to A direction. Data are represented as mean ± SD, n = 4. Statistical significance from 0.5 µM CSA or digoxin control is shown with asterisks, *, p < 0.05.

115

Effects of EGCG and xanthohumol on 3H-CSA and 3H-digoxin uptake in Caco-2 and MDCKII-MDR1 cells

Figures 3.4 and 3.5 show the results of the uptake studies performed with

CSA and digoxin using both Caco-2 and MDCKII-MDR1 cells, respectively, pretreated with increasing concentrations of EGCG and xanthohumol (0.003 through 100 µM). There was a surprisingly clear dose-dependent inhibition of

3H-CSA uptake with EGCG and xanthohumol with increasing concentrations over

5 logs rather than an increase in CSA uptake as expected (Figure 3.4A); in contrast, there was an increase in 3H-digoxin uptake with EGCG and xanthohumol (Figure 3.4B) presumably due to the inhibition of MDR1-mediated transport. These findings are surprising because the inhibition of B to A transport of CSA by EGCG or xanthohumol should result in an increased uptake of CSA.

Figure 5A shows that xanthohumol and EGCG affect the transport of CSA in a similar manner in the MDCKII-MDR1 cells, which is opposite of the response seen with that in the Caco-2 cells. However, there effects occurred at relatively higher concentrations compared with Caco-2 cells. On the other hand, there did not appear to be any significant effect of xanthohumol and EGCG on digoxin uptake in MDCKII-MDR1 cells (Figure 3.5B). 116

A. Cyclosporin A

100 CSA with Xanthohumol CSA with EGCG

90 * ** ** 80 ** ** ** ** ** ** 70 ** ** ** ** 60

** 50 CSA Uptake (% control dpm) control (% Uptake CSA 40

30 5 0 100 101 10 2 10 3 104 105 10 6 Log [Dose XN and EGCG]

B. Digoxin

500 Digoxin with Xanthohumol Digoxin with EGCG

400 **

300 ** * * 200 * Digoxin Uptake (% control dpm) 100

0 100 101 10 2 10 3 104 105 10 6 Log [Dose XN and EGCG]

Figure 3.4 The effects of increasing EGCG or xanthohumol (XN) concentrations (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 µM) on (0.1 µCi, 0.5 µM) 3H- cyclosporin A (CSA, Panel A) or 3H-digoxin (Panel B) uptake using Caco-2 cells. Data are represented as mean ± standard deviation, n = 8. Statistical significance from 0.5 µM CSA or digoxin control is shown with asterisks, **, p < 0.01, and *, p < 0.05. 117

A. Cyclosporin A

250 CSA with Xanthohumol CSA with EGCG 225 **

200 **

175

150

125

100

CSA Uptake (% control dpm) control (% Uptake CSA 75

25 5 0 100 101 10 2 10 3 104 105 10 6 Log [Dose XN and EGCG]

B. Digoxin

250 Digoxin with Xanthohumol Digoxin with EGCG 225

200

175

150

125 *

100

75 Digoxin Uptake(% controldpm) 50

25 5 0 100 101 10 2 10 3 104 105 10 6 Log [Dose XN and EGCG]

Figure 3.5 The effects of increasing EGCG or xanthohumol (XN) concentrations (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 µM) on (0.1 µCi, 0.5 µM) 3H- cyclosporin A (CSA, Panel A) or 3H-digoxin (Panel B) uptake using MDCKII- MDR1 cells. Data are represented as mean ± standard deviation, n = 8. Statistical significance from 0.5 µM CSA or digoxin control is shown with asterisks, **, p < 0.01, and *, p < 0.05. 118

Effects of Menohop® extract on the transport of 0.5 µM 3H-CSA and 0.5 µM 3H-digoxin in Caco-2 cells

Figure 3.6 shows the effect of Menohop® extract on the transport of CSA and digoxin in Caco-2 cells. Menohop® extract significantly decreased effective permeability of CSA in both B to A direction and A to B direction, the significant effect starts at 0.01 mg/ml (Figure 3.6A). In our previous data (Figure 3.7A), xanthohumol significantly decreased the effective permeability of CSA in B to A direction and A to B direction (data on A to B direction not shown) as well. The effect of Menohop® extract on transport of CSA in Caco-2 cells is probably due to the effect of xanthohumol because no significant effect of other component in

Menohop® extract (isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin and chalconaringenin) were seen on the transport of CSA in B to A direction. As for the digoxin transport, Menohop® extract decreased the permeability of digoxin in

B to A direction and the significant effect starts at 0.01 mg/ml (Figure 3.6B).

Compared to the effect seen on the transport of CSA, Menohop® extract has less effect on transport of digoxin in Caco-2 cells. In our previous data (Figure 3.7B), only isoxanthohumol significantly reduced efflux of digoxin in the Caco-2 cells.

The effect seen with the Menohop® extract on efflux of digoxin is likely due to the effect of isoxanthohumol. Meanwhile, the concentration of isoxanthohumol in

Menohop® extract is about 2.00%, which is not as high as the xanthohumol

(3.20%) in Menohop® extract. Thus, the inhibitory effect of Menohop® extract on the efflux of digoxin is not as potent as that on the efflux of CSA. 119

A. Cyclosporin A

8 B to A

) 7

-6 A to B 6

4 ** ** ** 3 (cm/sec x x 10 (cm/sec

e **

P ** 2 ** ** 1 ** ** ** ** ** 0 C 0.5 µM V 100 µM 0.01 0.025 0.05 0.1 0.25

Menohop® Extract (mg/ml)

B. Digoxin

5.5 5.0 B to A 4.5 A to B ) -6 4.0 ** 3.5 ** ** 3.0 * 2.5 ** ** ** ** 2.0 **

(cm/sec x 10 (cm/sec *

e ** 1.5

P ** 1.0 ** 0.5 ** 0.0 D 0.5 µM V 100 µM 0.01 0.025 0.05 0.1 0.25

Menohop® Extract (mg/ml)

Figure 3.6 Effects of Menhop® extract on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cell monolayers. Panel A: Effects of Menhop® extract (0.01, 0.025, 0.005, 0.1, 0.25 mg/ml) on CSA transport from basolateral (B) to apical (A) and A to B directions. C, CSA; V, verapamil. Data are represented as mean ± standard deviation, n = 4. Panel B: Effects of Menhop® extract (0.01, 0.025, 0.005, 0.1, 0.25 mg/ml) on digoxin transport from B to A and A to B directions. D, digoxin; V, verapamil. Data are represented as mean ± standard deviation, n = 4. Statistical significance from 0.5 µM CSA or digoxin is shown with asterisks, **, p < 0.01, and *, p < 0.05. 120

A. Cyclosporin A

6 ) -6 5

4 ** 3

(cm/sec x 10 x (cm/sec 2 e P 1

0 l l in in SA mo mo n n C hu ge ge ohu ringenin rin rin tho h n na ant o Xa x lc o enylna a Is renylna P -Pr Ch 6- 8

B. Digoxin

5 **

) 4 -6

2 ** (cm/sec x 10 x (cm/sec e

P 1

in in n x e enin o g g ig D rin rin a a lnaringenin n yln y o n alc Isoxanthohumol h Pre C 6- 8-Pren

Figure 3.7 Effect of xanthohumol and xanthohumol derivatives on transport of transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cell monolayers. Panel A: Effects of xanthohumol, isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin and chalconaringenin on CSA transport from basolateral (B) to apical (A) direction. Data are represented as mean ± standard deviation, n = 4 - 8. Panel B: Effects of isoxanthohumol, 6- prenylnaringenin, 8-prenylnaringenin and chalconaringenin on digoxin transport from B to A direction. Data are represented as mean ± standard deviation, n = 4. Statistical significance from 0.5 µM CSA or digoxin is shown with asterisks, **, p < 0.01. 121

Liquid Chromatography/Mass Spectrometry (LC/MS) of xanthohumol and Menohop® extract

Figure 3.8 and Figure 3.9 show the HPLC profile and the total ion chromatogram (mass spectrometric detection) obtained by passing xanthohumol standard and a Menohop® extract over a reverse-phase C18 column (see

Experimental section). The retention time and the molecular ion mass ([M+H]) corresponding to xanthohumol and peak1 in the extract are indicated. The peak 1 corresponding to m/z 354.98, is due to xanthohumol, confirmed by co-injection with commercial sample. It was also proved by mass spectrometric detection, the same m/z as berberine standard (354.98) was found in Menohop® extract (Figure

3.9B). 122

A. Xanthohumol standard (LC)

B. Xanthohumol Standard (MS at retention time 16.67-17.14 min)

Figure 3.8 High performance liquid chromatograph (HPLC) chromatograms and Mass Spectrometry (MS) of xanthohumol. Panel A: HPLC chromatogram of xanthohumol standard. Panel B: MS of xanthohumol standard at retention time 16.67-17.14 min. AU: arbitrary unit.

123

A. Menohop® Extract (LC)

Peak 1

B. Menohop® Extract (MS at retention time 16.83-17.09 min)

Figure 3.9 High performance liquid chromatograph (HPLC) chromatograms and Mass Spectrometry (MS) of Menohop® extract. Panel A: HPLC chromatogram of Menohop extract. Panel B: MS of Menohop® extract at retention time 16.83-17.09 min. AU: arbitrary unit.

124

3.5 Discussion

Use of dietary/herbal supplements may have beneficial effects by restoring health and well being in some individuals but may put others at risk for potential adverse drug-herbal interactions. A number of herbal preparations contain bioactive compounds such as flavonoids that may influence the absorption and pharmacokinetics of certain drugs. Dietary supplements that could enhance the bioavailability of anti-cancer drugs or immunosuppressive drugs such as CSA by promoting drug absorption would be considered beneficial to human health.

However, there are certain drugs such as digoxin that have a narrow therapeutic range such that dietary supplements which promote the absorption of these drugs may lead to abnormally high drug plasma levels. A number of dietary or herbal supplements contain various types of flavonoids whose effects on drug absorption are not well understood.

Flavonoids have been reported to inhibit drug efflux by directly interacting with various sites of P-gp (MDR1) (Conseil et al., 1998; Zhou et al., 2004) as well as interacting with the multidrug-associated resistance proteins (MRP1 and MRP2)

(Vaidyanathan and Walle, 2003). MDR1 recognizes a wide variety of structurally diverse lipophilic compounds (Shapiro and Ling, 1997) and has the ability to extract lipophilic substrates such as CSA from the lipid bilayer. Ferté et al (Ferte et al., 1999) demonstrated that the flavone or flavanone moiety, N-benzylpiperazine chain, was more potent than verapamil in inhibiting MDR1-mediated transport, 125 thus providing evidence of structure activity relationships in MDR1-mediated inhibition. However, other flavonoids such as catechins can increase MDR1- mediated drug transport by heterotropic allosteric mechanism (Zhou et al., 2004).

Inhibition of MDR1 by flavonoids may be important in reversing multidrug resistance in cancer cells, whereas the stimulation of MDR1 activity may lower intracellular concentrations of carcinogens resulting in reduced cancer risk.

Flavonoids can also modulate drug transport through MDR1-independent mechanisms. For example, quercetin has multiple MDR1-independent mechanisms, such as: i) be a substrate for the Na+-dependent glucose cotransporter

(Walgren et al., 2000); ii) increase short-circuit current (Cermak et al., 1998); and iii) has a dual effect on Cl- secretion (Sanchez de Medina et al., 1997). Flavonoids found in apple, orange, and grapefruit juices had an inhibitory effect on organic anion-transporting polypeptides (Dresser et al., 2002). In our previous study, several flavonoid compounds of diverse structures, such as EGCG, ECG, xanthohumol and quercetin, were found to inhibit the B to A transport of CSA but not of digoxin. However, some of these compounds such as EGCG and xanthohumol inhibited uptake of CSA and increased uptake of digoxin (Figure

3.4). These findings are surprising because the inhibition of B to A transport of

CSA by EGCG or xanthohumol should result in increased uptake of CSA.

Furthermore, increased uptake of digoxin could not be explained by inhibition of

MDR1-mediated efflux by EGCG or xanthohumol because these compounds did not inhibit the B to A transport of digoxin. Aside from MDR1, digoxin transport 126 could involve diffusional uptake and Na+-K+ pump-mediated endocytosis (Cavet et al., 1996). One possible explanation for the increased uptake of digoxin is that

EGCG or xanthohumol may be altering MDR1-independent transport. The capacity of flavonoids to block MDR1 activity may involve mechanisms described for other MDR/MRP modulators, for example, antiestrogens which interact with membrane phospholipids such that the packing density of the lipids are altered resulting in an altered diffusion rate (Scambia et al., 1994).

Xanthohumol, the most abundant flavonoid in Humulus lupulus, was chosen as the representative prenylated chalcone because studies demonstrated that it has antioxidative (Miranda et al., 2000a), anti-mutagenic (Miranda et al., 2000b), and chemopreventive activities (Miranda et al., 1999; Gerhauser et al., 2002).

Xanthohumol also inhibits nitric oxide production in mouse macrophage RAW

264.7 cells (Zhao et al., 2003) and inhibits triglyceride and apolipoprotein B secretion in HepG2 cells (Casaschi et al., 2004). EGCG is the most abundant flavonoid found in green or white tea and a common ingredient of several dietary supplements. It was found that xanthohumol and EGCG may increase intracellular levels of digoxin in cells expressing multiple drug transport proteins. It is important to elucidate the mechanisms and drug specificity of this efflux inhibition in order to evaluate the possible risks of consuming xanthohumol or EGCG containing foods and/or supplements when taking digoxin. Our previous and present study indicated that xanthohumol, was most effective in reducing the permeability of the MDR1 substrate, CSA, from B to A direction (Figures 3.1-3.3, 127 previous data not shown) of the flavonoids tested. These results suggest that xanthohumol may increase the bioavailability of CSA by inhibiting MDR1- mediated and MDR1-independent efflux in intestinal cells; thus, providing a potential approach to increase the bioavailability of CSA immunosuppressant therapy. However, xanthohumol also inhibited the uptake of CSA in Caco-2 cells

(Figure 3.4A) when these cells were no longer a polarized cell monolayer so it is unclear what effects xanthohumol might have in other settings such as chemoresistant tumors expressing MDR phenotypes. Although the mechanism by which xanthohumol alters the uptake and efflux of CSA in Caco-2 and MDCKII-

MDR1 cells is not yet known, this flavonoid may serve as a lead compound for structure-activity relationships aimed to discover potent inhibitors of MDR1.

The above studies only partially explain the differences in transport inhibition due to flavonoid interactions. The observation that grapefruit juice enhances absorption of CSA but not digoxin has been attributed to the inherent bioavailability of digoxin (Dresser and Bailey, 2003). Our results suggest that this difference may be due to distinct transport processes utilized by each drug.

Digoxin is widely recognized as a MDR1 substrate, but the results presented in

Figure 3.1B provide evidence to suggest that it may share substrate specificity with other transporter(s) such as MRP2 as well or interacting with MDR1 at a different site than xanthohumol such at xanthohumol has no effect of the transport of digoxin (Figure 3.2B). Unlike CSA, the efflux of digoxin was not effectively inhibited by xanthohumol and MK-571, CSA, and verapamil significantly reduce 128

the Pe of digoxin from the B to A direction (Figure 3.1B). The findings of Lowes’ et al. (Lowes et al., 2003) support our findings of the inhibitory effects of CSA and

MK-571 on the transport of digoxin in Caco-2 cells, and Lowes et al. found that

CSA inhibited the transport of digoxin but MK-571 did not in MDCKII-MDR1 cells. Unlike their studies, our studies showed that MK-571 had an apparent inhibition of the efflux for digoxin, albeit non-significant.

The additional studies of CSA uptake inhibition suggest that this is indeed the case because the inhibition of CSA uptake with various concentrations of xanthohumol and EGCG at equilibrium demonstrated the effect over five logs of dose (Figure 3.4A). These data suggest that the effects seen in the transport studies are the result of more than one transport mechanism and/or multiple transporter(s). Xanthohumol appeared to have a higher affinity for at least one of the transporters involved compared to EGCG. In addition, xanthohumol and

EGCG increased cellular uptake of digoxin in Caco-2 cells (Figure 3.4B). These results are perplexing because xanthohumol also inhibits MDR1 activity as evidenced by its ability to reduce the efflux of CSA in a competitive manner

(Figure 3.1A) and suggests an alternate efflux pathway. CSA uptake studies confirm that there are multiple mechanisms involved and we hypothesize that an alternate transport mechanism exists for digoxin in Caco-2 cells that is distinct from CSA and not inhibited by plant polyphenols tested in our study. In a study using Caco-2 cells, the Pe of digoxin was enhanced with increasing lemon juice concentrations while there was a decrease in the Pe with increasing grapefruit and 129 pummelo juice concentrations from the B to A direction (Xu et al., 2003).

However, all the juices increased the Pe of digoxin from the A to B direction.

Neither xanthohumol nor EGCG increased the A to B transport of digoxin in either the Caco-2 cells or MDCKII-MDR1 cells.

A large-scale screening of the flavonoid family for their ability to modulate

MDR1 showed that prenylated flavonoids: bind with high affinity; strongly inhibited ATP hydrolysis; and cause drug interactions (Di Pietro et al., 2002). Our previous study revealed that xanthohumol was the most potent inhibitor of B to A transport of CSA in Caco-2 cells (data not shown). However, the mechanism for the inhibition of CSA transport by xanthohumol is not clear. CSA is a MDR1 substrate and it is tempting to conclude that the effect of xanthohumol on CSA transport is through inhibition of MDR1 activity. Our previous data also suggest that xanthohumol competes with CSA (at 0.5 µM) for transport in a competitive manner. However, in this study, xanthohumol had no effect on B to A transport of another MDR1 substrate, digoxin (Figure 3.1B), or on the uptake of calcein-AM

(data not shown). Furthermore, uptake of CSA itself in Caco-2 cells was inhibited rather than increased by xanthohumol (Figure 3.4).

