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Characterization of Herb-Drug Interactions through Glucuronidation

Permanent Link: http://ufdc.ufl.edu/UFE0041440/00001

Material Information

Title: Characterization of Herb-Drug Interactions through Glucuronidation
Physical Description: 1 online resource (127 p.)
Language: english
Creator: Mohamed, Mohamed
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cohosh, cranberry, echinacea, egcg, garlic, ginkgo, ginseng, glucuronidation, herb, incubation, inhibition, interactions, intestine, liver, metabolism, microsomes, mpa, palmetto, pharmacokinetics, phytochemicals, raloxifene, saw, tea, thistle, ugt, ugt1a, ugt1a1, ugt1a4, ugt1a6, ugt1a9, valerian
Pharmaceutics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The use of herbal supplements has continued to increase over the last decade. Many herbal supplement users concomitantly take prescription and non-prescription drugs, raising the potential for herb-drug interactions. Cytochrome P450-mediated herb-drug interactions have been reported in many studies. By contrast, the effects of herbal extracts on UDP-glucuronosyl transferase (UGT) enzymes have not been adequately studied. The goal of this research project was to identify commonly used herbal extracts that have potential to inhibit the glucuronidation pathway. First, we studied the effects of Ginkgo biloba extract and its major constituents on mycophenolic acid (MPA) glucuronidation. Ginkgo extract and its main flavonoid aglycones, quercetin and kaempferol, inhibited MPA glucuronidation in human liver and intestinal microsomal incubates. By comparing IC50 values to expected physiologic concentrations of ginkgo compounds in different body compartments, ginkgo extract is likely to inhibit MPA glucuronidation in the human intestine. The second aim was to identify herbal extracts that can potentially inhibit UGT1A1-mediated drug metabolism. A screening in human liver microsomes (HLM) was performed with commonly used herbal extracts to assess the potential for inhibition of UGT1A1 activity. Milk thistle extract and the green tea catechin epigallocatechin gallate (EGCG) were found to be potential inhibitors of first pass metabolism of UGT1A1 substrates. Among the extracts screened, EGCG exhibited the most potent inhibition. Therefore, we examined the effect of EGCG on intrinsic intestinal clearance of raloxifene, a substrate for intestinal glucuronidation by UGT1A1. EGCG exhibited concentration-dependent inhibition of raloxifene in vitro intestinal clearance, suggesting that green tea extracts may increase raloxifene oral bioavailability if taken concomitantly. Lastly, we screened commonly used herbal supplements for their effects on UGT1A4, 1A6, and 1A9 activities in HLM. In vitro inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. In conclusion, this project shows that commonly used herbal supplements may inhibit UGT-mediated drug metabolism. Based on observed inhibitory potency and predicted or known concentrations, glucuronidation is more likely to be affected in the intestine than the liver. The observed herb-UGT interactions warrant further research to investigate the pharmacokinetic consequences and clinical significance.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mohamed Mohamed.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Frye, Reginald F.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041440:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041440/00001

Material Information

Title: Characterization of Herb-Drug Interactions through Glucuronidation
Physical Description: 1 online resource (127 p.)
Language: english
Creator: Mohamed, Mohamed
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cohosh, cranberry, echinacea, egcg, garlic, ginkgo, ginseng, glucuronidation, herb, incubation, inhibition, interactions, intestine, liver, metabolism, microsomes, mpa, palmetto, pharmacokinetics, phytochemicals, raloxifene, saw, tea, thistle, ugt, ugt1a, ugt1a1, ugt1a4, ugt1a6, ugt1a9, valerian
Pharmaceutics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The use of herbal supplements has continued to increase over the last decade. Many herbal supplement users concomitantly take prescription and non-prescription drugs, raising the potential for herb-drug interactions. Cytochrome P450-mediated herb-drug interactions have been reported in many studies. By contrast, the effects of herbal extracts on UDP-glucuronosyl transferase (UGT) enzymes have not been adequately studied. The goal of this research project was to identify commonly used herbal extracts that have potential to inhibit the glucuronidation pathway. First, we studied the effects of Ginkgo biloba extract and its major constituents on mycophenolic acid (MPA) glucuronidation. Ginkgo extract and its main flavonoid aglycones, quercetin and kaempferol, inhibited MPA glucuronidation in human liver and intestinal microsomal incubates. By comparing IC50 values to expected physiologic concentrations of ginkgo compounds in different body compartments, ginkgo extract is likely to inhibit MPA glucuronidation in the human intestine. The second aim was to identify herbal extracts that can potentially inhibit UGT1A1-mediated drug metabolism. A screening in human liver microsomes (HLM) was performed with commonly used herbal extracts to assess the potential for inhibition of UGT1A1 activity. Milk thistle extract and the green tea catechin epigallocatechin gallate (EGCG) were found to be potential inhibitors of first pass metabolism of UGT1A1 substrates. Among the extracts screened, EGCG exhibited the most potent inhibition. Therefore, we examined the effect of EGCG on intrinsic intestinal clearance of raloxifene, a substrate for intestinal glucuronidation by UGT1A1. EGCG exhibited concentration-dependent inhibition of raloxifene in vitro intestinal clearance, suggesting that green tea extracts may increase raloxifene oral bioavailability if taken concomitantly. Lastly, we screened commonly used herbal supplements for their effects on UGT1A4, 1A6, and 1A9 activities in HLM. In vitro inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. In conclusion, this project shows that commonly used herbal supplements may inhibit UGT-mediated drug metabolism. Based on observed inhibitory potency and predicted or known concentrations, glucuronidation is more likely to be affected in the intestine than the liver. The observed herb-UGT interactions warrant further research to investigate the pharmacokinetic consequences and clinical significance.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mohamed Mohamed.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Frye, Reginald F.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041440:00001


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CHARACTERIZATION OF HERB-DRUG INTERACTIONS THROUGH
GLUCURONIDATION




















By

MOHAMED-ESLAM F. MOHAMED


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

UNIVERSITY OF FLORIDA

2010

































2010 Mohamed-Eslam F. Mohamed

































To my parents









ACKNOWLEDGMENTS

First, I would like to express my gratitude to Dr. Reginald Frye, my advisor, for his

continuous guidance, teaching, and support since I joined his lab. For the rest of my

career, I will always be indebted to him for his mentorship. I would also like to thank Dr.

Butterweck, Dr. Hochhaus, and Dr. Shuster for serving on my committee and for their

helpful comments on my dissertation. I am deeply grateful to the person who

encouraged me to seek graduate studies, provided advice, and brotherly support when I

worked at Misr International University in Egypt-Dr. Sherief Khalifa. He is indeed one

of the people who directed my path.

I would like to thank the members of Frye lab who made the work in the lab

enjoyable. Special thanks to Cheryl Galloway and Melonie Stanton for creating a family

environment in the lab; I appreciate their kindness and support. I would also like to

thank Prajakta for her help when I first started the program. I am grateful to the

pharmacy students who helped me with this project: Stephen Harvey, Daniel Gonzales,

and Tiffany Tseng. Their hard work is an integral part of this project. To my colleagues

at the pharmacotherapy department, I appreciate your friendship, the conversations we

had, and the discussions in the journal clubs and seminars. At every little discussion, I

learned something that affected the way I approached my research.

I would like to show my gratitude to the person who provided an indispensable

emotional support and encouragement-my wife Rana-and to the little one whose

pure smiles and giggles soothed the toughness of graduate school-my daughter

Malak. Without the two of them, graduate school would have been much tougher.









Lastly, I would like to thank my parents who have been continuously praying for my

success and my brother and sisters for their invaluable support and for always being

there for me.









TABLE OF CONTENTS


page

A C K N O W LE D G M E N T S ......... ............................ ................................ .... ............... 4

LIS T O F TA B LE S .................................................................................................. 9

LIST O F FIG URES........................................... ............... 10

LIST OF ABBREVIATIONS ........................................... 11

A B ST R A C T .................... ........................................................................... ...... 12

CHAPTER

1 EFFECTS OF HERBAL SUPPLEMENTS ON DRUG GLUCURONIDATION.
REVIEW OF CLINICAL, ANIMAL, AND IN VITRO STUDIES ............... .............. 14

Introduction ................ ............... ... ............................. 14
Popularity of Herbal Supplement Use in the US ........................ ............... 15
Potential for Herb-Drug Interactions through Drug Metabolizing Enzymes ............. 16
Glucuronidation Enzymes .............................. ..... ............... 17
Glucuronidation as a Pathway for Drug Interactions ..................... ................ 18
Search Strategy .............................. ............ .................. .......... 19
Herbal Medicines Containing Substrates or Modulators of UGT Enzymes ............. 19
N o n i J u ic e ........................................................ ........... 1 9
G a rlic ......... ........................... .......................................... 1 9
M angosteen Juice ........... ......... ............................. ............. 20
G re e n T e a .......... ......... ......... .................. ..... ............... 2 0
Echinacea................................................ ............... 21
G in kg o ................................................................................ ............... 2 2
G in s e n g ............................................................................................... 2 2
M ilk Thistle ............................ ............................. 23
S o y ....................... ........ ................. .................................................. 2 4
Cranberry ...... ......... ............... ....... ........ 25
St. John's Wort .............. ........... ................... 26
A lo e ..................................................................................................... 2 7
V a le ria n ............................................................................................... 2 7
Conclusion and Summary........................................... ............... 28
Study O objectives ......... .... ............................................................................ 28








6









2 DETERMINATION OF MYCOPHENOLIC ACID PHENOLIC GLUCURONIDE IN
MICROSOMAL INCUBATES USING HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY.............................. 35

Introduction ...................................................................... ......... 35
Experim ental.......................... ..... ......... .............. 36
C hem icals and R agents ......................................................... .......... ..... 36
Chromatography Conditions........................ ..... ........................... 36
Mass Spectrometry Conditions................................................. 37
Stock Solutions, Standards, and Quality Controls (QCs) .............. .......... 38
Microsomal Incubation Conditions and Sample Preparation.......................... 38
M ethod V alidation ................................................ ........... 39
Calibration, Precision and Accuracy................. ....................... 39
Extraction Recovery, Matrix Effect, and Stability.................... ... .......... .. 39
Data Analysis ....................... ......... .... ...... ............... 40
Results ..... .................. ...... .. .... ............... 40
Chrom atographic M ethod ...................... ......... ........ ........... 40
Calibration, Precision, and Accuracy....................................... .................... 40
Extraction Recovery, Matrix Effect, and Stability......... ...... .. ........... 41
Characterization of Km and Vmax ...................... ................... 41
C o inclusion ............... ....................... ................ ........... ..... 4 1

3 INHIBITION OF INTESTINAL AND HEPATIC GLUCURONIDATION OF
MYCOPHENOLIC ACID BY GINKGO BILOBA EXTRACT AND FLAVONOIDS.... 47

Intro d uctio n ......... ...... ............ ................................. ........................... 4 7
Materials and Methods........................................ .......... 49
Chem icals and Reagents ... ... ............................................... .. .................. 49
Herbal Extracts ................. ............................ 50
Inhibition of M PA Glucuronidation Assay ............ ...................................... 50
Detection of MPA-7-O-glucuronide..................................... 51
E nzym e K inetics A nalysis................................ ................................ ......... 52
Results ................ ....... .... .. .. ........... .................. ............... 52
Inhibition of MPA Glucuronidation by Ginkgo biloba...................................... 53
Effect of Ginkgo Compounds on MPA Glucuronidation................................ 53
Inhibition Kinetics Analysis ............................................................. 54
D is c u s s io n .............. ..... ............ ................. ............................................. 5 4

4 INHIBITORY EFFECTS OF COMMONLY USED HERBAL EXTRACTS ON
UGT1A1 ENZYME ACTIVITY ... .................................................................... 64

Introduction ........... ....... ........................ ............ 64
Materials and Methods................................ ............... 66
Chem icals and Reagents ............................................................................... 66
Preparation of Herbal Working Solutions............... ....................... 66
In Vitro Incubations................... ...... ................ ............... 67
HPLC Analysis .......................... ......... ......... 68









Data Analysis ................ ........ ................. 69
Results ......... .. ...... ..................... ............ ................... 69
Screening Experiments: ... ................. ......... .......... 69
Confirmatory Experiments and Determination of Precise ICso Values: ........... 70
D iscu ss io n .......... ......... .................. ...... .................................... 7 0

5 INHIBITORY EFFECTS OF EPIGALLOCATECHIN GALLTE ON RALOXIFENE
IN VITRO CLEARANCE .......... .......... ......... ................ ........ ....... 80

Introd uctio n ............... .......... ........................... ........................... 80
Materials and Methods........................................ .......... 81
Chem icals and Reagents ........ ....................................................... ...... 81
Incubations w ith H IM ............. ................. ........................ .............. 81
HPLC-MS/MS Assay of Raloxifene ...... ................................ 82
Estimation of Non-specific Protein Binding....... ....... .. ...................... 83
D ata A analysis ....... .............. ................ ..... ................. .............. 83
Results and Discussion.......................................... ............... 83

6 INHIBITORY EFFECTS OF COMMONLY USED HERBAL EXTRACTS ON
UGT1A4, 1A6, AND 1A9 ENZYME ACTIVITIES.................................. ............... 86