Menohop® is commonly used to ease the menopause transition period in women and inhibits the proliferation of breast and uterine cancer cells (Heyerick et al., 2006) and it is also the first “hop” food supplement available on market.

Knowing its effect on altering the absorption of other drugs which are substrates for MDR1 is useful and necessary. Best to our knowledge, we are the first to 130 investigate its interactions of Menohop® with other drugs. In our results,

Menohop® extract is able to inhibit the efflux of CSA and digoxin and the significant effect starts at 0.01 mg/ml (Figure 3.6). Compared with our previous data looking at the effect of different pure compounds (may be the different components contained in Menohop® extract) on transport of CSA and digoxin

(Figure 3.7), the Menohop® effect is probably due to the effect of xanthohumol

(on transport of CSA) and isoxanthohumol (on transport of digoxin).

There may be clinical significance to inhibition of drug transport at concentrations that can be easily achieved in the intestinal lumen by the consumption of foods, beverages or dietary supplements. The possibility of interactions may need to be considered as dietary supplements containing phytochemicals are incorporated into treatment regimens that include concomitant administration of drugs that may utilize efflux or absorption pathways. Cautious use of plant polyphenols for such therapeutic actions in treatment regimens is warranted because there appears to be distinct mechanisms of transport from substrates that have been well accepted as MDR1 substrates. Clearly, more research into the function, specificity and regulation of drug and xenobiotic transporters is needed to fully understand the impact of plant polyphenols on these processes. 131

3.6 Appendix

Cytotoxicity of Menohop® extract in Caco-2 cells

In order to make sure all the concentrations used for the transport experiments were not toxic for the cells, cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme lactate dehydrogenase (LDH) into the medium and by assessing mitochondrial reduction of 3-(4,5-dimethythiazole-2yl)-2,5- diphenyl tetrazolium bromide (MTT). LDH catalyzes the reductive conversion of pyruvate to lactate with simultaneous oxidation of NADH to NAD+. The rate of disappearance of NADH in the presence of sodium pyruvate is directly proportional to LDH activity in the sample. Enzyme leakage into the medium was expressed as the percentage of total cellular LDH. The MTT assay is based on the reduction of the soluble yellow MTT tetrazolium salt to a blue MTT formazan product by mitochondrial dehydrogenases. It is a general measurement of mitochondrial dehydrogenase activity and cell viability. The detail description of

LDH and MTT, can be seen in Appendices (Protocol III).

Figure 3.10A shows MTT assay results of Menohop® extract in Caco-2 cells at 4 hours. Menohop® has an increase trend on % MTT reduction from 0.25

µg/ml to 250 µg/ml in Caco-2 cells (the statistic significant effect was seen at 100

µg/ml and 250 µg/ml). From 500 µg/ml to 1 mg/ml, Menohop® has a decrease on

% MTT reduction (the statistic significant effect starts at 750 µg/ml, from 100% to

68.82%). For the LDH assay, all the concentrations of Menohop® extract are not 132

A. MTT Assay

180 160 * * 140 120 100 4 hour 80 ** 60 **

% MTT Reduction 40 20 ** 0 (-) 0.25 0.5 1 µg/ml 2.5 5 µg/ml 10 25 50 100 250 500 750 1 75 µM control µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml mg/ml KT

B. LDH Assay

120

100 **

60 4 hour

40 **

% Total LDH Leakage Total % 20

0 tri-X (-) 0.25 0.5 1 µg/ml 2.5 5 µg/ml 10 25 50 100 250 500 750 1 75 µM control µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml mg/ml KT

Figure 3.10 Cytotoxicity (3-(4, 5-demethythiazole-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay results of Menohop® extract in Caco-2 cells at 4 hours. tri-X: Triton X-100. n = 5 - 8. Statistical significance from negative control is shown with asterisks, **, p < 0.01, *, p < 0.05.

133 toxic for the Caco-2 cells, except 1 mg/ml. It significantly increased the percentage of total LDH leakage compared with the negative control (from 2.8% to 30.26%) at 4 hours in Caco-2 cells. Therefore, concentrations of Menohop® did not exceed

500 µg/ml such that toxicity will not occur over the 4 hours transport period.

Effects of transport inhibitors on the transport of 0.5 µM 3H-CSA and 0.5 µM 3H-digoxin in LLC-MDR1 cells

Figure 3.11 shows the effect of transport inhibitors on transport of CSA and digoxin in LLC-MDR1 cells (overexpresses MDR1 gene). One hundred µM verapamil significantly increased the absorption of CSA and digoxin, whereas there was no significant effect seen in Pe of CSA and digoxin in B to A direction.

MK-571 significantly increased the Pe of CSA in A to B direction (p < 0.05) and digoxin in B to A direction (p < 0.01). Thirty µM XN, EGCG and ECG

-6 significantly inhibited the efflux Pe of CSA from 23.4 ± 11.0 × 10 cm/sec to 3.26

± 1.3 × 10-6 cm/sec, 6.65 ± 3.78 × 10-6 cm/sec, and 8.86 ± 2.75 × 10-6 cm/sec for

XN, EGCG and ECG, respectively (Figure 3.11A); whereas, there was no significant effect seen on digoxin transport (Figure 3.11B). 134

A. Cyclosporin A

60 * A to B 50 B to A ) -6 40

20 (cm/sec x 10 (cm/sec e P * 10 ** * ** 0

A il N m CG CS X E K-571 EGCG M µM M µM µ M 30 0 0.5 µ 3 0 30 µM µM verapa 1 0 0 1

B. Digoxin

12 11 A to B 10

) 9 B to A -6 8 7 6 5 4 (cm/sec x 10 (cm/sec e ** P 3 2 * ** 1 0 l in i G ox g M XN EC µ M M EGCG µ 30 µ 0 3 .5 µM di 30 0 10 µM MK-571 100 µM verapam

Figure 3.11 Effects of various transport inhibitors on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across LLC-MDR1 cell monolayers. Panel A: Effects of 100 µM verapamil, 50 µM MK-571, 30 µM xanthohumol (XN), EGCG and ECG on CSA transport from basolateral (B) to apical (A) (sink condition) and A to B (non-sink condition) directions. Data are represented as mean ± standard deviation, n = 4 - 6. Panel B: Effects of the treatment conditions on digoxin transport from the B to A and A to B directions. Data are represented as mean ± standard deviation, n = 4 - 6. Statistical significance from 0.5 µM CSA or digoxin is shown with asterisks, **, p < 0.01, and *, p < 0.05. 135

Endnotes

The vast majority of this chapter is published in Xenobiotica, January

2006, 36 (1): 41-58 (Rodriguez-Proteau et al., 2006).

136

4. BERBERINE AND BERBAMINE MODULATE P-GLYCOPROTEIN

Ying Fan & Rosita Rodriguez-Proteau

To be submitted to Xenobiotica, December, 2007

137

4.1 Abstract

Oregon Grape Root (OGR), Mahonia aquifolia, is native to the west coast of North America, and is also known by the names Mountain Grape and Holly-

Leaved Barberry. Berberine as an alkaloid found in the OGR as well as berbamine have been reported to have significant anticancer activity. The hypothesis of our study is that berberine and berbamine can modulate P-glycoprotein (P-gp), thereby, altering the apparent permeability of P-gp substrates, cyclosporine A

(CSA) and digoxin, across Caco-2 and MDCKII-MDR1 drug transport models.

Cytotoxicity was evaluated by measuring LDH leakage and mitochondrial function. Cells were exposed to various concentrations of OGR extract, berberine, or berbamine for 0 - 4 hour. There was no significant increase in LDH leakage or decrease in mitochondrial activity for OGR extract at 4 hour. However, there was a significant increase in LDH leakage and decrease in mitochondrial activity as early as 2 hour for 300 µM berbamine and 4 hour for 150 µM berberine (p < 0.01). 3H-

CSA/3H-digoxin (0.5 µM) transport experiments were performed with and without co-exposure of OGR extract, berberine, or berbamine. OGR extract inhibited the efflux of CSA and digoxin with a significant effect starting at 0.1 mg/ml. Ten µM berberine and 30 µM berbamine significantly reduced the apparent permeability

-6 -6 - coefficient (Papp) efflux of CSA (3.31 × 10 to 1.79 × 10 cm/sec and to 2.18 × 10

6 cm/sec for berberine and berbamine, respectively), while there was no measurable effect of berberine with digoxin. In MDCKII-MDR1 cells, 10 µM 138 berberine and 30 µM berbamine were capable of inhibiting the efflux of CSA

(13.1 × 10-6 to 5.34 × 10-6 cm/sec and 2.55 × 10-6 cm/sec, respectively) and digoxin (3.30 × 10-6 to 1.56 × 10-6 cm/sec and 1.71 × 10-6 cm/sec, respectively).

Thus, these data suggest that OGR extract, berberine, and berbamine can inhibit P- gp thereby may increase the bioavailability of drugs that are substrates of P-gp.

139

4.2 Introduction

Oregon grape root (Mahonia aquifolia), which is also known by the names barberry, mountain grape and holly-leaved barberry, has a direct action on the skin

(Succar, 1999). In folk medicine, it is used for chronic eruptions, rashes associated with pustules, and rashes associated with eating fatty foods (Dattner, 2003). It also has been reported to be used for gallbladder disease (Moga, 2003). Recently,

Oregon grape root extract has been shown to effectively treat inflammatory skin diseases, such as psoriasis and eczema (Bernstein et al., 2006; Donsky and Clarke,

2007).

Berberine is one of the alkaloids found in the Oregon grape root and has been used for many years in traditional Eastern medicine as an effective medication for the treatment of gastroenteritis and diarrhea (Chopra et al., 1932).

Later, many other pharmacological effects have been reported for berberine and its related proberine alkaloids, such as antimicrobial (Kaneda et al., 1991), antiarrhythmic (Sanchez-Chapula, 1996), anticancer (Iizuka et al., 2000), anti- inflammatory (Ckless et al., 1995; Kuo et al., 2004) and antiproliferative effects

(Jantova et al., 2007). Berbamine, another bisbenzylisoquinoline alkaloids, is widely used in traditional Chinese medicine as a source of leukogenics and have anti-arrhythmics, anti-hypertensives, and anticancer activity (Liu et al., 1983;

Shiraishi et al., 1987; Li et al., 1989). 140

Many studies have shown interaction of berberine and berbamine with P- glycoprotein (P-gp), which is the product of human ABCB1 (MDR1, multi-drug resistance 1) gene and a member of the ATP-binding cassette transporter family

(Tian and Pan, 1997; Lin et al., 1999a; Stermitz et al., 2000; He and Liu, 2002).

For example, it has been reported that 24 hour berberine treatment up-regulated P- gp expression in human and murine hepatoma cells (Lin et al., 1999a) as well as being able to up-regulate P-gp expression in cultured bovine brain capillary endothelial cells (He and Liu, 2002). Berbamine has been found to down-regulate expression of MDR1 mRNA and P-gp after 72 hour treatment in human erythroleukemic cells by using reverse transcriptase polymerase chain reaction

(RT-PCR) and flow cytometry (Han et al., 2003) as well as having similar activity to verapamil in reversing multi-drug resistance (MDR) in breast cancer cell line

(Tian and Pan, 1997). However, there are few publications showing the P-gp mediated drug-drug interaction with berberine (He and Liu, 2002; Maeng et al.,

2002; Pan et al., 2002), whereas there are no publications with berbamine or the crude extract Oregon grape root. This study’s hypothesis is that berberine, berbamine and Oregon grape root extract can modulate P-gp, thereby altering the apparent permeability of drugs that are substrates of P-gp, like cyclosporin A

(CSA) and digoxin.

Cyclosporin A is a potent immunosuppressive drug in the prevention of allograft rejection. It is metabolized by cytochrome P 450 3A4 to form hydroxylated and N-demethylated derivatives in both liver and intestine (Kronbach 141 et al., 1988; Combalbert et al., 1989; Kolars et al., 1991). Studies suggest that P-gp plays an important role in the oral bioavailability of cyclosporin A (Kaplan et al.,

1999; Masuda et al., 2003). Digoxin is an important cardiotonic drug with very narrow therapeutic window, 0.64 to 2.56 nM (Mooradian, 1988), and oral administration of P-gp mediated drugs, such as talinolol, have been shown to significantly increase the area under the concentration-time curve (AUC) from 0 to

72 hours of digoxin by 23% and significantly increased the maximum serum levels of digoxin by 45% in healthy human volunteers (Westphal et al., 2000).

P-gp was initially found in cancer cells, because it is overexpressed in cancer cells and it extrudes many anticancer drugs, such as doxorubicin and vinblastine, out of cancer cells (Dano, 1973; Juliano and Ling, 1976; Ueda et al.,

1987). Later, it has been observed that P-gp is also present in normal tissues, such as blood-brain barrier, intestine, kidney, liver, pancreas and adrenal gland. These organs are critical in removing toxins from the body or keeping toxins out of the body (Sun et al., 2004). In the intestine, P-gp acts as a secretory xenobiotic efflux pump, located almost exclusively within the brush border on the apical surface of mature enterocytes and plays an important role in limiting the absorption of xenobiotics in the intestine (Benet et al., 1999). Because P-gp has many substrates

(Table 4.1), P-gp limits the oral bioavailability of many drugs such as CSA

(Kaplan et al., 1999) and even the clinical relevant interactions between drugs and diets, such as indinavir and milk thistle (DiCenzo et al., 2003). Induction of 142

Table 4.1 Typical substrates of P-glycoprotein (P-gp)

Substrates

Anticancer drugs Actinomycin D, Daunonibicin, Docetacel, Doxorubicin, Etoposide, Imatinib, Paclitaxel, Tamoxifen, Teniposide, Vinblastine, Vincristine

Antibiotics Cefazolin, Erythromycin, Ofloxacin

Antihistamines Fexofenadine, Terfenadine

Ca2+-channel blockers Diltiazem, Verapamil

Cardiac drugs Digoxin, Digitoxin, Quinidine

Fluorencent dyes Rhodamine 123

HIV protease inhibitors Ritonavir, Inidnavir, Saquinavir

Immunosuppressants Cyclosporin A, Sirolimus, Tacrolimus Valspodar

Steroids Dexamethasone, Methylprednisolon

Miscellaneous drugs Amitryptiline, Colchicine, Itraconazole, Lansoprazole, Loperamide, Losartan, Morphine, Phenytoin, Rifampicin

References: (Ambudkar et al., 1999; Kerb et al., 2001; Litman et al., 2001; Dietrich et al., 2003; Ho and Kim, 2005) 143 intestinal P-gp of drugs such as rifampin decreased the bioavailability of P-gp substrate such as digoxin from 63% to 44% (p < 0.05) in healthy human volunteers

(Greiner et al., 1999). However, the molecular mechanisms of induction of P-gp are not understood. The role of the pregnane X receptor (PXR), a nuclear receptor superfamily of transcription factors, has been identified (Geick et al., 2001; Owen et al., 2004). A P-gp inducer can either activate cytoplasmic-nuclear translocation of constitutive androstane receptor (CAR) or directly activate PXR in the nucleus

(Handschin and Meyer, 2003). Both PXR and CAR can combine with the retinoid-

X-receptor (RXR) and bind to their respective response elements on target gene and increase the transcription of P-gp (Ambudkar et al., 1999; Handschin and

Meyer, 2003). Expression of human MDR1 (ABCB1) can be regulated by nuclear receptor PXR (Burk et al., 2005). It binds to the enhancer region located 8 kb upstream from the transcriptional initiation sites of ABCB1 and the activation of

PXR is known to increase the transcription of MDR1 gene and results in an increase in the expression of P-gp (Geick et al., 2001).

Caco-2 cells are derived from a human colon adenocarcinoma cell line and used to investigate the characteristics of intestinal absorption of drugs. There are many human genes functionally expressed in Caco-2 cells, such as MDR1, multidrug resistance associated protein 1 (MRP1), and multi-drug resistance associated protein 3 (MRP3) (Hirohashi et al., 2000; Taipalensuu et al., 2001). The Madin-

Darby Canine Kidney (MDCKII)-MDR1 cells, which overexpresses human MDR1

(Tang et al., 2002) have been well characterized and commonly used for MDR1- 144 mediated drug transport in comparison with Caco-2 and MDCKII wild-type cells

(Irvine et al., 1999; Guo et al., 2002). To investigate if P-gp is involved in the drug transport, the MDCKII-MDR1 cells were selected.

The expression of many transporters in Caco-2 cells is similar to that in the human gastrointestinal tract (Gottesman and Pastan, 1993; Goto et al., 2003).

However, the Caco-2 cells do not functionally express PXR (Thummel et al.,

2001). In addition, the LS-180 cells, were selected as a model of the human intestinal epithelium because it has abundant PXR (Mitin et al., 2004) and have been used for comparison of P-gp induction in Caco-2 cells (Perloff et al., 2001).

Therefore, we investigate the effects on gene expression of human MDR1

(ABCB1) in different cell models (Caco-2 and LS-180 cells) to elucidate the mechanism of P-gp modulation.

Interestingly, one report has evaluated the effect of berberine on P-gp in

Caco-2 cells and P-gp was found to modulate the efflux of berberine (Maeng et al.,

2002). Because Oregon grape root extract is known as a source of berberine and berbamine (Succar, 1999), we hypothesized that Oregon grape root extract will have similar effects as berberine and berbamine on modulating the transport of P- gp substrates. Therefore, berberine, berbamine and Oregon grape root extract modulation of P-gp in Caco-2 and MDCKII-MDR1 drug transport models will be evaluated using P-gp substrates, such as CSA and digoxin. 145

4.3 Material and method

4.3.1 Chemicals and reagents

Radiolabeled 3H-cyclosporin A (CSA, 7.0 Ci/mmol, 96.7% purity) and 3H- digoxin (23.5 Ci/mmol, 97% purity) were obtained from Amersham, Inc.

(Piscataway, NJ, USA). Berberine chloride, berbamine dihydrochloride, CSA, digoxin, verapamil, levothyroxine, rifampin, sodium pyruvate, L-lactic dehydrogenase (LDH), β-nicotinamide adenine dinucleotide, reduced form (β-

NADH), thiazolyl blue tetrazolium bromide and triethylamine (TEA) were purchased from Sigma, Inc. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Dulbecco’s Modified Eagle Medium

(DMEM) and non-essential amino acids were obtained from Invitrogen

Corporation (Grand Island, NY, USA). 0.01% penicillin/streptomycin was purchased from Mediatech, Inc. (Herndone, VA, USA). The 18s internal standard and MDR1 primers were purchased from Applied Biosystems (Foster City, CA,

USA). Solvent acetonitrile was HPLC grade and was purchased from Fisher

Scientific (Fair Lawn, NJ, USA). Ammonium acetate was purchased from

Mallinckrodt Baker Inc. (Paris, KY, USA). Oregon grape root extract 1 was a gift from Oregon’s Wild Harvest (Sandy, OR, USA). Oregon grape root extract 2 was purchased as a root powder from Health Herbs (Philomath, OR, USA). All other reagents were analytical grade.

146

4.3.2 Oregon grape root extract preparation

Oregon grape root extract 1 (E1) (liquid):

One ml of Oregon grape root extract 1 ethanol solution was transfered to two amber glass vials (3.5 cm × 6 cm) and 5 ml distilled water was added to each vial.

Then, the amber glass vial was stored at -80 ˚C for 1 hour. Afterwards, the frozen sample was using FreeZone® freeze dry system (Kansas City, MO, USA) to obtain E1 as a dryness powder. The final weight of product yield was 25 mg/ml.

To prepare the stock solution of the study, the E1 (100 mg) was transferred to a glass vial (0.6 cm × 2.75 cm), 0.25 ml of ethanol (200 proof purity) and 0.75 ml of distilled water were added. Then serial dilutions (0.05, 0.1, 0.5, 1, 2 mg/ml) were made with serum-free and antibiotics-free medium (cytotoxicity and real time RT-

PCR studies) or Hanks’ buffered salt solution with 10 mM HEPES and 25 mM D- glucose (transport studies). The highest percentage of ethanol was 0.15%. The stock solution was stored at -20 ˚C and the dilutions were prepared fresh on the day for each experiment and kept away from direct light.

Oregon grape root extract 2 (E2) (root powder):

Exactly 1.5 g of Oregon grape root extract 2 root powder was transferred in a 250 ml Erlenmeyer, 30 ml methanol was added, sonicate in water bath at 25 ˚C for 5 min, moderate hand-shaken for 5 min 2, 200 rpm, and was transferred to two 15 ml centrifuge tube and centrifuge (CentrificTM Centrifuge model 225, Fisher

Scientific, Pittsburgh, PA, USA) 5 min at 2, 200 rpm. The supernatant was taken and stored at 4 ˚C. To prepare the E2 powder, 1 ml supernatant was transferred to 147 two amber glass vials (3.5 cm × 6 cm) and 5 ml distilled water was added to each vial. Then, the amber glass vial was stored at -80 ˚C for 1 hour. Afterwards, the frozen sample was using FreeZone® freeze dry system (Kansas City, MO, USA) to obtain E2 as a dryness powder. To prepare the stock solution of the study, the

E2 (100 mg) was transferred to a glass vial (0.6 cm × 2.75 cm), 0.25 ml of ethanol

(200 proof purity) and 0.75 ml of distilled water were added. Then serial dilutions

(0.05, 0.1, 0.5, 1, 2 mg/ml) were made with serum-free and antibiotics-free medium (cytotoxicity studies and real time RT-PCR studies) or Hanks’ buffered salt solution with 10 mM HEPES and 25 mM D-glucose (transport studies). The highest percentage of ethanol was 0.15%. The stock solution was stored at -20 ˚C and the dilutions were prepared fresh on the day for each experiment and kept away from direct light.

4.3.3 Cell culture

The human intestinal epithelial cell line, LS-180 was purchased from

American Type Culture Collection (Manassas, VA, USA) and grown in Minimum

Essential Medium supplemented with 10% FBS. The Caco-2 cell line was obtained from American Type Culture Collection (Rockville, MD, USA).

MDCKII-MDR1 and MDCKII wild-type cell lines were a generous gift from Dr.

Piet Borst (The Netherlands Cancer Research Institute). Caco-2 cells were grown with DMEM, 10 % FBS, and 0.1 mM non-essential amino acids, and 0.01% 148 penicillin/streptomycin (Evers et al., 1996; Evers et al., 1998). MDCKII-MDR1 and MDCKII wild-type cell line was treated in DMEM, 10 % FBS, and 0.01% penicillin/streptomycin. All cell lines were maintained at 37 °C with 5% CO2, and

95% relative humidity. For the transport experiments, the cells were seeded, 300,

000 cells/well, onto 6-well Transwell® inserts 4.71 cm2 (Corning Costar,

Cambridge, MA, USA) and maintained until use on days 21 to 25 (Caco-2, passages 23 - 30) and days 6 to 7 (MDCKII-MDR1 and MDCKII wild-type). The integrity of the monolayers was assessed by measuring transepithelial electrical resistance (TEER) using a World Precision Instrument, EVOM (Sarasota, FL,

USA) and evaluating 14C-mannitol transport. The average TEER readings were

625.94 ± 75.47 Ωcm2 (Caco-2), 476.91 ± 101.21 Ωcm2 (MDCKII-MDR1) and

211.52 ± 22.33 Ωcm2 (MDCKII wild-type). Also, permeability studies with 14C- mannitol were performed and the transport rate was less than 1% per hour throughout the entire experiment. The apparent permeability (Papp) of mannitol was 2.09 ± 0.40 × 10 -6 cm/s. These data indicates that the cell monolayer was not compromised. For the real time RT-PCR, cells were seeded 600,000 cells/well

(Caco-2) and 1,000,000 cells/well (LS-180) onto 6-well Transwell® inserts 4.71 cm2 (Corning Costar, Cambridge, MA, USA) and grown for 7 (Caco-2) and 6 (LS-

180) days.

4.3.4 Chemical exposure 149

We initiated cytotoxicity experiments with the Caco-2, MDCKII-MDR1 and MDCKII-wild type cells on day 7 (Caco-2) and day 4 (MDCKII-MDR1 and

MDCKII-wild type) after the initial plating of the cells at 25, 000 cells/well in 48- well plates (Becton Dickinson and company, Franklin lakes, NJ, USA). Stock solutions of 10 mM berberine or berbamine were prepared by dissolving the compounds in DMSO (berberine) or distilled water (berbamine). Stock solutions of 100 mg/ml E1 or E2 were prepared by dissolving 100 mg E1 or E2 in 25% ethanol (200 proof purity) with 75% distilled water. Then, the stock solutions of berberine, berbamine, E1 and E2 were diluted with serum-free and antibiotics-free medium to yield final concentrations ranging from 0.03 µM to 300 µM berberine and berbamine, 0.05 mg/ml to 2 mg/ml E1 and E2. The final concentration of

DMSO was 0.15% (v/v) DMSO/culture medium and the final concentration of ethanol was 0.15% (v/v) ethanol/culture medium. Ketoconazole (75 µM) was used as positive control and no treatment (medium alone) was used as negative control for each experiment.

For the transport experiments, cells grown on Transwell® inserts were exposed to test compounds on 21-30 days (Caco-2) or 6-7 days (MDCKII-MDR1 and MDCKII-wild type). Stock solutions of 10 mM berberine or berbamine were prepared as described above. Stock solutions of 100 mg/ml E1 or E2 were prepared by dissolving the 100 mg E1 or E2 in 25% ethanol (200 proof purity) with 75% distilled water. Then, the stock solutions of berberine, berbamine, E1 and E2 were diluted with Hank’s buffered salt solution (HBSS) with 10 mM 150

HEPES and 25 mM D-glucose at a pH of 6.8 for the apical (A) compartment and

7.4 for the basolateral (B) compartment to yield final concentrations of 10 µM through 100 µM berberine and berbamine and 0.05 mg/ml to 1 mg/ml for E1 and

E2. The final treatment concentrations of DMSO was 0.15% (v/v) and of ethanol was 0.15% (v/v). The MDR1 transport inhibitor, 100 µM verapamil, was used as a positive control and no treatment was used as a negative control.

For the real time RT-PCR studies, the cells 600, 000 cells/well (Caco-2) or

1, 000, 000 cells/well (LS-180) were grown on Transwell® inserts and exposed to test compounds on day 7 (Caco-2) or day 6 (LS-180). Stock solutions of 100 mg/ml E1 and E2 were prepared by dissolving the compounds in 25% ethanol (200 proof purity) with 75% distilled water. Stock solutions of 100 µM levothyroxine or

10 µM rifampin were prepared by dissolving compounds in 50% DMSO with 50% distilled water. Then, the stock solutions of E1, E2, levothyroxine or rifampin were diluted with serum-free and antibiotics-free medium to yield final concentrations

0.1 mg/ml E1, 0.25 mg/ml E1, 0.1 mg/ml E2, 0.25 mg/ml E2 and 100 µM levothyroxine or 10 µM rifampin. The final treatment concentration of ethanol or

DMSO was 0.15% (v/v) ethanol or DMSO/culture medium. Levothyroxine 100

µM and rifampin 10 µM were used as positive controls for Caco-2 and LS-180 cells, respectively. No treatment (medium alone) was used as the negative control.

151

4.3.5 Cytotoxicity assays

MTT assay

The MTT (3-(4,5-dimethythiazole-2yl)-2,5-diphenyl tetrazolium bromide) reduction assay was done according to (Mosmann, 1983). The MTT assay is based on the reduction of the soluble yellow MTT tetrazolium salt to a blue MTT formazan product by mitochondrial dehydrogenases. It is a general measurement of mitochondrial dehydrogenase activity and cell viability. This assay was conducted immediately after the 2-, 4-, 6-, and 8-hour treatments. Briefly, MTT was dissolved in serum- and antibiotic-free culture medium at concentration of 0.5 mg/ml and filtered in 10 ml syringe (Becton Dickinson and Company, Franklin

Lakes, NJ, USA) with 0.2 µM pore size 25 mm diameter polythersulfone membrane (Whatman Inc., Florham Park, NJ, USA) to remove small amounts of insoluble residue. MTT-containing medium was added to each well in a volume of

0.25 ml and incubated for 2 hours. Thereafter, the supernatant was removed and

0.625 ml of 0.4 N-HCl-isopropanol (1:24, v/v) was added to solubilize the intracellular MTT formazan product for a period of 30 min at 25 ˚C in the dark.

Afterwards, 250 µl formazan solution was transferred to a 96-well plate (Falcon®,

Becton Dickinson and company, Franklin lakes, NJ, USA) and the absorbance was read at 570 nm and 630 nm on the SpectroMax 190 (Molecular Devices Co.,

Sunnyvale, CA, USA). The results were expressed as the percentage of negative controls.

152

Lactate dehydrogenase (LDH) assay

The LDH enzymatic activities were assayed using a procedure previously developed in our laboratory for cell cultures (Mitchell et al., 1980). Briefly, the reaction mixture contained 130 µl of warm 0.1 M phosphate buffer (pH 7.4), 25 µl

0.25% (w/v) reduced NADH solution, 25 µl 1.0% (w/v) sodium pyruvate solution, and a 25 µl test sample. The test samples were obtained from the media after the

2-, 4-, 6-, and 8-hour treatments with berberine, berbamine, E1 and E2. The reaction mixture was mixed and immediately placed into a SpectroMax 190 with

SOFTmax® PRO (Molecular Devices Co., Sunnyvale, CA, USA) at 37 ˚C.

Absorbance at 340 nm was recorded at intervals of 30 s for 3 min. LDH catalyzes the reductive conversion of pyruvate to lactate with simultaneous oxidation of

NADH to NAD+. The rate of disappearance of NADH in the presence of sodium pyruvate is directly proportional to LDH activity in the sample. Enzyme leakage into the medium was expressed as the percentage of total cellular LDH.

4.3.6 Transport studies

Transport of 3H labeled compounds (CSA and digoxin) were performed using a transport buffer consisting of HBSS with 10 mM HEPES and 25 mM D- glucose at a pH of 6.8 for the apical (A) compartment and 7.4 for the basolateral

(B) compartment. Experiments were performed at 37 °C, 5% CO2, and 95% relative humidity. Cells in the Transwell® inserts were washed 3 times with 153 transport buffer and allowed to equilibrate for 30 min prior to the addition of test compounds. The concentrations of MDR1 substrates were 0.5 µM for both CSA and digoxin. The concentrations of berberine and berbamine were 3 µM, 10 µM,

30 µM and 100 µM. For the Oregon grape root extract studies, the concentrations of E1 and E2 were 0.05, 0.1, 0.25, 0.5 and 1 mg/ml. The MDR1 transport inhibitor used was 100 µM verapamil. Berberine, berbamine, E1, E2 and verapamil were prepared in transport buffer at the appropriate pH (pH 6.8 for the A compartment and 7.4 for the B compartment) and allowed to incubate at 37 °C for 30 min prior to the start of the experiment. Berberine, berbamine, E1, E2 and verapamil were present in both the A and B chambers during the transport of the MDR1 substrates

(CSA and digoxin). To evaluate the efflux mechanism involved with berberine and berbamine, 100 µM of berberine and berbamine were placed in donor chamber and

100 µl samples were collected in the receiver chamber at 0.5, 1.0, 1.5, 2.0, 2.5, and

3.0 hour and analyzed by HPLC (see HPLC section for experimental details).

Ethanol was used as the solvent vehicle (0.15%v/v) for CSA and verapamil and

DMSO (0.15%v/v) was used as the solvent vehicle for digoxin and berberine.

Each experiment was performed in duplicate and repeated for each condition tested. For experiments performed in “non-sink” conditions, 100 µl aliquots were taken from the donor and receiver chambers at the beginning and from the receiver chamber at 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 hour. An equal volume of 100 µl was replaced in the chamber at each sample time point. ‘Non-sink’ conditions were performed when less than 10% of compound is transported across cells during the 154 transport experiment such that a concentration gradient was always present. For the “sink” condition experiments, the entire receiver volume was replaced at each time interval and a 100 µl aliquot was taken from the receiver volume for scintillation counting. A 100 µl sample was taken from the donor chamber at 3 hr in order to calculate the mass balance of radioactivity to determine if adsorption to the cell culture transport apparatus had occurred. Each sample was placed in 0.9 ml of scintillation fluor (Cytoscint ES, ICN, Cosa Mesa, CA, USA) and read on a

Beckman LS 6500 (Palo Alto, CA, USA) scintillation counter for 3H activity. For the transport of berberine and berbamine, 100 µl aliquot was assayed by HPLC.

The apparent permeability (Papp) was calculated using the following equation (Taub et al., 2005):

Papp = dQ/dt × 1/(A × C0)

Efflux ratio was determined by dividing the Papp in the B to A direction by the Papp in the A to B direction (Crivori et al., 2006):

Efflux ratio = Papp(B to A)/Papp(A to B)

4.3.7 Real time quantitative PCR analysis of MDR1 mRNA 155

Caco-2 or LS-180 cells were grown on Transwell® inserts, 4.71 cm2

(Corning Costar, Cambridge, MA, USA) for 7 (Caco-2) and 6 (LS-180) days. The cells were exposed to different concentration of E1, E2, levothyroxine or rifampin for 24 hours and 48 hours. Total RNA was isolated by adding 1 ml Trizol® reagent (Gibco-BRL, Carlsbad, CA, USA) to the cells and processed according to the manufacturer’s instructions. The concentration and purity of isolated RNA samples were measured using Quant-iTTM RiboGreen® RNA assay kit (Invitrogen,

Eugene, OR, USA). The RNA sample (0.06 µg) was reverse transcribed by a iScriptTM cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA).

Relative quantification of gene expression was performed by iTaqTM SYBR®

Green supermix with ROX (internal reference dye) (Bio-Rad Laboratories,

Hercules, CA, USA) using an ABI Prism 7500 Real Time PCR system (Applied

Biosystems, Foster City, CA, USA). Each experiment was performed in duplicate and repeated for each condition tested (n = 4). The mRNA levels of all genes were normalized using 18s as internal control (Toft-Hansen et al., 2007). Results are expressed as ratios of MDR1 to 18s expression.

4.3.8 High Performance Liquid Chromatography (HPLC) analysis

A Waters 2690 separations module with a Model 996 photodiode array detector (Waters Corporation, Milford, MA, USA) was used for HPLC analysis.

The system was operated with the Millennium 32 Chromatograph Manager 156 version 3.0 software (Waters Corporation, Milford, MA, USA). E1 (1 mg/ml), E2

(1 mg/ml), berberine standard (1 mg/ml), and berbamine standard (1 mg/ml) were prepared by dissolving 1 mg E1, E2, berberine or berbamine in 50% methanol and

50% distilled water. Then, all the samples and standards were filtered using a syringe with a 0.2 µm pore size Waterman® syringe filter (Whatman Inc., Clifton,

NJ, USA) before injection. The injection volume was 10 µl. The column used was a 250 × 4.6 mm i.d., 5 µM, Zorbax Eclipse XDB-C18 Column (Agilent

Technologies, Palo Alto, CA, USA) with a 3.9 × 20 mm i.d. Symmetry® C18 guard column (Waters Corporation, Milford, MA, USA), with an isocratic mobile phase (32 acetonitrile: 68 buffer). The buffer consisted of 30 mM ammonium acetate and 14 mM TEA, adjusted to pH 4.85 with acetic acid. The flow rate was 1 ml/min and the UV absorption spectrum at 254 nm was used for analysis. The retention time of berberine and berbamine was 7.84 min and 3.03 min, respectively. The cumulative amount transport was determined using the corresponding peak area for berberine or berbamine. The cumulative amount of berberine or berbamine transported across the cell membrane was evaluated as function of time. The apparent permeability was calculated by the following equation:

Papp = dQ/dt × 1/(A × C0)

2 Where A is the surface area of the monolayer (4.71 cm ) and C0 is the initial concentration of berberine or berbamine in the donor compartment. dQ/dt is the slope of the steady-state rate constant. 157

4.3.9 Liquid Chromatography/Mass Spectrometry (LC/MS) analysis

LS/MS analysis was performed using a Surveyor LC pump (Thermo

Finnigan, San Jose, CA, USA) with Surveyor PDA detector, coupled with

Surveyor autosampler system hyphenated to a LCQ advantage mass spectrometer

(Thermo Finnigan, San Jose, CA, USA) equipped with an electrospray ionization

(ESI) source. The separation was achieved using a reverse-phase C18 column 250

× 4.6 mm i.d., 5 µM, Zorbax Eclipse XDB-C18 (Agilent Technologies, Palo Alto,

CA, USA) with a 3.9 × 20 mm i.d. Symmetry® C18 guard column (Waters

Corporation, Milford, MA, USA), pumped isocratically with acetonitrile: buffer

(32:68). The buffer consisted of 30 mM ammonium acetate and 14 mM TEA, adjusted to pH 4.85 with acetic acid.