Introd uctio n ............... .......... ........................... ........................... 86
Materials and Methods........................................ .......... 87
Chem icals and Reagents ............................................................................... 87
Preparation of Herbal W working Solutions............... ....................... 88
Incubations of Herbal Extracts with TFP .................................... ................... 89
Chromatographic Analysis of TFP glucuronide (TFPG) ............................... 90
Incubations of Serotonin with Herbal Extracts ............... .............. .............. 90
Chromatographic Analysis of Serotonin Glucuronide................................... 91
Incubations of M PA with Herbal Extracts................................ .................... 92
M PAG LC-MS/MS Assay....... .......................................... ................. ....... 92
Data A analysis ............. ............................................................................... 93
Results ................ ....... ............................................. 93
Effect of herbal extracts on TFPG formation..................................... 94
Effect of herbal extracts on serotonin glucuronide formation.......................... 94
Effect of herbal extracts on MPAG formation...... ........ .................... 95
D iscu ss io n .......... ......... .................. ...... .................................... 9 5

7 CONCLUSION AND FUTURE DIRECTIONS.................... .................. 109

R E F E R E N C E S ............................................... ...... .......... ...... 113

BIOGRAPHICAL SKETCH .............. ............ ... ......................... 127









LIST OF TABLES


Table page

1-1 Top selling herbal supplements in the US in 2006................ ........ ............... 30

1-2 Summary of studies on glucuronidation of phytochemicals and modulation of
UGT enzymes by phytochemicals and herbal extracts.................................. 31

2-1 Precision (R.S.D. %) and accuracy (R.E. %) for MPAG in microsomal
incubations. .................. ............. ............ .. ......... ......... ..... 43

2-2 Assessment of extraction recovery, matrix effect, and stability of MPAG
analytical assay. .............. ....... .............. ................................ 43

3-1 Inhibition of MPA-7-O-glucuronidation by Ginkgo biloba extracts ............... ... 59

3-2 Inhibition of MPA-7-O-glucuronidation by ginkgo flavonoids. ........................... 60

4-1 List of Herbal extracts investigated for effect on UGT1A1............... ............ 75

4-2 Rough ICso and volume/dose index values for inhibition of estradiol-3-0-
glucuronidation by nine herbal extracts. ................... ............... 76

4-3 Precise ICso values for herbal extracts showing strongest inhibition of
estradiol-3-O-glucuronidation. ................. .......... ... ... ............... 77

5-1 Effect of green tea EGCG on raloxifene in vitro intrinsic clearance using
HIM ........... .... ..... ................ .... ........... 85

6-1 List of herbal extracts screened for UGT1A4, UGT1A6, and UGT1A9
inhibition. .............. ......... .............................................. 100

6-2 Effect of commonly used herbal extracts on UGT1A4, UGT1A6, and UGT1A9
a ctiv ity ................ ................................... ........................... 10 1

6-3 Determination of inhibitory potency of selected UGT1A4, UGT1A6, and
UGT1A9 herbal inhibitors ............................................................. 103









LIST OF FIGURES


Figure page

1-1 The growing interest in studying herbal supplements............... ............... 33

1-2 Expression of UGT enzymes in the liver and the small intestine. ................. 34

2-1 Chemical structures of analytes .............................. .............. 44

2-2 Extracted HPLC-MS/MS chromatograms of incubations and spiked MPAG
s a m p le s ......... ................................................. .................................... 4 5

2-3 Determination of apparent Km and Vmax for MPAG formation in human liver
m icrosomes. ............. .......... ...................... 46

3-1 Chemical structures of main ginkgo components, mycophenolic acid, and
M PA -7-O -glucuronide ........................................................ .............. 61

3-2 Effect of Ginkgo biloba extracts on mycophenolic acid 7-O-glucuronidation in
v itro .................. .................................. ....... ..... ...... 6 2

3-3 Inhibition of mycophenolic acid 7-O-glucuronidation by quercetin and
kaem pferol ............. ...... ........... ................................. ... 63

4-1 Effect of herbal extracts on E-3-G formation as an index for UGT1A1 activity
in HLM. .............. ......... ........... ... ......... ...... ................ 78

4-2 Inhibition of E-3-G formation by herbal extracts................................. ......... .. 79

5-1 Effect of green tea EGCG on raloxifene in vitro intrinsic clearance using HIM... 85

6-1 Effect of commonly used herbal extracts on UGT1A4, UGT1A6, and UGT1A9
enzyme activities. ............................. ....... ............... ............... 105

6-2 Inhibitory effect of green tea catechin EGCG on TFPG formation in HLM........ 106

6-3 Inhibition of serotonin glucuronide formation by saw palmetto and milk thistle
extracts. ............. .......... ... .. ............ ................................. 107

6-4 Inhibition of MPAG formation by cranberry and milk thistle extracts................. 108











AUC

CLint, u

CYP

E-3-G

ECG

EGC

EGCG

HIM

HLM

HPLC

ICso

Ki

Km

LC-MS/MS

MPA

MPAG

MS

TFP

TFPG

UDPGA

UGT

V/D


LIST OF ABBREVIATIONS

Area under the plasma concentration-time curve

Unbound intrinsic clearance

Cytochrome P450

Estradiol-3-O-glucuronide

(-)-epicatechin-3-gallate

Epigallocatechin

(-)-epigallocatechin-3-gallate

Human intestine microsomes

Human liver microsomes

High-performance liquid chromatography

Concentration of inhibitor that results in 50% inhibition of reaction

Dissociation constant for binding of inhibitor to enzyme

Concentration of substrate that produces half-maximal velocity

Liquid Chromatography/tandem mass spectrometry

Mycophenolic acid

Mycophenolic acid 3-D-glucuronide

Mass spectrometry

Trifluoperazine

Trifluoperazine-N-glucuronide

Uridine diphosphate glucuronic acid

U DP-glucuronosyltransferase

Volume per dose index


Maximum enzyme velocity


Vmax









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

CHARACTERIZATION OF HERB-DRUG INTERACTIONS THROUGH
GLUCURONIDATION


By


Mohamed-Eslam F. Mohamed

August 2010

Chair: Reginald F. Frye
Major: Pharmaceutical Sciences

The use of herbal supplements has continued to increase over the last decade.