4.3.10 Statistical analysis

(ANOVA) followed by a Dunnett’s multiple comparison post-test which compare all the treatments with control. Differences between A and B transport were compared using a t-test. A probability of difference less than 0.05 (p < 0.05) was considered to be statistically significant. 158

4.4 Results

Cytotoxicity of berberine, berbamine, and Oregon grape root extract

Table 4.2 represents the cytotoxicity data of berberine and berbamine in Caco-2,

MDCKII-MDR1 and MDCKII wild-type cells assessed by the MTT and LDH assay. After four hours of exposure, 150 µM and 300 µM berberine significantly decreased the percentage MTT reduction (100 ± 0.92% to 76.37 ± 2.13%, p <

0.0001, and to 58.25 ± 9.24%, p < 0.0001, for 150 µM and 300 µM, respectively) in Caco-2 cells. The percentage total LDH leakage was significantly increased by berberine at 150 and 300 µM as well in Caco-2 cells (3.80 ± 1.34% to 25.93 ±

4.78%, p < 0.0001, and to 44.88 ± 2.09%, p < 0.0001, for 150 µM and 300 µM, respectively). Three hundred µM berbamine significantly decreased the percentage

MTT reduction from 100 ± 11.59% to 64.99 ± 11.46% (p < 0.0001), and significantly increased the percentage total LDH leakage from 4.78 ± 0.72% to

43.88 ± 7.44% (p < 0.0001). All concentrations of berberine and berbamine in

MDCKII-MDR1 and MDCKII wild-type cells were not toxic after 4 hours exposure, except for 300 µM berberine and berbamine in the MDCKII wild-type cells. Therefore, all berberine and berbamine concentrations were equal or less than 100 µM to investigate the effects of P-gp-mediated transport in the Caco-2,

MDCKII-MDR1 and MDCKII wild-type cells. In addition to the dose-response, a time-response was also performed at 2, 4, 6, and 8 hours. There was a significant 159

Table 4.2 Dose-response cytotoxicity data of berberine and berbamine from the 3- (4, 5-demethythiazole-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays in Caco-2, MDCKII-MDR1 and MDCKII wild-type (MDCKII-WT) cells after 4 hours exposure. Values represent percentage of control.

Caco-2 cells MDCKII-MDR1 cells MDCKII-WT cells

MTT assay LDH assay MTT assay LDH assay MTT assay LDH assay (%) (%) (%) (%) (%) (%)

Berberine 0.03 µM □ ■ □ ■ □ ■ 3.0 µM □ ■ □ ■ □ ■ 30 µM □ ■ □ ■ □ ■ 100 µM □ ■ □ ■ □ ■ 150 µM 76.37 ± 2.13↓ 25.93 ± 4.78↑ □ ■ □ ■ 300 µM 58.25 ± 6.37↓ 44.88 ± 2.09↑ □ ■ 24.82 ± 15.91 ↓ ■ Berbamine 0.03 µM □ ■ □ ■ □ ■ 0.3 µM □ ■ □ ■ □ ■ 30 µM □ ■ □ ■ □ ■ 100 µM □ ■ □ ■ □ ■ 150 µM □ ■ □ ■ □ ■ 300 µM 64.99 ± 11.46↓ 43.88 ± 7.44↑ □ ■ 64.32 ± 13.1 ↓ ■

□: %MTT reduction is not significantly decreased compared with the negative control; ■: % Total LDH leakage is not significantly increased compared with the negative control. ↓: %MTT reduction is significantly decreased compared with the negative control (p < 0.01); ↑: % Total LDH leakage is significantly increased compared with the negative control (p < 0.01). n = 6.

160

increase in LDH leakage and decrease in MTT reduction as early as 2 h for 300

µM berbamine (p < 0.0001) and 4 h for 150 µM berberine (p < 0.0001) (data not shown).

Cytotoxicity studies of Oregon grape root extract 1 (E1) and Oregon grape root extract 2 (E2) using MTT and LDH assay in Caco-2 and LS-180 cells are shown in Table 4.3. After 4 hours of exposure, all concentrations evaluated for E1 and E2 were not toxic in the Caco-2 cells except for 2 mg/ml. Two mg/ml E1 and

E2 significantly decreased percentage MTT reduction (98.23 ± 12.14% to 54.26 ±

2.15%, p < 0.0001, and to 32.76 ± 10.76%, p < 0.0001, for E1 and E2, respectively). It also significantly increased % total LDH leakage (2.40 ± 0.49% to

9.99 ± 0.94%, p < 0.0001, and to 7.38 ± 1.31%, p < 0.0001, for E1 and E2, respectively). Therefore, in the transport studies, the highest concentration used was 1 mg/ml E1 and E2 in Caco-2 cells. For the real time RT-PCR studies, 24 and

48 hour exposures were performed with concentrations equal or less than 0.25 mg/ml in the Caco-2 and LS-180 cells for doing pretreatment for real time RT-

PCR studies.

161

Table 4.3 Dose-response cytotoxicity data of Oregon grape root extract (E1) and Oregon grape root extract (E2) from the 3-(4, 5-demethythiazole-2yl)-2, 5- diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays in Caco-2 and LS-180 cells. Values are expressed as a percentage of control values.

Caco-2 Cells LS-180 Cells 4 hours 24 hours 48 hours 24 hours 48 hours

MTT LDH MTT LDH MTT LDH MTT LDH MTT LDH (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)

E1 0.05 mg/ml □ ■ □ ■ □ ■ □ ■ □ ■ 0.1 mg/ml □ ■ □ ■ □ ■ □ ■ □ ■ 0.25 mg/ml □ ■ □ ■ □ ■ □ ■ □ ■ 0.5 mg/ml □ ■ □ ■ □ 12.56↑ □ ■ □ ■ 1 mg/ml □ ■ 30.16↓ 11.11↑ 4.73 ↓ 19.28↑ 52.06↓ 27.55↑ 20.35↓ 25.93↑ 2 mg/ml 54.26↓ 9.99↑ 1.41↓ 16.54↑ 2.57↓ 19.10↑ 29.46↓ 27.96↑ 11.98↓ 29.49↑ E2 0.05 mg/ml □ ■ □ ■ □ ■ □ ■ □ ■ 0.1 mg/ml □ ■ □ ■ □ ■ □ ■ □ ■ 0.25 mg/ml □ ■ □ ■ □ ■ □ ■ □ ■ 0.5 mg/ml □ ■ □ ■ □ 10.21↑ □ ■ □ ■ 1 mg/ml □ ■ 16.98↓ 8.32↑ 21.48↓ 12.92↑ 35.24↓ 23.86↑ 33.21↓ 25.85↑ 2 mg/ml 32.76↓ 7.38↑ 14.29↓ 9.92↑ 31.70↓ 19.72↑ 19.82↓ 36.13↑ 19.26↓ 40.26↑

Transepithelial transport of berberine and berbamine in Caco-2 and MDCKII-MDR1 cells

In order to investigate whether carrier-mediated transport is involved in the transepithelial transport of berberine and berbamine, the transport of 100 µM berberine and berbamine were determined as a function of time in Caco-2 cells

(Figure 4.1) and MDCKII-MDR1cells (Figure 4.2). Because berberine has been shown to be actively effluxed by Caco-2 cells (Maeng et al., 2002), the efflux mechanism speculated to be involved in berberine and berbamine transport was evaluated by measuring their permeability and the amount transported across the

Caco-2 and MDCKII-MDR1 cells from the B to A and the A to B direction. Figure

4.1 shows that the transport of berberine and berbamine was faster from the B to A direction (1.45 nmol/min, berberine, and 5.94 nmol/min, berbamine) than that in the A to B direction (0.18 nmol/min, berberine, and 0.39 nmol/min berbamine) in

Caco-2 cells. Similar results were seen in MDCKII-MDR1 cells as well (Figure

4.2). The Papp of berberine and berbamine from the B to A direction was also significantly greater than the A to B direction in Caco-2 and MDCKII-MDR1 cells

(Figures 4.1 4.2 insets). For example, approximately a 4-fold increase of Papp was observed in the B to A direction (10.4 × 10 -6 cm/s) compared with the A to B direction (2.35 × 10 -6 cm/s) for 100 µM berberine (Figure 4.1A) and about 30-fold

-6 increase of Papp was seen in the B to A direction (60.3 × 10 cm/s) compared with the A to B direction (2.49 × 10 -6 cm/s) for 100 µM berbamine (Figure 4.1B). This suggests that there is an efflux mechanism involved in the transport of both 163

Caco-2

A. Berberine

11 B to A 10 A to B 9 250 B to A 8 7

A to B (cm/sec) 6 **

200 -6 5 **

x 10 4 3 150 app P 2 1 100 (nmole) 0

0 Cumulative Amount Transport Transport Amount Cumulative 0 50 100 150 200 Time (min)

B. Berbamine t 65 B to A 1400 B to A 60 55 A to B 1200 A to B 50 45 40

1000 (cm/sec) 35 ** -6 30 ** 25

800 x 10 20

app 15 600 P 10 (nmole) 5 400 0 200 0

Cumulative Amount Transpor 0 50 100 150 200 Time (min)

Figure 4.1 Time course of the transport of 100 µM berberine (Panel A) or 100 µM berbamine (Panel B) from the apical to the basolateral (A to B, □) and the B to A (♦) across Caco-2 cells. The inset shows the apparent permeability (Papp) of berberine 100 µM and berbamine 100 µM from the A to B and the B to A direction. Data are presented as mean ± standard deviation, n = 4. 164

MDCKII-MDR1 A. Berberine

400 B to A 17.5 B to A A to B 350 15.0 A to B 300 12.5

10.0 (cm/sec) ** 250 -6 ** 7.5

200 x 10 5.0 app P (nmole) 150 2.5

100 0.0

Cumulative Amount Transport Transport Amount Cumulative 0 0 50 100 150 200 Time (min)

B. Berbamine

900 45 B to A B to A 40 A to B 800 A to B 35 30 700

(cm/sec) 25 ** -6 ** 600 20

x 10 x 15

500 app

P 10 400 5

(nmole) 0 300 200 100

Cumulative Amount Amount Transport Cumulative 0 0 50 100 150 200 Time (min)

Figure 4.2 Time course of the transport of 100 µM berberine (Panel A) or 100 µM berbamine (Panel B) from the apical to basolateral (A to B, □) and the B to A (♦) across MDCKII-MDR1 cells. The inset shows the apparent permeability (Papp) of berberine 100 µM and berbamine 100 µM from the A to B and the B to A direction. Data are presented as mean ± standard deviation, n = 4. 165

berberine and berbamine in the Caco-2 cells. In MDCKII-MDR1 cells, there was a greater efflux seen for berberine. Approximately 12-fold increase of Papp was observed in the B to A direction (15.6 × 10 -6 cm/s) compared with the A to B direction (1.3 × 10 -6 cm/s) (Figure 4.2A). For berbamine, less efflux effect was seen in MDCKII-MDR1 cells compared with that in Caco-2 cells (Figure 4.2B).

Effect of berberine and berbamine on transport of 3H-CSA and 3H- digoxin

In Caco-2 cells, the B to A transport of 0.5 µM 3H-CSA was decreased by berberine and berbamine with the significance seen as low as 10 µM for berberine

-6 -6 (Papp B to A of CSA from 3.31×10 cm/s to 1.79 ×10 cm/s, p < 0.0001) and 30

-6 -6 µM for berbamine (Papp B to A of CSA from 2.83 ×10 cm/s to 2.18 ×10 cm/s, p < 0.0001) (Figure 4.3 A, Table 4.4). For digoxin, berbamine significantly

3 -6 inhibited efflux of 0.5 µM H-digoxin (Papp B to A of digoxin from 2.78 ×10 cm/s to 1.79 ×10 -6 cm/s for 30 µM berbamine, p = 0.0253, and to 1.23 ×10 -6 cm/s for 100 µM berbamine, p = 0.0021), whereas there were no significant effects of berberine on digoxin transport (Figure 4.3B, Table 4.4). In MDCKII-MDR1 cells, berberine and berbamine significantly decreased the efflux of 0.5 µM 3H-CSA

-6 -6 (Papp B to A of CSA from 13.10 ×10 cm/s to 5.34 ×10 cm/s for 10 µM berberine, p < 0.0001, and to 2.55 ×10 -6 cm/s for 30 µM berbamine, p < 0.0001)

3 -6 and the efflux of 0.5 µM H-digoxin (Papp B to A of digoxin from 3.30 ×10 cm/s to 1.56 ×10 -6 cm/s for 10 µM berberine, p = 0.0232, and to 1.71 ×10 -6 cm/s for 30

µM berbamine, p = 0.0248) (Figure 4.4, Table 4.4). Similar results were seen in 166

Caco-2

A. Cyclosporin A

3.5 B to A 3.0 A to B

2.5 **

2.0 (cm/sec) ** **

-6 ** ** ** 1.5

x 10 1.0 app P 0.5

0.0 CSA 0.5 µM + V 100 µM + 10 µM + 30 µM + 30 µM + 100 µM Berberine Berbamine B. Digoxin

3 B to A A to B

2 * (cm/sec) -6 *

x 10 1 ** app P

0 dig 0.5 µM V 100 µM + 10 µM + 30 µM + 30 µM + 100 µM

Berberine Berbamine

Figure 4.3 Effects of berberine and berbamine on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) in the Caco-2 cells. Panel A: Effects of berberine (10 and 30 µM), berbamine (30 and 100 µM) and 100 µM verapamil on transport of CSA from basolateral the (B) to apical (A) direction. Panel B: Effects of berberine (10 and 30 µM), berbamine (30 and 100 µM) and 100 µM verapamil on transport of digoxin from the B to A direction. All data represents mean ± standard deviation, n = 4. V: verapamil. Statistical significance from 0.5 µM CSA or 0.5 µM digoxin is shown with asterisks, **, p < 0.01, *, p < 0.05.

167

MDCKII-MDR1

A. Cyclosporin A

15 B to A

10 (cm/sec) -6 ** ** ** 5 x 10 ** ** app P 0 CSA 0.5 µM+ V 100 µM + 10 µM + 30 µM + 30 µM + 100 µM

berberine berbamine

B. Digoxin

4 B to A 3

(cm/sec) **

-6 * 2 * * **

x 10 1 app P 0 dig 0.5 µM+ V 100 µM + 10 µM + 30 µM + 30 µM + 100 µM

berberine berbamine

Figure 4.4 Effects of berberine and berbamine on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII- MDR1 cells. Panel A: Effects of berberine (10 and 30 µM) and berbamine (30 and 100 µM) and 100 µM verapamil on transport of CSA from the basolateral (B) to apical (A) direction. Panel B: Effect of berberine (10 and 30 µM) and berbamine (30 and 100 µM) on transport of digoxin from the B to A direction. Data are represented as mean ± standard deviation, n = 4. V: verapamil. Statistical significance from 0.5 µM CSA or 0.5 µM digoxin is shown with asterisks, **, p < 0.01, *, p < 0.05. 168

MDCKII Wild-Type

A. Cyclosporin A

7.5 B to A

5.0 ** ** ** ** (cm/sec) ** -6

2.5 x 10 app P 0.0 CSA 0.5 µM+ V 100 µM + 10 µM + 30 µM + 30 µM + 100 µM

berberine berbamine

B. Digoxin

8 B to A 7 6 ** 5 ** (cm/sec) ** -6 4 3

x 10 2

app 1 P 0 dig 0.5 µM + V 100 µM + 10 µM + 30 µM + 30 µM + 100 µM

berberine berbamine

Figure 4.5 Effects of berberine and berbamine on the transport of 0.5 µM 3H- cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across MDCKII wild- type (MDCKII-WT) cells. Panel A: Effects of berberine (10 and 30 µM) and berbamine (30 and 100 µM) and 100 µM verapamil on transport of CSA from the basolateral (B) to apical (A) direction. Panel B: Effect of berberine (10 and 30 µM) and berbamine (30 and 100 µM) on transport of digoxin from the B to A direction. Data are represented as mean ± standard deviation, n = 4. V: verapamil. Statistical significance from 0.5 µM CSA or 0.5 µM digoxin is shown with asterisks, **, p < 0.01, *, p < 0.05. 169

Table 4.4 Effect of berberine and berbamine on apparent permeability (Papp) of [3H] cyclosporine A (CSA) and [3H] digoxin in Caco-2, MDCKII-MDR1 and MDCKII wild-type (MDCKII-WT) cells from basolateral to apical direction.

Data are represented as mean ± standard deviation. Statistical significance from 0.5 µM [3H] CSA and 0.5 µM [3H] digoxin shown with asterisks (*, p < 0.05, **, p < 0.01), n = 4.

Compound µM Caco-2 MDCKII-MDR1 MDCKII-WT

[3H]CSA [3H]digoxin [3H]CSA [3H]digoxin [3H]CSA [3H]digoxin -6 -6 -6 Papp ( 10 × cm/sec) Papp ( 10 × cm/sec) Papp ( 10 × cm/sec)

Berberine 0 3.31 ± 0.32 2.24 ± 0.70 3.10 ± 1.93 3.30 ± 1.28 6.85 ± 0.88 7.55 ± 0.65 3 2.54 ± 0.28 2.12 ± 0.16 ------10 1.79 ± 0.16** 2.37 ± 0.26 5.34 ± 1.56** 1.56 ± 0.43** 4.07 ± 0.56** 7.55 ± 0.46 30 1.52 ± 0.14** 2.26 ± 0.29 5.17 ± 0.92** 1.57 ± 0.81** 4.34 ± 0.29** 6.95 ± 0.71 100 1.60 ± 0.45** 2.93 ± 0.07 ------Berbamine 0 2.83 ± 0.28 2.78 ± 0.33 3.10 ± 1.93 3.30 ± 1.28 6.85 ± 0.88 7.55 ± 0.65 3 3.18 ± 0.25 3.10 ± 0.28 ------10 2.71 ± 0.08 3.04 ± 0.21 ------30 2.18 ± 0.46** 1.79 ± 0.04** 2.55 ± 1.14** 1.71 ± 0.66** 4.23 ± 0.54** 4.71 ± 0.80** 100 1.75 ± 0.21** 1.23 ± 0.02** 2.23 ± 0.95** 1.54 ± 0.72** 4.26 ± 1.01** 4.25 ± 0.30**

170

MDCKII wild-type cells to that in Caco-2 cells (Figure 4.5, Table 4.4).

Effect of Oregon grape root extract on transport of 3H-CSA and 3H-digoxin

Figure 4.6 and 4.7 demonstrate the effect of E1 (0.05, 0.1, 0.25, 0.5 and 1 mg/ml) and E2 (0.05, 0.1, 0.25, 0.5 and 1 mg/ml) on transport of 0.5 µM 3H-CSA and 0.5 µM 3H-digoxin in Caco-2 cells. The B to A transport of 3H-CSA was inhibited by E1 in a dose response manner with significance effect seen as low as

-6 -6 0.1 mg/ml (the Papp B to A of CSA from 5.88 ×10 cm/s to 4.53 × 10 cm/s, p =

0.0439, Figure 4.6A, Table 4.5). E2 also inhibited the efflux of 3H- CSA, whereas the significance was seen as low as 0.05 mg/ml (the Papp B to A of CSA from 6.36

×10 -6 cm/s to 3.99 × 10 -6 cm/s, p = 0.0007) (Figure 4.7A, Table 4.5). For digoxin,

E1 and E2 inhibited the efflux of 3H-digoxin with significance seen also as low as

-6 -6 0.1 mg/ml for E1 (the Papp B to A of digoxin from 5.68 ×10 cm/s to 2.96 × 10 cm/s, p < 0.0001) (Figure 4.6B, Table 4.5), and 0.05 mg/ml for E2 (the Papp B to A of digoxin from 4.46 ×10 -6 cm/s to 2.96 × 10 -6 cm/s, p < 0.0001) (Figure 4.7B,

Table 4.5). In addition, E1 increased the absorption of digoxin with significant

-6 - effect seen as low as 0.05 mg/ml (the Papp A to B from 1.1 ×10 cm/s to 1.95 ×10

6 cm/s, p = 0.0005).

To compare the effect of E1 and E2 modulating the efflux of CSA and digoxin, efflux ratios of the Papp of B to A and the A to B transport in Caco-2 cells were determined (Table 4.5). E1 and E2 significantly decreased the efflux ratios of

CSA and digoxin. The effect was seen as low as 0.1 mg/ml E1 (4.67 to 2.93, p = 171

Oregon grape root extract 1

A. Cyclosporin A

7 B to A 6 A to B

5 *

(cm/sec) 4

-6 * 3 ** * x 10 2 ** app

P 1

0 C 0.5 µM + V 100 µM + 0.05 + 0.1 + 0.25 + 0.5 + 1

Oregon Grape Root Extract (E1) (mg/ml)

B. Digoxin

6 B to A A to B 5

(cm/sec) ** ** **

-6 3 ** ** ** ** 2 ** ** ** ** ** x 10

app 1 P

0 D 0.5 µM + V 100 µM + 0.05 + 0.1 + 0.25 + 0.5 + 1

Oregon Grape Root Extract (E1) (mg/ml)

Figure 4.6 Effect of Oregon grape root extract 1 (E1) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cells. Panel A: The effect of 100 µM verapamil and E1 (0.05, 0.1, 0.25 0.5 and 1 mg/ml) on transport of CSA from the apical (A) to basolateral (B) and the B to A direction. Panel B: The effect of 100 µM verapamil and E1 (0.05, 0.1, 0.25 0.5 and 1 mg/ml) on transport of digoxin from the A to B and the B to A direction. Data are represented as mean ± standard deviation, n = 4. C: CSA, D: digoxin, V: verapamil. Statistical significance from 0.5 µM CSA or 0.5 µM digoxin is shown with asterisks, **, p < 0.01, *, p < 0.05. 172

Oregon grape root extract 2

A. Cyclosporin A

7 B to A 6 A to B

5 **

(cm/sec) 4 -6 3 ** ** x 10 2

app ** **

P 1 **

0 C 0.5 µM + V 100 µM + 0.05 + 0.1 + 0.25 + 0.5 + 1

Oregon Grape Root Extract (E2) (mg/ml)

B. Digoxin

5 B to A A to B 4 * * 3

(cm/sec) ** ** -6 ** 2

x 10 ** ** ** app 1 P

0 D 0.5 µM + V 100 µM + 0.05 + 0.1 + 0.25 + 0.5 + 1

Oregon Grape Root Extract (E2) (mg/ml)

Figure 4.7 Effect of Oregon grape root extract 2 (E2) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) and 0.5 µM 3H-digoxin (1 µCi) across Caco-2 cells. Panel A: The effect of 100 µM verapamil and E2 (0.05, 0.1, 0.25 0.5 and 1 mg/ml) on transport of CSA from the apical (A) to basolateral (B) and the B to A direction. Panel B: The effect of 100 µM verapamil and E2 (0.05, 0.1, 0.25 0.5 and 1 mg/ml) on transport of digoxin from the A to B and the B to A direction. Data are represented as mean ± standard deviation, n = 4. C: CSA, D: digoxin, V: verapamil. Statistical significance from 0.5 µM CSA or 0.5 µM digoxin is shown with asterisks, **, p < 0.01, *, p < 0.05. 173

Table 4.5 Comparison of apparent permeability (Papp), efflux ratio and IC 50 of [3H] cyclosporin A (CSA) and [3H] digoxin with various concentrations of Oregon grape root extract 1 (E1) and Oregon grape root extract 2 (E2) in Caco-2 cells.

Data are represented as mean ± standard deviation. Statistical significance from 0.5 µM [3H] CSA and 0.5 µM [3H] digoxin shown with asterisks (* p < 0.05, ** p < 0.01), n = 4.

[3H] CSA [3H] Digoxin

Papp (B to A) Papp (A to B) Efflux Ratio IC50 Papp (B to A) Papp (A to B) Efflux Ratio IC50 mg/ml (× 10 -6) (× 10 -6) (mg/ml) (× 10 -6) (× 10 -6) (mg/ml)

E1 0 5.88 ± 1.98 1.23 ± 0.15 4.67 0.35 5.68 ± 0.30 1.10 ± 0.23 5.31 0.25 0.05 5.22 ± 1.31 1.47 ± 0.46 3.62 4.16 ± 0.55 1.95 ± 0.16** 2.13 ** 0.1 4.53 ± 0.72* 1.66 ± 0.60 2.93* 2.96 ± 0.54** 1.73 ± 0.22** 1.72 ** 0.25 3.15 ± 0.17* 1.61 ± 0.55 2.19** 2.95 ± 0.57** 1.75 ± 0.26** 1.67 ** 0.5 2.13 ± 0.15* 1.01 ± 0.33 2.27** 2.98 ± 0.37** 1.84 ± 0.29** 1.67 ** 1 1.68 ± 0.37**1.65 ± 0.93 1.29 ** 2.12 ± 0.26** 2.50 ± 0.25** 0.89 **

E2 0 6.36 ± 0.38 1.18 ± 0.04 5.39 0.21 4.46 ± 0.93 0.59 ± 0.11 7.87 0.17 0.05 3.99 ± 0.06** 1.22 ± 0.05 3.27 ** 2.96 ± 0.83* 0.94 ± 0.14 3.24 ** 0.1 2.28 ± 0.14** 1.61 ± 0.42 1.42 ** 2.86 ± 0.99* 0.65 ± 0.18 4.70 * 0.25 0.83 ± 0.02** 1.28 ± 0.32 0.65 ** 2.05 ± 0.84** 0.64 ± 0.22 3.28 ** 0.5 1.09 ± 0.48** 1.30 ± 0.10 0.84 ** 2.37 ± 0.95** 0.82 ± 0.10 2.92 ** 1 1.26 ± 0.01** 1.73 ± 0.11 0.72 ** 2.02 ± 0.23** 1.26 ± 0.13 1.60 **

174

0.0349) and 0.05 mg/ml E2 (5.39 to 3.27, p < 0.0001) for CSA. For the digoxin, significance occurred at 0.1 mg/ml E1 (5.31 to 2.13, p = 0.0002) and 0.05 mg/ml

E2 (7.87 to 3.24, p = 0.0134). These results suggest that E2 has slightly greater effect in inhibiting the efflux of digoxin as well as the IC50 results (Table 4.5). IC50 of CSA was 0.35 mg/ml for E1 and 0.21 mg/ml for E2, whereas 0.25 mg/ml (E1)

and 0.17 mg/ml (E2) was the IC50 for digoxin.

HPLC chromatogram and Mass Spectrometry (MS) of berberine, berbamine and Oregon grape root extract

Figures 4.8 and 4.9 show the HPLC profile and the total ion chromatogram

(mass spectrometric detection) obtained by passing berberine standard, berbamine standard and a crude methanol Oregon grape root extract (E1 and E2) over a reverse-phase C18 column. The retention time and the molecular ion mass ([M+H]) corresponding to berberine, berbamine, and peak1, peak 2 in the extract are indicated. The peak 2 (Figure 4.8C, D) corresponding to m/z 336.13, is due to berberine (Figure 4.9A), confirmed by co-injection with the standard sample. It was also proved by mass spectrometric detection, the same m/z as berberine standard (336.13) was found in E1 and E2 (Figure 4.9D, F). However, in that period of retention time (2.98 min to 3.25 min for E1, 2.87 min to 3.44 min for E2) of peak 1 (Figure 4.8C, D), the same mass as berbamine can not be found in either

E1 or E2 (Figure 4.9C, E).

175

A. Berberine Standard

996 at 254.00 0.30

AU 0.20 RT : 7.84 min 0.10

0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes B. Berbamine Standard

996 at 254.00

0.15 AU RT : 3.03 min 0.10

0.05

0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes

C. Oregon Grape Root Extract 1 (E1)

996 at 254.00 0.80

0.60 Peak 1 RT : 3.11 min AU 0.40 Peak 2 RT : 7.78 min 0.20

0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes

D. Oregon Grape Root Extract 2 (E2) 0.80 996 at 254.00

0.60

AU 0.40 Peak 2 RT : 7.74 min 0.20 Peak 1 RT : 3.16 min

0.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes

Figure 4.8 High performance liquid chromatograph (HPLC) chromatograms of berberine (Panel A), berbamine (Panel B), Oregon grape root extract 1 (E1) (Panel C) and Oregon grape root extract 2 (E2) (Panel D). RT: retention time. AU: arbitrary units.

176

A. Berberine (RT: 7.84 min)

B. Berbamine (RT: 3.03 min)

Figure 4.9 Mass Spectrometry (MS) of berberine, berbamine, E1 and E2. Berberine at retention time 7.84 min (Panel A), berbamine at retention time 3.03 min (Panel B), E1 at retention time 2.98 min to 3.25 min (Panel C), E1 at retention time 7.39 min to 8.05 min (Panel D), E2 at retention time 2.87 min to 3.44 min (Panel E), E2 at retention time 7.12 min to 8.36 min (Panel F). RT: retention time. RA: relative abundance. 177

C. Oregon Grape Root Extract 1 (E1) (RT: 2.98 min to 3.25 min)

D. Oregon grape root extract 1 (E1) (RT: 7.39 min to 8.05 min)

(Continued)

178

E. Oregon Grape Root Extract 2 (E2) (RT: 2.87 min to 3.44 min)

F. Oregon grape root extract 2 (E2) (RT: 7.12 min to 8.36 min)

(Continued)

179

Effect of aqueous and organic portion of Oregon grape root extract on transport of 3H-CSA and 3H-digoxin

In the present study, Oregon grape root extract has inhibitory effect on the efflux Papp of both digoxin and CSA (Figure 4.6, 4.7) in Caco-2 cells. Berberine, as one of the component in Oregon grape root extract (Figure 4.8, 4.9), does not have effect on inhibiting the efflux Papp of digoxin (Figure 4.3B) in Caco-2 cells.

Berbamine, which was not found in Oregon grape root extract (Figure 4.8, 4.9), has inhibitory effect on the efflux Papp of both digoxin and CSA (Figure 4.3) in

Caco-2 cells. In order to investigate which portion of Oregon grape root extract cause the effect on transport of CSA and digoxin, the aqueous portion and organic portion of Oregon grape root extract 1 (EA1, EO1) and of Oregon grape root extract 2 (EA2, EO2) were separated and their effects on efflux Papp of CSA and digoxin were performed in Caco-2 cells. The concentrations of EA1, EO1 and

EA2 and EO2 were determined based on the percentage of aqueous portion or organic portion in Oregon grape root extract, which was 70.18%, 21.45%, 87.27% and 8.4% for EA1, EO1, EA2, and EO2, respectively. Figure 4.10, 4.11 demonstrate the effect of EA1 and EO1 on transport of CSA and digoxin in Caco-

2 cells. EA1 inhibits the efflux Papp of CSA and digoxin with the significance effect seen as low as 0.18 mg/ml (from 5.39 × 10 -6 cm/sec to 3.58 × 10 -6 cm/sec, p = 0.0040, and from 7.41 × 10 -6 cm/sec to 5.19 × 10 -6 cm/sec, p = 0.0044, for

CSA and digoxin, respectively), which is about 33.58% and 29.86% inhibition on the efflux Papp of CSA and digoxin, whereas, no effect was seen for EO1 on efflux 180

Papp of either CSA or digoxin. Similarly, EA2 inhibited the efflux Papp of CSA and digoxin with the significant effect seen as low as 0.22 mg/ml (from 5.20 × 10 -6 cm/s to 3.45 × 10 -6 cm/s, p = 0.0025, and from 5.50 × 10 -6 cm/s to 3.97 × 10 -6 cm/s, p= 0.0010, for CSA and digoxin, respectively, Figure 4.12A, 4.13A), whereas, no effect was seen for EO1 on efflux Papp of CSA and digoxin (Figure

4.12B, 4.13B). Compared to the results of E1 and E2 on transport of CSA and digoxin (Figure 4.6, 4.7), 0.25 mg/ml E1 inhibited the efflux Papp of CSA and digoxin from 5.88 × 10 -6 cm/s to 3.15 × 10 -6 cm/s, and 5.68 × 10 -6 cm/s to 2.95 ×

10 -6 cm/s, for CSA and digoxin, respectively, which is about 46.43% (CSA) and

48.06% (digoxin) inhibition. The results suggested that the effect seen in the E1 on the efflux Papp of CSA and digoxin is primarily due to the effect of EA1. For E2,

-6 0.1 mg/ml E2 inhibited the efflux Papp of CSA and digoxin from 6.36 × 10 cm/s to 2.28 × 10 -6 cm/s, and 4.46 × 10 -6 cm/s to 2.86 × 10 -6 cm/s, for CSA and digoxin, respectively, which is about 64.15% (CSA) and 35.87% (digoxin) inhibition. Compared to the inhibitory effect of EA2 on the efflux Papp of CSA and digoxin (33.65% and 27.82% for CSA and digoxin, respectively), the effect of E2 on transport of digoxin may be primarily due to the effect of EA2, whereas, the inhibitory effect on efflux of CSA is probably due to the combination effect of

EA2 and EO2. 181

A Oregon Grape Root Extract 1 Aqueous Portion (Cyclosporin A)

6.5 B to A 6.0 5.5 5.0 4.5 4.0 **

(cm/sec) 3.5 -6 3.0 ** ** 2.5 x 10 ** 2.0 ** app

P 1.5 1.0 0.5 0.0 C 0.5 µM V 100 µM 0.07 0.18 0.36 0.72

Oregon Grape Root Extract aqueous (EA1) (mg/ml)

B Oregon Grape Root Extract 1 Organic Portion (Cyclosporin A)

B to A 6.5 6.0 5.5 5.0 4.5 4.0

(cm/sec) 3.5 -6 3.0 2.5

x 10 ** 2.0 app

P 1.5 1.0 0.5 0.0 C 0.5 µM V 100 µM 0.02 0.05 0.1 0.2

Oregon Grape Root Extract Organic (EO1) (mg/ml)

Figure 4.10 Effects of Oregon grape root extract 1 aqueous portion (EA1) and organic portion (EO1) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) across Caco-2 cells. Panel A: The effect of 100 µM verapamil and EA1 on transport of CSA from the basolateral (B) to apical (A) direction. Panel B: The effect of 100 µM verapamil and EO1 on transport of CSA from the B to A direction. Data are presented as mean ± standard deviation, n = 4. C: CSA, V: verapamil. Statistical significance from 0.5 µM CSA is shown with asterisks, **, p < 0.01.