Many herbal supplement users concomitantly take prescription and non-prescription

drugs, raising the potential for herb-drug interactions. Cytochrome P450-mediated

herb-drug interactions have been reported in many studies. By contrast, the effects of

herbal extracts on UDP-glucuronosyl transferase (UGT) enzymes have not been

adequately studied. The goal of this research project was to identify commonly used

herbal extracts that have potential to inhibit the glucuronidation pathway. First, we

studied the effects of Ginkgo biloba extract and its major constituents on mycophenolic

acid (MPA) glucuronidation. Ginkgo extract and its main flavonoid aglycones, quercetin

and kaempferol, inhibited MPA glucuronidation in human liver and intestinal microsomal

incubates. By comparing ICso values to expected physiologic concentrations of ginkgo

compounds in different body compartments, ginkgo extract is likely to inhibit MPA

glucuronidation in the human intestine. The second aim was to identify herbal extracts

that can potentially inhibit UGT1Al-mediated drug metabolism. A screening in human









liver microsomes (HLM) was performed with commonly used herbal extracts to assess

the potential for inhibition of UGT1A1 activity. Milk thistle extract and the green tea

catechin epigallocatechin gallate (EGCG) were found to be potential inhibitors of first

pass metabolism of UGT1A1 substrates. Among the extracts screened, EGCG

exhibited the most potent inhibition. Therefore, we examined the effect of EGCG on

intrinsic intestinal clearance of raloxifene, a substrate for intestinal glucuronidation by

UGT1A1. EGCG exhibited concentration-dependent inhibition of raloxifene in vitro

intestinal clearance, suggesting that green tea extracts may increase raloxifene oral

bioavailability if taken concomitantly. Lastly, we screened commonly used herbal

supplements for their effects on UGT1A4, 1A6, and 1A9 activities in HLM. In vitro

inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw

palmetto for UGT1A6, and cranberry for UGT1A9. In conclusion, this project shows that

commonly used herbal supplements may inhibit UGT-mediated drug metabolism.

Based on observed inhibitory potency and predicted or known concentrations,

glucuronidation is more likely to be affected in the intestine than the liver. The observed

herb-UGT interactions warrant further research to investigate the pharmacokinetic

consequences and clinical significance.









CHAPTER 1
EFFECTS OF HERBAL SUPPLEMENTS ON DRUG GLUCURONIDATION.
REVIEW OF CLINICAL, ANIMAL, AND IN VITRO STUDIES

Introduction

In the last decade, interest in studying the pharmacologic effects of herbal

supplements, including their potential to interact with drug metabolizing enzymes, has

grown. The number of publications citing herbal supplements has increased by nearly

five-fold in the 2000s compared to the 1990s (Figure 1-1A). This upsurge coincided

with an escalation in use of herbal supplements, which raised concern by health

professionals regarding the potential for herbs to adversely affect drugs

pharmacokinetics and pharmacodynamics (Gardiner et al., 2008).

Several milestone events have lead to the development of interest in studying herb-

drug interactions as summarized in Figure 1-1B. These events shaped the current

widespread use of herbal supplements and highlighted the knowledge deficiency

regarding their safety. In 1994, the United States Congress passed the Dietary

Supplement Health and Education Act. Under the provisions of this law, dietary

supplements, including herbals, are exempt from regulations applied to drugs, including

premarketing safety and efficacy studies (Gurley, 2010). Concurrently, the Internet

became widely accessible and was commonly used to market herbal products, which

lead to a boost in the use of herbal supplements in the mid to late 1990s (Morris and

Avorn, 2003). In 1998, Congress established the National Center for Complementary

and Alternative Medicine with the goal of funding research on complementary and

alternative medicine, including herbal supplements (www.nih.gov). Two years later, a

milestone case study published in The Lancet described a possible interaction between

St. John's wort, an herbal supplement commonly used for depression, with the









immunosuppressant drug cyclosporine (Ruschitzka et al., 2000). This case study

sparked a wave of clinical, in vitro, and animal studies addressing St. John's wort

interactions with drug metabolizing enzymes (Shord et al., 2009). Meanwhile, reports

emerged associating ephedra use with heart attacks, which eventually lead to the

withdrawal of all products containing ephedra from the US market (Haller and Benowitz,

2000). These events set off an alarm that research was needed to characterize the

safety of herbal supplements as well as their potential to interact with conventional

drugs. Recently, the scientific community has requested that the FDA play a more

rigorous role in evaluating safety and efficacy of herbal supplements with calls for

premarketing safety data and studies on interactions with drug metabolizing enzymes

(Tsourounis and Bent, 2010).

Several case studies, reports, and review articles have described the potential of

herbal supplements and phytochemicals to modulate cytochrome P450 (CYP) enzymes.

On the other hand, the effect of herbal extracts on glucuronidation, a major conjugative

metabolism pathway, has not been sufficiently studied. The aim of this review is to

summarize evidence regarding the potential of the top 20 selling herbal supplements to

interact with UGT enzymes.

Popularity of Herbal Supplement Use in the US

The herbal supplement market has grown continuously in the last decade. Table 1-1

lists the top selling herbal supplements in the US in 2006 (NBJ, 2007). In 2006,

Americans spent $4.6 billion dollars on herbal supplements, representing a 4% growth

in sales from 2005 (NBJ, 2007). Survey studies show that about 20% of Americans use

at least one herbal supplement. Meanwhile, one in four herbal supplements users takes

one or more prescription drugs, raising the potential for herb-drug interactions









(Eisenberg et al., 1998; Bardia et al., 2007). In addition, patients with chronic diseases,

which are likely to be treated by multiple drugs, use herbal supplements more frequently

than the general population, thereby increasing the risk for interactions (White et al.,

2007; Miller et al., 2008).

Potential for Herb-Drug Interactions through Drug Metabolizing Enzymes

Enzymatic biotransformation (i.e., metabolism) plays a major role in disposition of

endogenous and exogenous compounds including both drugs and herbal constituents.

Biotransformation reactions are generally divided into two groups, phase I and phase II;

each encompasses a wide range of enzymes and catalytic activities (Crettol et al.,

2010). Phase I reactions involve hydrolysis, reduction, and oxidation and usually result

in only a small increase in hydrophilicity (Parkinson, 2001). In phase I, CYP enzymes

rank first in terms of clinical importance and number of substrates. On the other hand,

phase II reactions include conjugation of compounds with a hydrophilic group producing

a more hydrophilic and easily excreted product (except for acetylation and methylation).

Phase II reactions may or may not be preceded by phase I reactions. For some

substrates, such as morphine and mycophenolic acid, phase II conjugation with

glucuronic acid represent the chief metabolic pathway (Parkinson, 2001).