182

A Oregon Grape Root Extract 1 Aqueous Portion (Digoxin)

8 B to A 7

6 ** 5 **

(cm/sec) **

-6 4

3 x 10 **

app 2 P

0 D 0.5 µM V 100 µM 0.07 0.18 0.36 0.72

Oregon Grape Root Extract aqueous (EA1) (mg/ml)

B Oregon Grape Root Extract 1 Organic Portion (Digoxin)

B to A 10.0

7.5 (cm/sec)

-6 5.0 ** x 10

app 2.5 ** P

0.0 D 0.5 µM V 100 µM 0.02 0.05 0.1 0.2

Oregon Grape Root Extract Organic (EO1) (mg/ml)

Figure 4.11 Effects of Oregon grape root extract 1 aqueous portion (EA1) and organic portion (EO1) on the transport of 0.5 µM 3H-digoxin (digoxin, 1 µCi) across Caco-2 cells. Panel A: The effect of 100 µM verapamil and EA1 on transport of digoxin from the basolateral (B) to apical (A) direction. Panel B: The effect of 100 µM verapamil and EO1 on transport of digoxin from the B to A direction. Data are presented as mean ± standard deviation, n = 4. D: digoxin, V: verapamil. Statistical significance from 0.5 µM digoxin is shown with asterisks, **, p < 0.01.

183

A Oregon Grape Root Extract 2 Aqueous Portion (Cyclosporin A)

6 B to A

4 * (cm/sec)

-6 3 ** ** x 10 2 ** ** app P 1

0 C 0.5 µM V 100 µM 0.087 0.22 0.44 0.88

Oregon Grape Root Extract aqueous (EA2) (mg/ml)

B Oregon Grape Root Extract 2 Organic Portion (Cyclosporin A)

B to A 6

4 (cm/sec)

-6 3

x 10 2 ** app P 1

0 C 0.5 µM V 100 µM 0.0084 0.021 0.042 0.084

Oregon Grape Root Extract Organic (EO2) (mg/ml)

Figure 4.12 Effects of Oregon grape root extract 2 aqueous portion (EA2) and organic portion (EO2) on the transport of 0.5 µM 3H-cyclosporin A (CSA, 1 µCi) across Caco-2 cells. Panel A: The effect of 100 µM verapamil and EA1 on transport of CSA from the basolateral (B) to apical (A) direction. Panel B: The effect of 100 µM verapamil and EO2 on transport of CSA from the B to A direction. Data are presented as mean ± standard deviation, n = 4. C: CSA, V: verapamil. Statistical significance from 0.5 µM CSA is shown with asterisks, *, p < 0.05, **, p < 0.01.

184

A Oregon Grape Root Extract 2 Aqueous Portion (Digoxin)

6 B to A

5 * 4 ** (cm/sec) **

-6 3

x 10 2 ** app P 1

0 D 0.5 µM V 100 µM 0.087 0.22 0.44 0.88

Oregon Grape Root Extract aqueous (EA2) (mg/ml)

B Oregon Grape Root Extract 2 Organic Portion (Digoxin)

B to A 8

6 * 5 (cm/sec)

-6 4

3 x 10 x **

app 2 P 1

0 D 0.5 µM V 100 µM 0.0084 0.021 0.042 0.084

Oregon Grape Root Extract Organic (EO2) (mg/ml)

Figure 4.13 Effects of Oregon grape root extract 1 aqueous portion (EA2) and organic portion (EO2) on the transport of 0.5 µM 3H-digoxin (digoxin, 1 µCi) across Caco-2 cells. Panel A: The effect of 100 µM verapamil and EA2 on transport of digoxin from the basolateral (B) to apical (A) direction. Panel B: The effect of 100 µM verapamil and EO2 on transport of digoxin from the B to A direction. Data are presented as mean ± standard deviation, n = 4. D: digoxin, V: verapamil. Statistical significance from 0.5 µM digoxin is shown with asterisks, *, p < 0.05, **, p < 0.01. 185

Induction of P-gp by Oregon grape root extract

Oregon grape root extract up-regulated mRNA level of human MDR1 gene in both Caco-2 and LS-180 cells (Figure 4.14). Levothyroxine was used as a positive control in Caco-2 cells because it has been found to up-regulate human

MDR1 gene through PXR independent manner (Mitin et al., 2004). It increased the mRNA level of human MDR1 gene in a time-dependent manner. In Caco-2 cells,

E1 increased the mRNA level of human MDR1 at 3.28 ± 0.78 fold with the significant effect of E1 seen as low as 0.25 mg/ml at 48 hours (p = 0.0312), whereas for E2, the significant effect was seen as low as 0.1 mg/ml at 24 hours

(increased the mRNA level of human MDR1 gene at 3.96 ± 0.29 fold, p < 0.0001,

Figure 4.14A). Therefore, the significant effect of E2 was seen at lower concentration and earlier time compared with E2 in Caco-2 cells. In LS-180 cells, rifampin was used as the positive control because it is a well known P-gp inducer and modulates P-gp through PXR dependent pathway (Mitin et al., 2004; Huang et al., 2006). E1 increased the mRNA level of human MDR1 at 4.04 ± 1.25 fold in

LS-180 cells with the significant effect seen as low as 0.1 mg/ml at 48 hours (p =

0.0015, Figure 4.14B). This effect was seen at lower concentrations in LS-180 cells than in the Caco-2 cells. The significant effect of E2 still was seen at 0.1 mg/ml at 24 hours in LS-180 (p = 0.0017) with a greater effect at 48 hours

(increased the mRNA level of human MDR1 at 11.10 ± 3.63 fold, p < 0.0001) than that in Caco-2 cells. Therefore, E1 and E2 probably up-regulated MDR1 gene through both PXR dependent and PXR independent pathway. 186

A. Caco-2

10.0 **

7.5 **

5.0 ** * * 2.5

MDR1/18s Fold Change Fold MDR1/18s 0.0 C24 C48 L24 L48 E1 24 E1 48 E2 24 E2 48 E1 24 E1 48

0.1 mg/ml 0.25 mg/ml

B. LS-180 ** 13 12 11 10 9 8 7 6 5 ** * 4 ** 3 ** 2 1 MDR1/18s Fold Change MDR1/18s Fold 0 C24 C 48 R 24 R 48 E1 24 E1 48 E2 24 E2 48

0.1 mg/ml

Figure 4.14 Oregon grape root extract 1 (E1) and Oregon grape root extract 2 (E2) up-regulation of mRNA level of human MDR1 (ABCB1) in Caco-2 cells (Panel A) and LS-180 cells (Panel B). C: control. 24: 24 hours. 48: 48 hours. L: levothyroxine 100 µM. R: rifampin 10 µM. Bars indicate mean ± standard deviation, n = 4. Statistical significance from control is shown with asterisks (treatments at 24 hours were compared with the control at 24 hours, treatment at 48 hours were compared with the control at 48 hours), **, p < 0.01, *, p < 0.05. 187

4.5 Discussion

Drug resistance is a major obstacle in cancer chemotherapy. There are many mechanisms involved in drug resistance. Multi-drug resistance is the most important cause because it is associated with the expression of many efflux transporters, such as P-gp, multidrug resistance associated proteins (MRPs) and canalicular multispecific organic anion transporter (Guo et al., 2002).

P-gp is a 170 kDa phosphorylated glycoprotein which is encoded by the human MDR1 gene. It has been found not only in tumor cells, but also in epithelial cells of normal tissues (Thiebaut et al., 1987; Arboix et al., 1997). It is responsible for the systemic disposition of a large number of structurally and pharmacologically unrelated lipophilic and amphipathic drugs, carcinogens, and other xenobiotics in many organs, such as the intestine, liver, kidney, and brain

(Lechapt-Zalcman et al., 1997; Bodo et al., 2003; Schwab et al., 2003).

Overexpression of P-gp is associated with the clinical multi-drug resistance phenotype for many human cancers. Some clinically important herb-drug interactions were reported and many of them altered efficacy or level of adverse effect of the drug (Fugh-Berman, 2000; Fugh-Berman and Ernst, 2001). Therefore, inhibition of P-gp by herbal constituents may provide a novel approach for reversing multi-drug resistance in tumor cells, whereas the stimulation of P-gp expression may have implication for chemoprotective enhancement by herbal medicines. 188

Natural products have been used by the general populations and according to the reports, almost 40% of Americans use complementary and alternative medicine during their life time (Eisenberg et al., 2001; Kessler et al., 2001) .

Because drug concentrations can be altered by co-administration of herbs, which partly is due to induction or inhibition of drug transporter, such as P-gp (Walter-

Sack and Klotz, 1996; Wilkinson, 1997; Evans, 2000; Ioannides, 2002; Izzo,

2005), it is extremely important to identify the potential P-gp modulator(s) from the herbal medicine.

In previous studies, herbal products such as St. John’s wort (Wang et al.,

2002b; Dresser et al., 2003), garlic (Kaye et al., 2000; Piscitelli et al., 2002), ginko

(Rosenblatt and Mindel, 1997) and milk thistle (Gaedeke et al., 1996; von

Schonfeld et al., 1997), have been found to interact with drug efflux proteins, such as P-gp. They altered the bioavailability of the drugs which are substrates of P-gp, such as CSA (St. John’s wort) (Barone et al., 2000), saquinavir (garlic) (Piscitelli et al., 2002), and indinavir (milk thistle) (DiCenzo et al., 2003).

Bisbenzylisoquinoline alkaloids, which are found in several herbal products, such as golden seal (Hydrastis Canadensis), berberies and Oregon grape root (Mahonia aquifolia), have been shown to interact with P-gp and have potential drug-diet interaction (Wakusawa et al., 1992; Fu et al., 2001; Jakubikova et al., 2002). For example, Fu et al. (2001) screened potential MDR modifiers from a series of naturally occurring bisbenzylisoquinoline alkaloids that were isolated from natural plants using MCF-7/adr and KBv200 tumor cells. Many of these 189 natural compounds were shown potent activities to decrease the resistant of tumor cells to P-gp substrates, such as doxorubicin and vincristine, by using MTT assay.

In addition, these compounds also increased intracellular drug accumulation of

[3H] vincristine by 3.3-fold. Their results suggest that the mechanism of these compounds to reverse MDR was probably linked to increased intracellular drug accumulation by inhibiting the activity of P-gp (Fu et al., 2001). PK11195, an isoquinoline carboxamide ligand of the mitochondrial benzodiazepine receptor, has been reported to increase drug uptake and facilitated drug-indued apoptosis in human mutidrug-resistant leukemia cells in vitro (Jakubikova et al., 2002). The investigation has been also conducted on the effects of seven isoquinoline derivatives in overcoming resistance to vinblastine in adriamycin-resistant mouse leukemia P388/ADR cells and human myelogeneous leukemia K562/ADR cells

(Wakusawa et al., 1992). The compounds that effectively reversed the resistance to vinblastine inhibited [3H] vinblastine efflux, thus suggesting that the isoquinoline derivatives reverse multidrug resistance by inhibiting the binding of drug to P-gp

(Wakusawa et al., 1992).

Berberine, a benzylisoquinoline alkaloid, was effluxed by Caco-2 cells

(Figure 4.1A). This is consistent with a previous study which showed that the Papp of berberine from the B to A direction was much greater than that from the A to B direction in Caco-2 cells (Maeng et al., 2002). Moreover, the present study is the first to show that berberine is effluxed in the MDCK-MDR1 cells and the efflux 190

Papp is higher in MDCKII-MDR1 cells than the Caco-2 cells (Figure 4.2A), which suggest that berberine may be actively effluxed by P-gp.

In our study, berberine was also found to modulate the transport of P-gp substrates, such as CSA and digoxin (Figure 4.3). CSA and digoxin were chosen because they are known substrates for the efflux transporters on the apical epithelial cells of intestine in Caco-2 cells and in vivo (Augustijns, 1996). We found that berberine was able to inhibit the efflux of 0.5 µM CSA in Caco-2 cells, and the significant effect was seen as low as 10 µM berberine (Figure 4.3A).

However, no effect of berberine was seen on the efflux of 0.5 µM digoxin in Caco-

2 cells (Figure 4.3B). This is surprising because CSA and digoxin are both substrates of P-gp. If berberine can modulate the transport of CSA, a similar effect should be seen with the transport of digoxin. Thus, similar experiments were performed using MDCKII-MDR1 and MDCKII wild-type cells. Berberine was able to inhibit the Papp efflux of both CSA and digoxin in the B to A direction in

MDCKII-MDR1 cells (Figure 4.4). Similar results were also seen with the

MDCKII wild-type cells compared with Caco-2 cells (Figure 4.5). Therefore, the effect of berberine on transport of digoxin in the Caco-2 cells is probably due to the combination effect of berberine on digoxin transport. Digoxin is the substrate of P-gp, but is not specific because it has also been reported to be a substrate of the human organic anion-transporting polypeptide 8 (OATP8) (Kullak-Ublick et al.,

2001; Tsujimoto et al., 2006), and OATP4C1 (Mikkaichi et al., 2004; Yao and

Chiou, 2006). Because OATP4C1 is not expressed in Caco-2 cells (Sai et al., 191

2006; Maubon et al., 2007), berberine might induce some influx transporters such as OATP8 or other unidentified transport systems that is involved in the transport of digoxin. The unidentified transport system of digoxin was also suggested in the study of Tian et al. (2005). In their study, P-gp inducers did not reduce the intracellular 3H-digoxin in LS-180 cells which can not be explained by the P-gp mediated transport induction. In addition, the mRNA level of OATP8, another possible transporter involved in digoxin transport, can not be changed by P-gp inducers. Their results suggest that some other unidentified transport system may induced by these inducers (Tian et al., 2005).

Based on the current results, berberine has the potential effect on modulating the P-gp. This can explain clinical study by Wu et al. 2005. In their study, the mean AUC of CSA was increased by 34.5% (p < 0.05) after coadministration of berberine for 12 days in six renal-transport patients (Wu et al.,

2005). In addition, the current results are consistent with other studies in vitro and in vivo (Lin et al., 1999b; He and Liu, 2002; Pan et al., 2002). For example, berberine absorption in the intestine of rat was improved from 2.5% to 14.8% for

CSA, and to 17.2% for verapamil, respectively (Pan et al., 2002). In Lin et al.’s

(1999b) study, 24 hour treatment with berberine up-regulated the P-gp expression in different cancer cell lines (gastric, colon cancer cell lines) by using flow cytometry. In another study, berberine was able to increase the intracellular accumulation of rhodamine 123 (a P-gp substrate) in bovine brain capillary endothelial cells (He and Liu, 2002). 192

Berbamine, a bisbenzylisoquinoline alkaloid, was chosen for this study because berbamine and its derivative, have been found to modulate the expression of P-gp (Tian and Pan, 1997; Han et al., 2003; Zhu and Liu, 2003; Han et al.,

2004). The current results show that there is an efflux mechanism involved in berbamine transport in Caco-2 and MDCKII-MDR1 cells (Figure 4.1B).

Furthermore, there was about 5-fold difference in the amount of berbamine be effluxed than that of berberine (Figure 4.1). In MDCKII-MDR1 cells, the efflux ratio of berbamine (27.44 ± 5.11) is similar to that in Caco-2 cells (24.22 ± 4.49), which indicates berbamine may interact with other efflux transporters such as

MRPs (Figure 4.2B). Berbamine was able to inhibit the efflux of CSA and digoxin transport in the Caco-2 (Figure 4.3), MDCKII-MDR1 (Figure 4.4) and MDCKII wild-type cells (Figure 4.5). Therefore, berbamine has the potential to modulate P- gp. This is consistent with Han et al. (2003) study which shows that berbamine increased intracellular concentration of adriamycin and down-regulated expression level of MDR1 mRNA and P-gp in K562/A02 cells. A different cell line (MCF7) also obtained the similar results (Han et al., 2004). However, the effect of berbamine on modulating the transport of CSA and digoxin in the B to A direction was not as potent as berberine in MDCKII-MDR1 cells with the significant effect seen as low as 30 µM for berbamine whereas 10 µM for berberine (Table 4.4).

This is probably because there are more berbamine be effluxed by cells than berberine (Figure 4.1 and Figure 4.2), which can also be used to explain that 193 berbamine was less cytotoxic when compared with berberine in cytotoxicities studies (Table 4.2).

Also, the Papp of digoxin (B to A) in MDCKII-WT cells was higher than that in MDCKII-MDR1 cells (Table 4.4, Figure 4.4, Figure 4.5) which is contradictory to Taub et al. (2005) study. This is probably because the TEER for

MDCKII-MDR1 cells in our study (476.91 ± 101.21 Ω cm2) was lower than their study (>1000 Ω cm2). TEER has been used to measure the integrity of membrane barrier, and to evaluate the damage caused by drugs such as fenadine.HCl (Lin et al., 2007). In addition, TEER is also reported to indicate the permeability of cell monolayers and their barrier properties (Grasset et al., 1984; Hidalgo et al., 1989).

The cells which have lower TEER may form less tight junctions, thereby may contain less efflux transport proteins (Kannan et al., 2000; Bertilsson et al., 2001).

Therefore, in the present study, less amount of digoxin was effluxed by the cells, which leads to the decrease on Papp efflux of digoxin in MDCKII-MDR1 cells.

Oregon grape root extract is commonly used by Native American as a dietary supplement for treating the skin disease, gall bladder disease and gastrointestinal disorder (Moga, 2003; Bernstein et al., 2006; Donsky and Clarke, 2007).

Knowing its effect on altering the absorption and bioavailability of drugs, such as anticancer or immunosuppressive drugs, is useful and necessary because it may have the beneficial effects to human health by promoting drug absorption. On the other hand, it may lead to abnormally high drug plasma levels for some drugs such as digoxin that have narrow therapeutic window, which would be considered a risk 194 for potential adverse drug-dietary interactions. Currently, there are no publications evaluating its interactions with other drugs. In addition, only one analytical study showed the HPLC chromatogram of Oregon grape root, but did not quantify any of the components in the extract (Weber et al., 2003). Therefore, whether Oregon grape root extract actually contains berberine and berbamine still needs to be determined. In the current study, berberine was present in both E1 and E2 at the similar retention time as berberine standard (Figure 4.8A, 4.8C, 4.8D). It also has been proved by LC-MS, peak 2 has the same m/z ratio as berberine standard in both E1 and E2 (Figure 4.8C, 4.8D, 4.9A, 4.9D, 4.9F). Berberine has been quantified to be 15% in E1 and 17% in E2. However, based on the LC-MS data, berbamine is not present in the extracts. Some compounds with similar mass to berbamine could be its derivatives (Figure 4.9B, 4.9C, 4.9E). For further confirmation of identification of these compounds, MS-MS and nuclear magnetic resonance (NMR) spectroscopy should be performed.