Herbal supplements contain a myriad of natural chemicals that share the same

metabolic pathways with prescription drugs (Zhou et al., 2007). This may result in

activation or inhibition of the metabolism of concomitantly taken drugs, over or under-

exposure to drugs, and consequently, treatment failure or toxicity. At least 30 clinically

proven herb-drug interactions mediated through CYP enzymes have been described

(Skalli et al., 2007; Izzo and Ernst, 2009). Induction of CYP2C19, for example, by

Ginkgo biloba resulted in subtherapeutic levels of anticonvulsant drugs, which









precipitated fatal seizures (Kupiec and Raj, 2005). St. John's wort has the most

documented evidence of pharmacokinetic drug interactions with more than 100

publications in the last 10 years on its interactions with prescription drugs (Izzo and

Ernst, 2009). For example, induction of CYP3A4 and P-glycoprotein by St. John's wort

resulted in decreased exposure to midazolam (144%), tacrolimus (159%), alprazolam

(152%), verapamil (180%), and cyclosporine A (152%), respectively (Whitten et al.,

2006). In contrast, interactions through glucuronidation have not been adequately

characterized.

Glucuronidation Enzymes

Conjugation with glucuronic acid (glucuronidation) represents the main phase II

reaction and one of the most essential detoxification pathways in humans (Dutton,

1980). The UDP-glucuronosyl transferases (UGT) are a superfamily of enzymes which

constitutes two families, UGT1 and UGT2, and three subfamilies, UGT1A, 2A, and 2B

comprising at least 18 different enzymes (Figure 1-2) (Owens et al., 2005). UGT

enzymes are widely and differentially expressed throughout the human body

(Guillemette et al., 2010). Although the majority of UGT enzymes are expressed in the

liver, UGT1A7, 1A8, and 1A10 are expressed exclusively extrahepatically, mainly in the

intestine (Izukawa et al., 2009; Ohno and Nakajin, 2009). UGT1A9, 2B7, and 2B11 are

expressed at relatively high quantities in the kidney. Figure 1-2 displays the difference

in UGT expression between the liver and intestine, which are the main sites for

xenobiotic glucuronidation.

UGT enzymes conjugate a wide range of endogenous compounds, drugs,

environmental compounds, and phytochemicals (Tukey and Strassburg, 2000; Ouzzine

et al., 2003). Although UGT enzymes generally display broad and overlapping









substrate specificities, selective probes have been identified for the main hepatic UGT

enzymes, 1A1, 1A4, 1A6, 1A9, 2B7, 2B15, and 2B17 (Burchell et al., 2005; Court,

2005). Identification of selective probes, development of analytical assays, and the

commercial availability of human liver microsomes (HLM) have facilitated the in vitro

evaluation of glucuronidation interactions (Court, 2005).

Glucuronidation as a Pathway for Drug Interactions

Several reports have commented on the clinical significance of interactions through

UGT enzymes. The glucuronidation pathway has been frequently described as a low

affinity pathway, with relatively small impact on substrate exposure in vivo as a result of

inhibition (Williams et al., 2004; Burchell et al., 2005). This has been observed for

substrates that have alternative metabolic pathways and relatively low affinity for UGT

enzymes. However, if the substrate is metabolized mainly through glucuronidation,

inhibition can result in a significant increase in exposure. For example, exposure to

zidovudine, a substrate for UGT2B7, increased by 31% and 74% due to inhibition of

glucuronidation by atovaquone and fluconazole, respectively (Sahai et al., 1994; Lee et

al., 1996). Moreover, rash, which could be life-threatening, resulted from inhibition of N-

glucuronidation of lamotrigine by valproic acid (Kiang et al., 2005). In addition to

inhibition, interactions with glucuronidation can occur through induction of UGT

enzymes. Studies have reported that rifampicin and lopinavir/ritonavir induced

lamotrigine glucuronidation, which required a doubling of the dose to achieve a

therapeutic plasma concentration (Ebert et al., 2000; van der Lee et al., 2006). These

examples show that drug-drug interactions through modulation of glucuronidation can

be clinically significant. Similarly, since many phytochemicals are substrates for UGT

enzymes, herb-drug interactions may occur through this pathway.









Search Strategy

Systematic literature searches were conducted in MEDLINE (through PubMed) and

Google Scholar databases through March 2010. The search terms used were each of

the 20 top-selling herbal supplements (Table 1-1) or their main secondary metabolites in

combination with the terms 'glucuronidation' or 'UGT'. Only articles written in English

were included. No other restrictions were imposed. The herbal supplements below are

listed in the order of their 2006 sales (Table 1-1).

Herbal Medicines Containing Substrates or Modulators of UGT Enzymes

Noni Juice

Noni juice (Morinda citrifolia) has a long history of being used as a medicinal plant for

a wide range of indications including hypertension, menstrual cramps, gastric ulcers,

and many others (Potterat and Hamburger, 2007). Noni juice contains several classes

of secondary metabolites, including polysaccharides, fatty acid glycosides, iridoids,

anthraquinones, and flavonoids (Potterat and Hamburger, 2007). Many of these are

phenolic compounds that may be substrates for UGT enzymes and may compete with

metabolism of drugs. However, no studies were found regarding the glucuronidation of

compounds in noni juice. In a study in rats, noni juice inhibited ex-vivo p-nitrophenol

glucuronidation, which is mainly catalyzed by UGT1A enzymes, by 35% at a dose of 2.1

mg/kg and 49% at a dose of 21 mg/kg. However, there was no inhibition at a higher

dose of 210 mg/kg (Mahfoudh et al., 2009).

Garlic

Garlic (Alium sativum) bulbs have been used for over 4000 years as a medicinal

plant for a variety of ailments including headache, bites, intestinal worms and tumors

(Corzo-Martinez et al., 2007). Garlic is rich in organo-sulphur compounds such as alliin,









and y-glutamylcysteines, diallyl sulphide, diallyl disulphide, and others (Corzo-Martinez

et al., 2007). These compounds are not known to be substrates for glucuronidation.

Gwilt et al. (1994) studied the effect of garlic on acetaminophen metabolism in healthy

subjects. Subjects were given 10 mL garlic extract daily (equivalent to six to seven

cloves of garlic) for three months. Garlic consumption did not have a significant effect

on acetaminophen or acetaminophen glucuronide pharmacokinetic parameters.

Mangosteen Juice

Mangosteen (Garcinia mangostana) juice is well-known for its anti-inflammatory

properties and it is traditionally used in the treatment of skin infections and wounds

(Obolskiy et al., 2009). Mangosteen juice is rich in phenolic compounds called

xanthones, mainly a, 3, and y-mangostin (Obolskiy et al., 2009). Bumrungpert et al.

(2009) showed that a-mangostin was conjugated by phase II enzymes in caco-2 cells.

In their study, one third of a-mangostin was conjugated after 4-6 hours of incubation

with cells. Conjugation was measured by hydrolysis using snail enzyme that possesses

both glucuronidase and sulfatase activity. Therefore, it was not possible to determine

the relative contribution of glucuronidation and sulfation.

Green Tea

Green tea (Camellia sinensis) has gained increased popularity as a beverage and an

herbal supplement with many attributed health benefits including reduction in the risk of

cardiovascular disease and certain cancers (Cabrera et al., 2006). Green tea extract is

rich in polyphenolic compounds called catechins. The major green tea catechins are:

(-)-epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate (ECG), (-)-

epigallocatechin (EGC), (-)-epicatechin, (+)-gallocatechin, and (+)-catechin (Gupta et

al., 2002). EGCG is believed to be the most biologically active and most abundant









catechin in green tea extract (Feng, 2006). In vitro, animal, and human studies provide

evidence that green tea catechins are metabolized by methylation, sulfation and

glucuronidation in (Feng, 2006). Lu et al. (2003) reported that EGCG was conjugated

by UGT1A1, 1A8, and 1A9 and that glucuronidation of EGCG was much higher than

EGC.

In terms of interactions, a study in rats showed that consumption of green tea extract

for four weeks enhanced hepatic glucuronidation of 2-nitrophenol, a substrate for

UGT1A enzymes. However, the effect was not dose dependent (Bu-Abbas et al.,

1998). Zhu et al. investigated the effect of administration of green tea extract for 18

days on hepatic glucuronidation activity in female Long-Evans rats. Green tea extract

stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by

30-37%, 15-27% and 26-60%, respectively (Zhu et al., 1998). The same authors

reported that green tea polyphenols, including EGCG, inhibited estradiol and estrone

glucuronidation in vitro using rat liver microsomes with ICso values of 10-20 pg/mL (Zhu

et al., 1998). In HLM, green tea catechins inhibited the glucuronidation of SN-38, the

active metabolite of the anticancer drug irinotecan, in a concentration-dependent

manner (Mirkov et al., 2007). However, in human hepatocytes, a significant decrease in

glucuronide was observed in only 33% (EGCG), 44% (ECG), and 44% (EGC) of the

hepatocyte preparations. Therefore, the authors concluded that at pharmacologically

relevant concentrations, catechins are unlikely to inhibit the formation of irinotecan

inactive metabolites when administered concomitantly (Mirkov et al., 2007).

Echinacea

Echinacea products refer to herbs or roots of Echinacea purpurea, Echniacea

angustifolia, or Echinacea pallida, or a combination of any of them (Gale Group., 2001).









The herbs and roots of these different species have different composition and medicinal

properties. Among the common compounds in echinacea are polyphenolic compounds

including cichoric acid and echinacoside. Jia et al. (2009) studied phase II metabolites

of echinacoside in rats and isolated two glucuronide metabolites for echinacoside (Jia et

al., 2009). In vitro studies using HLM or expressed UGT enzymes are needed to

characterize the contribution of UGT to echinacoside metabolism

Ginkgo

Ginkgo (Ginkgo biloba) leaf extract is commonly used for its perpetual benefits on

memory and circulation. The primary active constituents of ginkgo are terpene lactones

(ginkgolides and bilobalide) and flavone glycosides, which are hydrolyzed in vivo to

flavone-aglycones (e.g., quercetin, kaempferol, and isorhamnetin) (Chan et al., 2007).

Ginkgo flavonoids are substrates for intestinal and hepatic UGT enzymes, primarily

UGT1A9 and, to a lesser extent, UGT1A3 (Oliveira and Watson, 2000; Zhang et al.,

2007; Chen et al., 2008b).

There is in vitro and animal evidence that flavonoids modulate UGT enzymes. In a

study using HLM, quercetin inhibited UGT1A1 activity with an IC50 value higher than

50 pM (Williams et al., 2002; Moon et al., 2006). In contrast, quercetin and kaempferol

increased testosterone glucuronidation by almost 2.5- and 4-fold, respectively, in a

prostate cancer cell line (Sun et al., 1998). In a study done in rats, quercetin induced 4-

nitrophenol glucuronidation activity by 1.5- to 4-fold in rat liver and different parts of the

intestine (Van der Logt et al., 2003).

Ginseng

Ginseng typically refers to roots of Panax ginseng or Panax quinquefolium, which are

used as general tonics and adaptogens (Chen et al., 2008a). The most important









bioactive components contained in ginseng are a group of saponins called ginsenosides

(Chen et al., 2008a). No reports of ginsenosides glucuronidation were found in the

literature. In a pharmacokinetic study in which ginsenoside Rd was administered

intravenously to volunteers, no glucuronide conjugates were detected in plasma (Yang

et al., 2007). Another in vitro study on metabolism of ginsenoside Rg3 using rat S9 liver

fraction did not detect any glucuronidated metabolites (Cai et al., 2003). In a

pharmacokinetic interaction study, 10 healthy volunteers received 300 mg of

zidovudine, a UGT2B7 substrate, orally before and after 2 weeks of treatment with 200

mg American ginseng extract twice daily. American ginseng did not significantly affect

the pharmacokinetic parameters of zidovudine or zidovudine glucuronide (Lee et al.,

2008).

Milk Thistle

Milk thistle (Silybum marianum) is commonly used to treat hepatotoxicity (Shord et

al., 2009). Extract of milk thistle is rich in flavonolignans, primarily silybin, silydianin,

and silychristine, which are collectively known as silymarin (Dhiman and Chawla, 2005).

There is evidence on glucuronidation of silymarin flavonolignans from both animal and

human studies. In a study in rats, silybin A, silychristin, and silydianin were excreted as

glucuronides (Miranda et al., 2008). Moreover, silibinin mono- and di-glucuronides were

detected in human plasma following ingestion of silibinin phytosome capsules in

colorectal carcinoma patient (Hoh et al., 2006).

In vitro experiments using recombinant enzymes and hepatocytes showed inhibitory

effects of milk thistle compounds on UGT enzymes. Silybin inhibited recombinant

UGT1A1, 1A6, 1A9, 2B7 and 2B15 with IC50 values of 1.4, 28, 20, 92, and 75 pM,

respectively using 7-Hydroxy-4-(trifluoromethyl)coumarin as a substrate for the different









UGT enzymes (Sridar et al., 2004). In hepatocytes, silymarin inhibited glucuronidation

of 4-methylumbelliferone, a substrate for UGT1A6 and 1A9, by about 80% and 90% at

concentrations of 100 and 250 pM, respectively (Venkataramanan et al., 2000). In

another in vitro study using HLM and estradiol-3-O-glucuronidation as an index for

UGT1A1 activity, silymarin inhibited UGT1A1 at estradiol concentrations of 50 and

100 pM, while results at lower concentrations showed mixed inhibition and activation

(Williams et al., 2002). On the other hand, in a pharmacokinetic study in cancer

patients, 4-day and 12-day administration of milk thistle showed no significant effects on

the pharmacokinetics of the anticancer drug irinotecan (van Erp et al., 2005).