To the best of our knowledge, this is the first report of the interaction of

Oregon grape root extract with P-gp. In order to know if Oregon grape root extract modulates P-gp, transport and real time RT-PCR studies were performed. E1 and

E2 both inhibited the transport of CSA and digoxin in the B to A direction in

Caco-2 cells and the significant effect was seen at 0.1 mg/ml for E1 and 0.05 mg/ml for E2 (Figure 4.6, 4.7). This suggests that E2 probably has slightly greater effect than E1 in inhibiting the efflux of CSA and digoxin as well as the efflux ratio and IC50 results (Table 4.5). From the transport data of aqueous and organic 195 portion of E1 and E2 (Figure 4.10-4.13), it is suggested that the inhibitory effects of E1 and E2 are primarily due the aqueous portion of Oregon grape root extract.

In the real time RT-PCR study, E1 and E2 are both able to up-regulate the mRNA level of human MDR1 gene (ABCB1) in Caco-2 and LS-180 cells (Figure 4.14).

The significant effect was seen as low as 0.25 mg/ml E1 at 48 hours and 0.1 mg/ml

E2 at 24 hours in Caco-2 cells. In the LS-180 cells, the significant effect was seen at 0.1 mg/ml at 48 hours for E1 and 24 hours for E2. Caco-2 cells and LS-180 cells were chosen because those two cell lines are commonly used for comparison in P- gp mechanism studies (Mitin et al., 2004; Bertelsen et al., 2006; Zimmermann et al., 2006; Maier et al., 2007). Because Caco-2 cells lack PXR receptor and LS-180 cells have it, E1 and E2 probably modulate P-gp through PXR-independent and

PXR-dependent manner. According to the dosing information, Oregon grape root extract (750 mg) should be taken three times a day. Based on previous reports, the intestinal concentration of Oregon grape root extract is likely to be approximately

0.75 mg/ml (Perloff et al., 2001; Tian et al., 2005). Therefore, the concentrations used in the study are clinically relevant.

In conclusion, this study is the first to demonstrate that Oregon grape root extract is able to modulate P-gp. Berberine and berbamine have been proved to inhibit the transport of P-gp substrates. Dietary supplements, such as berberine, berbamine, Oregon grape root extract or other isoquinoline alkaloids compounds, may affect the drug efflux or absorption in the intestine. Use of diet supplement 196 which contains ispquinoline alkaloids should be carefully considered when some drugs of P-gp substrates are taken.

197

5. GENERAL CONCLUSION

In the ketoconazle (KT) studies, an efflux mechanism was found to be involved in transport of KT. KT was able to modulate the apparent permeability

(Papp) of P-glycoprotein (P-gp) substrates, cyclosporin A (CSA) and digoxin, by interacting with MDR1 protein. In addition, our data suggests that KT may also modulate multidrug resistance associated proteins (MRPs)-mediated transport.

Dietary flavonoids, EGCG and xanthohumol, were able to modulate the transport and uptake of KT, which indicates significant KT-dietary interactions with diet supplements, food or beverages which contains EGCG or xanthohumol.

The flavonoids studies demonstrated that xanthohumol can inhibit the efflux of CSA, but not of digoxin transport; EGCG and ECG were able to inhibit the efflux of CSA in Caco-2 cells, but not in MDCKII-MDR1 cells; xanthohumol and EGCG inhibited the uptake of CSA, but increased the uptake of digoxin.

These data suggest that xanthohumol may increase the bioavailability of CSA by inhibiting MDR1-mediated efflux, whereas digoxin may share substrate specificity with other transporters such as MRP2, or interact with MDR1 at a different site than xanthohumol. Therefore, the intestinal transport of these P-glycoprotein substrates is probably more complex than previously reported. In addition, EGCG and ECG may inhibit other efflux transport proteins besides P-glycoprotein, such as MRPs. In clinical practice, cautious use of these dietary flavonoids needs to be 198 taken into consideration when drugs that use efflux or absorption pathways are administered.

Lastly, an efflux mechanism appears to be involved in the transport of berberine and berbamine. Also, berberine and berbamine can modulate the transport of P-gp substrates, CSA and digoxin, across Caco-2 and MDCKII-MDR1 drug transport models. Oregon grape root extract, one of the reported sources of berberine and berbamine, was shown to inhibit the efflux of CSA and digoxin as well in both drug transport models. In addition, Oregon grape root extract up- regulated mRNA levels of human MDR1 gene in pregnane X receptor independent and dependent pathway in Caco-2 and LS-180 cells, respectively. Therefore, repeated administration of Oregon grape root extract may reduce the intestinal absorption of P-gp substrates because they are being effluxed back into the gut lumen. Our data suggests that the dietary supplements which contain berberine, berbamine or other isoquinoline alkaloids should be carefully considered when drugs that are P-gp substrates are taken.

All studies provide important information regarding drug-drug and drug- diet interaction associated with P-gp or MRPs. Administration of pharmaceuticals, such as KT, or dietary supplements, food or beverages containing flavonoids

(EGCG, ECG, xanthohumol) or isoquinoline alkaloids (berberine or berbamine) may alter the bioavailability, therapeutic efficacy and safety of the drugs that are

P-gp or MRPs substrates. Understanding the drug transport, knowing the effect of other pharmaceuticals and natural products on multidrug resistance mediated 199 transport and the mechanism of modulating the drug transport proteins are useful for predicting the clinical drug-drug and drug-diet interaction in humans. These findings suggest that KT, EGCG, ECG, xanthohumol, berberine and berbamine may have significant effects on drug transport and in the future, more research about the function, specificity and regulation of drug and xenobiotic transporters is needed in order to fully understand the impact of pharmaceuticals or natural products on drug transport processes.

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243

APPENDICES

244

Protocol A: Real time Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Objective: To investigate the gene expression of human MDR1, MRP1 and MRP2 after pretreatment of Oregon grape root extract.

A.1 Instructions for pretreatment:

Seed cells:

Caco-2 or LS-180 cells were seeded 600,000 cells/well (Caco-2) or 1,000,000 cells/well (LS-180) in 6 well Transwell® inserts (Corning # 3414) and grown for 7

(Caco-2) or 6 (LS-180) days

Prepare solution:

Levothyroxine 100 µM preparation:

1. Weight 0.0088 gram levothyroxine (Sigma # T2501), dissolve it in 500 µl

DMSO (Sigma # 154938), add 500 µl distilled water to obtain 10 mM levothyroxine stock solution

2. Take 150 µl 10 mM levothyroxine stock solution and dissolve it in 15 ml

Dulbecco’s Modified Eagle Medium (DMEM, Giboco # 12800-017) prepared solution, obtain 100 µM levothyroxine solution

Rifampin 10 µM preparation 245

1. Weight 0.00823 gram rifampin (Sigma # R3501), dissolve it in 500 µl DMSO, and 500 µl distilled water obtain 10 mM rifampin stock solution

2. Take 15 µl 10 mM rifampin stock solution and dissolve it in 15 ml DMEM prepared solution, obtain 10 µM rifampin solution

Preparation of Oregon grape root extract 1 (E1, Oregon’s Wild Harvest) 0.1 mg/ml, 0.25 mg/ml:

1. Take 25 mg E1 powder and dissolve it in 250 µl ethanol, add 250 µl distilled water, obtain 50 mg/ml stock solution

2. Take 30 µl 50 mg/ml stock solution of E1, dissolve it in 15 ml DMEM, obtain

0.1 mg/ml E1

Take 75 µl 50 mg/ml stock solution of E1, dissolve it in 15 ml DMEM, obtain 0.25 mg/ml E1

Preparation of Oregon grape root extract 2 (E2, Health Herbs # 5156) 0.1 mg/ml,

0.25 mg/ml:

Same as E1 0.1 mg/ml and E1 0.25 mg/ml preparation

Start pretreatment:

1. Caco-2 or LS-180 cells were washed twice with warm Phosphate buffered saline

(PBS) buffer at time zero and measure transepithelial electrical resistance (TEER) using a World Precision Instrument, EVOM (Sarasota, FL, USA) 246

2. 1 ml Trizol® reagent (Invitrogen # 15596-018) was added in negative control at

0 hour treatment wells and collected to 1.5 ml centrifuge tube (VWR # 20170-038) and stored at -20 °C

3. DMEM prepared solution alone (negative control for 48 hours treatment), 0.1,

0.25 mg/ml E1, 0.1, 0.25 mg/ml E2, and 100 µM levothyroxine or 10 µM rifampin for 48 hours treatments were added to the wells.

4. Put completed media to the rest of the wells

5. Next day, same time, wash cells (for 24 hour treatment) twice with PBS

6. DMEM alone (negative control for 24 hours treatment), 0.1, 0.25 mg/ml E1, 0.1,

0.25 mg/ml E2, and 100 µM levothyroxine for 24 hours treatments were added to the wells

247

A.2 Instructions for Sample collection and RNA separation:

Sample collection:

1. All treatments were removed and cells were washed twice with PBS

2. Take TEER reading again and compare with the previous one (TEER reading at the beginning)

3. Remove PBS, and all cells were collected by adding 1 ml of Trizol® reagent to each well and pipetting the cells with reagent to a 1.5 ml centrifuge tube

(Eppendorf # 02236411). The collected samples were then stored at -20 °C.

RNA separation:

1. The samples were taken out of the -20 °C and 200 µl of chloroform (EM

Science # 3150) was added to each sample. Mix well and centrifuge 15 min at

12,000 rpm using Eppendorf centrifuge (Eppendorf # 5415C)

2. Take the aqueous layer (top) and transfer to eppendorf 1.5 ml centrifuge tube

(The remaining of the sample, protein and DNA, put in -20 °C or -80 °C)

3. Add 500 µl isopropanol (Ricca Chemical # 4210-1) to aqueous layer, centrifuge

8 min at 12,000 rpm (12 samples/time for centrifuge, put rest of the samples in the ice)

4. Remove isopropanol with a drawn out pastuer pipette (VWR # 14673-043) (can see the small pallet, RNA)

5. Add 75% ethanol 200 µl (wash the pallet) and centrifuge for 8 min at 12,000 rpm 248

6. Remove the 75% ethanol with the drawn out pasture pipette

7. Repeat 5, 6 twice

249

A.3 Instructions for RNA quantification:

The Quant-iTTM RiboGreen® RNA reagent kit (Invitrogen # R11490) was used for

RNA quantification.

Preparation of Diethyl Pyrocarbonate (DEPC) water: add 0.5 ml DEPC (EM

Science # 3660) to 500 ml distilled water (0.1% v/v DEPC: water), stir overnight to dissolve. Autoclave to remove the rest RNA in the bottle

Make 1 × TE buffer: Take 1 ml 20 × TE (from RNA quantification kit) in 19 ml

DEPC water

Make RNA standard solution: Take 20 µl RNA solution (from RNA quantification kit) in 980 µl 1 × TE buffer

Make RNA reagent solution: Take 50 µl RNA reagent (from RNA quantification kit) in 10 ml 1 × TE buffer

Procedure:

1. Add 50 µl DEPC water to the sample, mix well and dissolve the pallet

2. Take 5 µl out from the sample, put in 995 µl DEPC water (1.5 ml eppendorf tube), do 1 to 500 fold dilution (or 2 µl to 998 µl DEPC water)

The rest sample (about 45 µl or 47.5 µl), put in the sample bag and store them in

-80 °C

250

3. Put 100 µl of diluted sample in the 96 well black fluorescence plate (Nunc TM,

Denmark) (each sample do triplicate, do 24 samples, need 24 × 3=72 wells)

4. Make RNA standard curve:

100 µl RNA standard solution (2 µg/ml) + 0 µl 1 × TE

50 µl RNA standard solution (1 µg/ml) + 50 µl 1 × TE

20 µl RNA standard solution (0.4 µg/ml) + 80 µl 1 × TE

10 µl RNA standard solution (0.2 µg/ml) + 90 µl 1 × TE

2 µl RNA standard solution (0.04 µg/ml) + 98 µl 1 × TE

0 µl RNA standard solution (0 µg/ml) + 100 µl 1 × TE

5. Add 100 µl reagent solution in each well (includes RNA standard curve)

6. Cover the plate using parafilm® and foil, take it to the ALS room 1155, code

5194291

7. The plate was read on the Gemini fluorometer (Spectromax Gemini Sunnyvale,

CA, USA) at excitation 480 nm and emission 520 nm (SpectriMax software, mix, set up the template and set up the wave length, print out the result)

8. In excel sheet, the concentration of RNA in each sample was calculated based on the standard curve. 251

A.4 Instructions for cDNA synthesis:

The iScriptTM cDNA synthesis kit (Bio-Rad # 170-8890, can buy from ALS room

2061) was used for cDNA synthesis (for 40 µl reactions).

For each sample, take different amount of mRNA out in 0.5 ml eppendorf tube

(Enpendorf # 022363719), add different amount of DEPC water (total volume 30

µl, preparation of DEPC water: see I.3 Instructions for RNA quantification), make each sample contain the same amount of mRNA (2 µg/ml)

Preparation:

5 × iScript reaction mix: 8 µl × 26 = 208 µl iScript reverse transcriptase: 2 µl × 26 = 52 µl

Mix them in a 1.5 ml eppendorf tube (Eppendorf # 02236411), take 10 µl mixture out, put in 0.5 ml eppendorf tube (contains 30 µl), total volume 40 µl

Procedure:

The reactions were done for 40 µl total, using 8 µl of 5 × iScript reaction mix, 2 µl of iScript reverse transcriptase, 2 µg/ 30µl of sample RNA per reaction. The reaction was incubated at 25 °C for 5 minutes, then 42 °C for 30 minutes and 85

°C for 5 minutes then held at -20 °C until needed for quantitative PCR. 252

A.5 Instructions for Quantitative PCR reaction:

For reservation the instrument:

Online website: http://www.cgrb.oregonstate.edu/cgi- bin/webevent/webevent.cgi?cmd=login&ncmd=startup&de=1&tf=0&sib=1&sb=0

&sa=0&ws=0&stz=Default&sort=e,m,t&swe=1&cf=cal&set=1&m=10&d=11&y

=2007

User name: quantpcr

Password: 7500

For using the instrument:

Location: ALS 3012

Room code: 49402*

Password for the computer: 7500

This was done with the iTaq TM SYBR® Green supermix with Rox (Bio-Rad #

170-8850). Two separate supermixes were made up one with the MDR1 human primer (Applied Biosystems #277805) or MRP1 (Applied Biosystems # 495708) or MRP2 (Applied Biosystems # 433182) and one with the 18s ribosomal control primer (Applied Biosystems #430448604025, forward; #430449004026, reverse).

Then 75 µl of supermix was added to 0.5 µl eppindorf tubes and 5 µl of sample was added and pipetted to mix well. Then 25 µl of this was pipetted to three separate wells on the 96 well PCR plate (Midsci # B70501). The plate was covered 253 with optically transparent sealing films (ThermalSeal RT TM, Excel Scientific #

TS-RT2-100) before run.

These samples were run on an ABI 7500 Real Time PCR system (Applied

Biosystems, Foster City, CA, USA)

The details are as follows:

1. Preparation:

Supermix: For primer MDR1, MRP1 or MRP2

Water: 33.5 µl × 10 (8 + 2 extra) = 335 µl

SYB: 40 µl × 10 = 400 µl (easy to have bubbles, careful)

Primer: 1.5 µl × 10=15 µl

Supermix: For 18 s

Water: 32.6 µl × 10 = 326 µl

SYB: 40 µl × 10 = 400 µl

Reverse: 1.2 µl × 10 = 12 µl

Forward: 1.2 µl × 10 =12 µl

2. Do dilution:

18s: needs to be diluted, usually 1:100 dilution (5 µl in 500 µl water), set up 24 1.5 ml Eppendorf tube

Set up 32 0.5 ml Eppendorf tubes (each primer has 8 samples, 18 s has 8 samples)

3. Start reaction:

For primer MDR1, MRP1 and MRP2: 254

Add 5 µl original cDNA to the 0.5 ml eppendorf tube, then add 75 µl supermix

(primer) to it

For primer 18s:

Add 5 µl diluted cDNA to 0.5 ml eppendorf tube, then put 75 µl supermix (18s) to it

4. In the 96 well PCR plate (Midsci # B70501), do each sample triplicate, so 25 µl for each well

5. Cover the plate with optically transparent sealing films (ThermalSeal RT TM,

Excel Scientific # TS-RT2-100)

6. Run the PCR plate on an ABI 7500 Real Time PCR system (Applied

Biosystems, Foster City, CA, USA)

255

Protocol B: Ketoconaozle Multidrug Resistance (MDR) assay

Objective: To evaluate the interaction of ketoconazole with multidrug resistance proteins

MDR assay was conducted by using Vybrant TM Multidrug Resistance Assay Kit from Molecular Probe (#V-13180)

Prepare different concentrations of ketoconazole (KT)

1. Weight 0.0053 g ketoconazole (Jassen Biotech N.V. # R41400) and dissolve in

1 ml Phosphate buffered saline (PBS), adjust pH to 5, make the stock solution 10 mM and 1 mM KT

2. Prepare serial dilutions of KT using PBS (pH = 5): 0.004, 0.012, 0.04, 0.12, 0.4,

1.2, 4, 12, 40, 120, and 400 µM (the concentrations are 4-fold higher than the final concentrations)

3. The day before the experiment, typsinized the cells (Caco-2 or MDCKII-MDR1 cells) and transfer to another flask.

Procedure

1. Use PBS wash cells 3 times, 10 ml/time, remain 10 ml in the flask after the last time wash. 256

2. Scrape all the cells on the flask by using scraper (Corning # 3010) and transfer them to 15 ml centrifuge tube (VWR # 21008-216)

3. Count the cells, do some dilution if needed, make cells 5 x 10 5/100 µl

4. Centrifuge (CentrificTM Centrifuge model 225, Fisher Scientific, Pittsburgh, PA,

USA), remove the PBS, add 10 ml media to the centrifuge tube

5. Prepare 1 µM calcein AM (from Vybrant TM multidrug resistance assay kit): take 10 µl calcein AM from original tube (1 mM) and dissolve it in 10 ml PBS, do

1: 1000 dilution, make the final concentration 1 µM calcein AM

6. Add KT different concentrations 50 µl/well in 96 well plate (Falcon® # 35-

3911), PBS alone in the control wells.