Soy

There has been increasing interest in soy isoflavones, especially genistein and

daidzein, due to their wide range of potential biological activities (Nielsen and

Williamson, 2007). In vitro and clinical studies provide evidence that soy isoflavones

are substrates for UGT enzymes. Despite being structurally similar, genistein and

daidzein conjugation exhibit preferences for different UGT enzymes. UGT1A1, 1A4,

1A6, 1A7, and 1A9 catalyzed 7- and 4'-glucuronidation of both genistein and daidzein,

while UGT 1A10 was selective for genistein. The authors also reported that genistein,

but not daidzein, was conjugated in human colon microsomes (Doerge et al., 2000).

The glucuronide was the predominant circulating form for both genistein (69-98%) and

daidzein (40-62%), with smaller amounts of the aglycone and sulfate. This indicates

that glucuronidation is the primary route of metabolism for these soy isoflavones.

Pfeiffer et al. (2005) reported that daidzein and genistein as well as several

structurally related isoflavones modulated UGT1A1 activity in vitro using HLM. Daidzein

(25 pM) stimulated estradiol-3-glucuronidation, a marker for UGT1A1 activity, by about









50%. In contrast, genistein (25 pM) inhibited the 3-glucuronidation by about 80%. The

17-glucuronidation of estradiol was not affected by either compound. In another study

in HLM, unhydrolyzed and hydrolyzed soy extracts inhibited dihydroestosterone

glucuronidation, an index for UGT2B15 activity, with ICso values of 4.6 and 6.1 pg/mL,

respectively (Anderson et al., 2003).

In a study in mice, genistein and daidzein only slightly decreased UGT activities in

some tissues (Froyen et al., 2009). The effect was sex and duration dependent. In this

study, genistein and daidzein inhibited glucuronidation of 3-methyl-2-nitrophenol in the

small intestine of male mice after five days of isoflavone administration by about 50%

and 40%, respectively. This effect did not reproduce in the liver and the kidneys, or in

female mice.

Cranberry

Cranberry (Vaccinium macrocarpon) is commonly consumed in the US to prevent

urinary tract infections with potential activity as an antibacterial and anticancer (Neto,

2007). Cranberry juice contains a high content of flavonoids and phenolic acids.

Among the cranberry flavonoids, quercetin is the most abundant (Neto, 2007). As

mentioned under ginkgo, quercetin is conjugated by UGT1A9 and, to a lesser extent,

UGT1A3 (Oliveira and Watson, 2000; Zhang et al., 2007; Chen et al., 2008b) In

addition to flavonoids, cranberry juice contains resveratrol, which is also found in grapes

and red wine (Wang et al., 2002). In vitro studies show that resveratrol is

glucuronidated to two major glucuronide conjugates, resveratrol-3'-glucuronide and

resveratrol-4'-glucuronide. The major enzymes that catalyze resveratrol glucuronidation

are UGT1A1 and UGT1A9 (Brill et al., 2006; Iwuchukwu and Nagar, 2008). No studies

on effects of cranberry juice on UGT enzyme activities were found.









St. John's Wort

St. John's wort (Hypericum perforatum) extract is a commonly used herbal therapy

for insomnia and depression (Gaster and Holroyd, 2000). Flavonol glycosides are the

major class of compounds found in St. John's wort extract, with rutin, hyperoside,

isoquercitrin, quercetrin (qercetin 3-rhamnoside), and miquelianin being the main

compounds. Other components include hypericin, pseudohypericin, and hyperforin

(Butterweck and Schmidt, 2007). As mentioned for ginkgo, quercetin is known to be a

substrate and modulator of UGT1A enzymes (Oliveira and Watson, 2000; Chen et al.,

2008b). No studies regarding glucuronidation of other St. John's wort components were

found.

In a recent study, Volak et al. (2010) reported that hypericin inhibited UGT1A6-

mediated glucuronidation of acetaminophen in human colon cells and serotonin in

UGT1A6-expressing insect cells with ICso values of 7.1 and 0.59 pM, respectively. The

authors concluded that the mechanism of this interaction was through inhibition of

UGT1A6 phosphorylation by protein kinase C, which is considered a novel mechanism

of drug-drug interaction.

In an animal study, effects of St. John's wort on irinotecan pharmacokinetics were

measured after 3 and 14 days of daily St. John's wort administration. Long-term (14-

day) exposure to St. John's wort significantly decreased Cmax of irinotecan by 39.5%

and SN-38 by 38.9%, but didn't significantly affect SN-38 glucuronide plasma

concentrations. On the other hand, short-term (3-day) administration of St. John's wort

did not significantly alter the pharmacokinetics of CPT-11 and SN-38, but decreased the

AUCo0_ and the elimination tl/2 of SN-38 glucuronide by 31.2% and 25.8%, respectively

(Hu et al., 2007). In the same study, St. John's wort extract (5pg/mL) decreased SN-38









glucuronidation by 45% in rat liver microsomes, while pre-incubation of St. John's wort

extract in hepatoma cells significantly increased SN-38 glucuronidation. These results

indicate that St. John's wort may affect pharmacokinetics of SN-38.

Aloe

Aloe vera leaf extract is used as an herbal supplement due to its attributed biological

benefits, including antiviral, antibacterial, laxative, and immunostimulatory effects (Ni et

al., 2004). Aloe extract contains several classes of phytochemicals that have been

thoroughly described (Dagne et al., 2000). Among the different classes, Aloe vera

extract is rich in anthracene derivatives (e.g., aloe-emodin). There is evidence that

glucuronidation is the primary route of metabolism of aloe-emodin in rats (Shia et al.,

2009). Characterization of aloe-emodin glucuronidation has not been performed.

Valerian

Valerian (Valeriana officinalis) extract is commonly used as an herbal supplement to

treat sleeping disorders, restlessness, and anxiety (Pato ka and Jakl, 2010). Alkaloids,

organic acids, terpenes, and valepotriates are among the major classes of

phytochemicals found in valerian extract. In terms of interactions with UGT enzymes,

valerian methanolic extract inhibited UGT1A1 and UGT2B7 in HLM using estradiol and

morphine as probe substrates, respectively. In the same study, valerenic acid, a

monoterpene in valerian extract, inhibited glucuronidation of acetaminophen, estradiol,

and morphine with both HLM and expressed UGT enzymes (Alkharfy and Frye, 2007).