7. Add cells in 96 well plate, 100 µl/well

8. Add calcein AM in 96 well plate, 50 µl/well

9. Mix and incubate at 37 ˚C for 15 min

10. Centrifuge 5 min at 200 × g (Beckman, Model TJ-6 Centrifuge)

11. Remove supernatant, add 200 µl ice cold media

12. Repeat 10, 11 twice

13. Measure the calcein retention as calcein-specific fluorescence by using fluorometer (Spectromax Gemini Sunnyvale, CA, USA). The absorption maximum for calcein is 494 nm, the emission maximum is 517 nm.

257

Protocol C: Cytotoxicity Studies

Objective: To make sure all the concentrations of test compounds or extracts we used in other studies are not toxic for the cells in certain time period.

1. Seed Caco-2, MDCKII-MDR1 or MDCKII-wild type cells at 25,000 cells/well in 48 well plate (Falcon® 35-3078, (Becton Dickinson and company, Franklin lakes, NJ, USA) grown for 7 days (Caco-2) and 4 days (MDCKII-MDR1 and

MDCKII-wild type)

2. Add different concentrations of test compounds or extracts at different time point

3. Start Cytotoxicity studies

Cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium and by assessing mitochondrial reduction of 3-(4,5-dimethythiazole-2yl)-2,5-diphenyl tetrazolium bromide

(MTT).

C.1 Instruction for LDH assay:

Preparation of buffer and reagents: 258

1. Prepare 0.1 M phosphate buffer (pH 7.4): Weight 2.1761 gram KH2PO4 (Fisher

# P285-500) and 22.4658 gram Na2HPO4 · 7H2O (Fisher # S373-500) to 500 ml distilled water. Adjust pH to 7.4 by using 1M NaOH or HCl. Then make volume to

1000 ml with distilled water.

2. Prepare 2.5 mg/ml (0.25% w/v) NADH (β-nicotinamide adenine dinucleotide, reduced form, Sigma # N-8129) solution in 10 ml of 0.1 M phosphate buffer and place on ice.

3. Prepare sodium pyruvate (Sigma # P2256) solution 1 mg/ml in 10 ml of 0.1 M phosphate buffer and place on ice.

Procedure:

1. 130 µl of warm 0.1 M phosphate buffer

2. Add 25 µl culture medium test sample.

3. Add 25 µl NADH solution.

4. Add 25 µl sodium pyruvate solution.

5. Mixture was mixed and immediately placed into a SpectroMax 190 (Molecular

Devices Co., Sunnyvale, CA, USA) at 37 ˚C

6. Absorbance at 340 nm was recorded at intervals of 30 s for 3 min.

259

C.2 Instructions for MTT assay:

The assay was conducted immediately after test compounds or extracts at different concentrations and different time points treatment.

Procedure:

1. 3-(4,5-dimethythiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma #

M5655) was dissolved in serum- and antibiotic-free culture medium at concentration 0.5 mg/ml and filtered through 10 ml syringe (Becton Dickinson and

Company, Franklin Lakes, NJ, USA) with 0.2 µM pore size 25 mm diameter polythersulfone membrane (Whatman Inc., Florham Park, NJ, USA) to remove small amounts of insoluble residue.

2. MTT-containing medium was added to each well in a volume of 0.25 ml and incubated for 2 hours

3. Remove supernatant and add 625 µl 0.4N-HCL-isopropanol(1:24,v/v), wait for

30 min at room temperature in the dark.

4. 250 µl formazan solution was transferred to the 96-well plate (Falcon®, Becton

Dickinson and company, Franklin lakes, NJ, USA) and the absorbance of the formazan solution was read at a wavelength of 570 nm and 630 nm on the

SpectroMax 190 (Molecular Devices Co., Sunnyvale, CA, USA). 260

Protocol D: Menohop® Liquid Chromatography/Mass Spectrometry LC/MS

Objective: To investigate if Menohop® extract contains xanthohumol

Sample Preparation: xanthohumol standard: 1 mg/ml (dissolve in 50% methanol (Fisher # A452-4) and

50% water)

Menohop® extract: 1 mg/ml (dissolve in 50% DMSO (Sigma # 154938) and 50% water)

Column: Phenomenex® Luna 5 µ C18 (2)

P/No. 00F-4252-E0

100°A particle material diameter

Size: 150 × 4.60 mm

S/No. 337484-10

Guard column: Phenomenex ® C18,

Dimensions: 4 mm × 3 mm ID

PDA set up:

Wave length for channels: 212, 370, 258 nm

PDA scan: from 200 to 400 nm

261

Survey Autosampler set up:

Needle height from bottom: 2.0 mm

Syringe speed: 8.0 µl/s

Flush volume: 400 µl

Flush speed: 250 µl/s

LCQ Advantage MS set up:

Mass range: 150-800

Run time: 35 min

Mobile phases: A: acetonitrile (ACN, Fisher # A998-4), B: 1% formic acid (Fisher

# A119-500)/H2O

Gradient: 40% ACN to 100% ACN in 1% aqueous formic acid over 30 min, at 35 min, the % ACN was returned to 40% in 2 min, and the column was equilibrated for 15 min prior to the next injection. Mass spectra were recorded on a mass spectrometer using atmospheric-pressure chemical ionization (APCI) in positive mode.

Flow rate: 0.8 ml/min

Volume of injection: 10 µl

262

Protocol E: Oregon Grape Root Extract Liquid Chromatography/Mass Spectrometry (LC/MS)

Objective: To investigate if Oregon grape root extract contains berberine and berbamine

Sample Preparation:

Berberine standard (Sigma # B3251): 1 mg/ml (dissolve in 50% methanol (Fisher

# A452-4), and 50% water)

Berbamine standard (Sigma # 547190): 1 mg/ml (dissolve in 50% methanol

(Fisher # A452-4), 50% water)

Oregon grape root extract 1 (Originally from Oregon’s Wild Harvest) and Oregon grape root extract 2 (Originally from Health herbs # 5156): 1 mg/ml (dissolve in

50% methanol (Fisher # A452-4), 50% water)

Column: Zorbax Eclipse XDB-C18

P/No. 990967-902

Size: 250 × 4.60 mm

S/No. USNH010427

Guard column: Symmetry ®

C18, dimensions: 3.9 mm × 20 mm

Part No. WAT054225

263

PDA set up:

Wave length for channels: 235, 254, 345 nm

PDA scan: from 200 to 400 nm

Survey Autosampler set up:

Needle height from bottom: 2.0 mm

Syringe speed: 8.0 µl/s

Flush volume: 400 µl

Flush speed: 250 µl/s

LCQ Advantage MS set up:

Mass range: 150-800

Run time: 20 min

Mobile phases: A: acetonitrile (ACN, Fisher # A998-4), B: buffer solution (30 mM ammonium acetate , 14 mM Triethylamine), isocratic A:B = 32%: 68%

Flow Rate: 1.0 ml/min

Volume of injection: 10 µl

Preparation of the buffer solution:

Take 2 ml from Triethylamine (TEA, Sigma # T0886) to 800 ml distilled water,

Add 2.3 gram ammonium acetate (Mallinckrodt, # 3484), mix well

Adjust pH to 4.85 by using acetic acid (Fisher # A507-500) 264

Adjust volume to 1000 ml with distilled water

Calculation for the percentage of berberine in Oregon grape root extract:

Oregon grape root extract 1 (E1):

Total area: 6365603 + 4797205 + 942500 + 496890 + 446499 + 4800712 +

105182 + 463550 + 1345863+ 169248 + 3651244 + 7138 + 7280 +13254 + 15818

= 23627986

Berberine area: 3651244

The percentage of berberine in Oregon grape root extract 1:

3651244/23627986 = 15.45%

Oregon grape root extract 2 (E2):

Total area:

10841503 + 9309344 + 1378469 + 1435458 + 1229183 + 1110156 + 9202622 +

2506197 + 7971589 = 44984521

Berberine area: 7971589

The percentage of berberine in Oregon grape root extract 2:

7971589/44984521 = 17.72%

265

Protocol F: Oregon Grape Root Extract Purification

Objective: To purify different component in Oregon grape root extract

Procedure:

1. Prepare Oregon grape root extract 1 (E1) and Oregon grape root extract 2 (E2)

E1 preparation:

One ml of Oregon grape root liquid extract (Oregon’s Wild Harvest) was transfered to two amber glass vials (3.5 cm × 6 cm), and 5 ml distilled water was added to each vial. Then, the amber glass vial was put it in -80 ˚C freezer for 1 hour. Afterwards, the frozen sample was using FreeZone® freeze dry system

(Kansas City, MO, USA) to obtain E1 as a dryness powder. The final weight of product yield was 25 mg/ml.

E2 preparation:

Exactly 1.5 g of Oregon grape root extract powder (Health Herbs # 5156) was transferred in a 250 ml Erlenmeyer, 30 ml methanol was added, sonicate in water bath at 25 ˚C for 5 min at 2, 200 rpm, moderate hand-shaken for 5 min, and was transferred to two 15 ml centrifuge tube and centrifuge (CentrificTM Centrifuge model 225, # 04-978-50, Fisher Scientific, Pittsburgh, PA, USA) 5 min at 2, 200 rpm. The supernatant was taken and stored in 4 ˚C refrigerator. To prepare E2 powder, 1 ml supernatant was transferred to two amber glass vials (3.5 cm × 6 cm), and 5 ml distilled water was added to each vial. Then, the amber glass vial 266 was put it in -80 ˚C freezer for 1 hour. Afterwards, the frozen sample was using

FreeZone® freeze dry system (Kansas City, MO, USA) to obtain E2 as a dryness powder.

2. Add different amount of methanol and make the final concentration 12.5 mg/ml for E1 and E2

3. Run HPLC (Waters 2690)

The HPLC was used a Waters 2690 separations module with a Model 996 photodiode array detector (Waters Corporation, Milford, MA, USA). The system was operated with the Millennium 32 Chromatograph Manager version 3.0 software (Waters Corporation, Milford, MA, USA). The samples were filtered using 1 ml syringe (Becton Dickinson and Company, Franklin Lakes, NJ, USA) with a 0.2 µm pore size Waterman® syringe filter (Whatman Inc., Clifton, NJ,

USA) before injection. The analysis was performed through a 250 × 4.6 mm i.d., 5

µM, Zorbax Eclipse XDB-C18 Column (Agilent Technologies, Palo Alto, CA,

USA) with a 3.9 × 20 mm i.d. Symmetry® C18 guard column (Waters Corporation,

Milford, MA, USA), pumped isocratically with acetonitrile: buffer (32:68). The buffer consisted of 30 mM ammonium acetate and 14 mM TEA (2 ml TEA in 1L distill water, add 2.3 g ammonium acetate), adjusted to pH 4.85 with acetic acid.

The flow rate was adjusted to 1 ml/min. The UV absorption spectrum of each signal was recorded between 200 to 400 nm and the chromatograms were 267 extracted at 254 nm. The total run time for HPLC analysis was 20 min. Injection volume was 20 µl, do 5 times injection, then use 20% water 80% ACN wash. Each peak was collected for about 25 times.

4. The collected samples were using roto- evaporator (evaporate organic portion) and FreeZone freeze dry system (evaporate aqueous portion) to obtain the dry powder for each peak

5. Add 1 ml methanol to dissolve the powder

6. Repeat 3, 4 until obtain the clean peak for each component

268

Protocol G: Oregon Grape Root Extract Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC/MS/MS) Analysis

Objective: To characterize different components in Oregon grape root extract 1 and Oregon grape root extract 2

Sample preparations and the column description are the same as that in the protocol for Oregon grape root extract LC/MS (See Appendix E)

The details for the MS instrument set up are as follows:

MS Detector set up:

Segment 1:

Time: 4 min

Scan event: 3

Event 1: parent mass: 342.13 (m/z), isolation width: 1.0 (m/z), normalized collision energy: 80%

Event 2: parent mass 639.27 (m/z), isolation width: 1.0 (m/z), normalized collision energy: 80%

Event 3: parent mass 625.27 (m/z), isolation width: 1.0 (m/z), normalized collision energy: 80%

Segment 2 : 269

Time: 2.5 min

Scan event: 1

Parent mass 338.13 (m/z), isolation width: 1.0 (m/z), normalized collision energy:

80%

Segment 3:

Time: 10 min

Scan event: 1

Parent mass 352.13 (m/z), isolation width: 1.0 (m/z), normalized collision energy:

80%

Segment 4

Time: 12.5 min

Parent mass 336.13 (m/z), isolation width: 1.0 (m/z), normalized collision energy:

80%

Survey PDA method:

Spectra:

Start wavelength: 200 nm to 400 nm

Units: wavelength/absorbance

Wavelength step (nm): 5

Sample rate (HZ): 5.0 270

Filter bandwidth (nm): 1

Channel: 3

Channel A: 235 nm, filter bandwidth (nm): 9

Channel B: 254 nm, filter bandwidth (nm): 9

Channel C: 345 nm, filter bandwidth (nm): 9

Survey LC pump:

Gradient program:

Time (min) Flow (ml/min) buffer ACN

0 1 68% 32%

20 1 68% 32% 271

Protocol H: Protein Separation

Objective: To separate the protein from the cells after different pretreatments

Preparation:

1. Preparation for 0.3 M guanidine chloride in 95% ethanol:

Take 15 ml from the original bottle of 8 M guanidine chloride (Pierce # 24115), add 385 ml 95% ethanol/distill water

Preparation for 1% SDS:

Weight 5 g SDS powder (Fisher # BP166-500), dissolve it in 500 ml distilled water

2. Add Phosphate buffered saline (PBS) to the cells, wash twice.

3. Remove PBS, and all cells were collected by adding 1 ml of Trizol® reagent

(Invitrogen # 15596-018) to each and pipetting to a 1.5 ml centrifuge tube (VWR #

20170-038). The collected samples were then stored at -20 °C.

Procedure:

1. The cells were taken out of the -20 °C and 200 µl of chloroform (EM Science #

3150) was added to each sample. Mix well and centrifuge 15 min at 12,000 rpm by using Eppendorf centrifuge (Eppendorf # 5415C) 272

2. Remove the aqueous portion (RNA)

3. Add 100% ethanol 300 µl to the original 1.5 ml centrifuge tube. Mix it and wait for 2-3 min

4. Centrifuge for 5 min at 2-3 × 1000/ min, suspend down DNA (in the bottom)

5. Prepare two 1.5 ml Eppendorf centrifuge tubes (Eppendorf # 022364111)

6. Take about 490 µl (aqueous portion) from the original 1.5 ml centrifuge tube to the new tube (490 µl/tube)

6. Add 750 µl isopropanol (Ricca Chemical # 4210-1) to each tube

7. Wait for 10 min

8. Centrifuge at 12,000 g for 10 min

9. Remove the aqueous portion by using drawn pipette (VWR # 14673-043) (we can see the white pellet on the bottom)

10. Add 1 ml 0.3 M guanidine chloride to each tube

11. Wait for 20 min

12. Centrifuge at 10,000 g for 5 min

13. Remove guanidine chloride by using drawn pipette

14. Repeat 10, 11, 12, 13 twice

15. Add 1 ml 100% ethanol to each tube, vortex them (mix, lift the pellet from the bottom) using Fisher Vortex (Fisher # 12-812)

16. Wait for 20 min

17. Centrifuge at 10,000 g for 5 min

18. Remove the ethanol with drawn pipette 273

19. Vacuum dry the protein pellet for 10 min

20. Add 5% SDS 200 µl for dissolving the pellet (if not dissolve, incubate them at

50 ˚C, sediment any insoluble material by centrifugation at 10,000 g for 10 min at

2˚C to 8˚C, and transfer the supernatant to a fresh tube).

21. The sample is stored at -20˚C for future use. 274

Protocol I: Protein Determination

Objective: To determine the protein concentrations in the different pretreatment samples

Standard curve: using Bovine Serum Albumin (BSA)

Prepare stock solution of BSA: Take 2 mg of BSA (Sigma # A2153) and dissolve it in 1 ml distill water, make the stock solution 2 mg/ml

Standard curve is from BSA 125 µg/ml to 2000 µg/ml (2 mg/ml), each concentration do triplicate (5 µl each)

Standard volume Solvent used for dissolve Concentration of BSA and source the protein (1% or 2% SDS) 1 20 µl 2 mg/ml BSA 0 2000 µg/ml (2 mg/ml)

2 30 µl 2 mg/ml BSA 10 µl 1500 µg/ml (1.5 mg/ml) 3 20 µl 2 mg/ml BSA 20 µl 1000 µg/ml (1 mg/ml

4 20 µl 1500 µg/ml 20 µl 750 µg/ml (tube 2) 5 20 µl 1000 µg/ml 20 µl 500 µg/ml (tube 3) 6 20 µl 500 µg/ml 20 µl 250 µg/ml (tube 5) 7 20 µl 250 µg/ml 20 µl 125 µg/ml (tube 6) 8 0 20 µl 0

In 96 well plate (Falcon® Becton # 35-3072), put 5 µl protein sample or BSA from each tube to well, each sample do triplicate 275

Add 25 µl reagent A (BioRad # 500-0113) and reagent S (BioRad # 500-0115) mixture (50:1, v:v) to each well

(Total for 96 wells: 25 µl × 100 well = 2.5 ml, approximately 3 ml reagent A + 60

µl reagent S)

Add 200 µl reagent B (BioRad # 500-0114) to each well

(Total for 96 wells: 200 µl × 100 well = 20 ml reagent B)

Take the plate to the Spectromax 190 (Molecular Devices Co., Sunnyvale, CA,

USA) and mix for 5 sec

Wait for 15 min

Read at 790 nm