ICso values for inhibition with valerenic acid were 9.24 pM for acetaminophen

glucuronidation, 8.79 pM for estradiol-3-O-glucuronidation, 2.33 pM for estradiol-17-0-

glucuronide, 4.96 pM for morphine-3-glucuonide, and 47.31 pM for testosterone

glucuronide. The clinical significance of this in vitro interaction is yet to be determined.









Conclusion and Summary

The studies reviewed provide evidence on the potential for modulation of UGT-

mediated drug metabolism by commonly used herbal supplements. Flavonoid

compounds were the most studied class of phytochemicals for metabolism by and

interactions with UGT enzymes. Based on in vitro and animal studies, flavonoid-rich

supplements may affect metabolism of UGT drug substrates. However, this effect has

not been studied in a clinical pharmacokinetics study. Overall, no studies were found

for 6 out of the top 20 reviewed herbs regarding their glucuronidation or modulation of

UGT enzymes. Moreover, only 3 clinical studies investigating the effect of herbal

supplements on the pharmacokinetics of UGT drug substrates were published (Gwilt et

al., 1994; van Erp et al., 2005; Lee et al., 2008). Taken together, there is a scarcity of

information on glucuronidation of majority of phytochemicals and their potential to

interact with UGT-mediated drug metabolism.

The overall goal of this work was to characterize the effects of commonly used herbal

supplements on glucuronidation reactions in vitro.

Study Objectives

* Study the effect of Ginkgo biloba leaf extract and its major flavonoid and terpene
lactone components on MPA glucuronidation using human liver and intestine
microsomes. This aim was constructed based on the finding that quercetin and
kaempferol, the major ginkgo falvonoid aglycones, are metabolized through
UGT1A9, the main enzyme metabolizing MPA.

* Characterize the effects of commonly used herbal extracts on UGT1A1 activity in
HLM and determine inhibitory potency for potential inhibitors. Our hypothesis
was that herbal extracts would inhibit UGT1A1 due to the high content of
polyphenolic phytochemicals, which can be substrates and inhibitors of UGT1A1.

* Assess the effect of green tea catechin EGCG on raloxifene intrinsic clearance
using human intestine microsomes. Our hypothesis was that EGCG, having
been identified as a UGT1A1 inhibitor, would inhibit raloxifene intrinsic clearance
in vitro.









* Screen commonly used herbal extracts for inhibition of UGT1A4, UGT1A6, and
UGT1A9 using HLM and characterize the inhibitory potency of the potential
inhibitors. Our aim was to identify potential inhibitors among herbal extracts that
may interact with drugs metabolized through these enzymes.









Table 1-1. Top selling herbal supplements in the US in 2006. Source: NBJ's
Supplement Business Report, October 2007.
Top Herbs 2006 sales
($millions)
1 Noni Juice 257
2 Garlic 155
3 Mangosteen Juice 147
4 Green Tea 144
5 Saw Palmetto 134
6 Echinacea 129
7 Ginkgo Biloba 106
8 Ginseng 98
9 Milk Thistle 93
10 Psyllium 85
11 Soy 69
12 Cranberry 68
13 Maca 66
14 Goji 65
15 Green Foods 64
16 St. John's wort 60
17 Aloe 60
18 Stevia 58
19 Black Cohosh 57
20 Valerian 55









Table 1-2. Summary of studies on glucuronidation of phytochemicals and modulation of UGT enzymes by
phytochemicals and herbal extracts.


Herb

Noni Juice

Garlic
Mangosteen

Green Tea



Saw Palmetto
Echinacea
Ginkgo Biloba







Ginseng


Milk Thistle


Phytochemicals studied for In vitro
glucuronidation


a-mangostin

EGCG>> EGC




Echinacoside
Flavonoids


Flavonolignans


Polyphenols:
JUGT1A


Interaction Studies
Animal

Noni juice:
JUGT1A


Clinical



+UGT1A6


Green tea:
IUGT1A


----------------------------No studies reported--------------------------


Flavonoids:
tUGT2B1 7
JUGT1A1


,UGT1A1
iUGT 1A6
iUGT 1A9
,UGT 2B7
,UGT 2B15


Flavonoids:
tUGT1A6


-UGT2B7


+UGT1A1


References

(Mahfoudh et al.,
2009)
(Gwilt et al., 1994)
(Obolskiy et al.,
2009)
(Bu-Abbas et al.,
1998; Zhu et al.,
1998; Lu et al.,
2003)


(Sun et al., 1998;
Oliveira and
Watson, 2000;
Williams et al.,
2002; Van der
Logt et al., 2003;
Chen et al.,
2008b)
(Cai et al., 2003;
Yang et al., 2007;
Lee et al., 2008)
(Venkataramanan
et al., 2000;
Williams et al.,
2002; Sridar et
al., 2004; van Erp
et al., 2005; Hoh










Psyllium
Soy





Cranberry





Maca
Goji
Green Foods
St. John's wort






Aloe
Stevia
Black Cohosh


et al., 2006)


Isoflavones





Flavonoids
Resveratrol







Flavonoids


Aloe-emodin


----------------------------No studies reported----------------------------
Genistein: *-UGT1A
TUGT1A1
Daidzein:
jUGT1A1
Soy extract:
jUGT2B15
Quercetin: Quercetin:
jUGT1A1 IUGT1A6


----------------------------No studies reported----------------------
----------------------------No studies reported--------------------- -
------------------------------Various contents -------------------
SJW : Quercetin:
jUGT1A1 IUGT1A6
Quercetin:
jUGT1A1
Hypericin:
jUGT1A6


----------------------------No studies reported----------------------
----------------------------No studies reported----------------------


(Doerge et al.,
2000; Anderson
et al., 2003;
Pfeiffer et al.,
2005; Froyen et
al., 2009)
(Oliveira and
Watson, 2000;
Williams et al.,
2002; Van der
Logt et al., 2003;
Brill et al., 2006)



(Oliveira and
Watson, 2000;
Williams et al.,
2002; Hu et al.,
2007; Chen et al.,
2008b; Volak,
2010)
(Shia et al., 2009)


Valerian Valerian & (Alkharfy and
valerenic acid: Frye, 2007)
jUGT1A1
jUGT1A6
JUGT2B7
,, inhibition of UGT; f, activation or induction; "-, no effect on UGT activity; ND, No glucuronides detected in metabolism studies.


























-0Q
1994:
DSH EApassed
HS Exempt from
drug regulations


NCCAM



Wide spread
use of internet

Sharp increase
In use of herbal
supplements


Lancetcase
study
St. John's wort
leads to graft
rejection


0-

Calls for rigorous
FDArole in monitoring
adverse effects of HS
& interactions with drugs


> 24 clinical
studies followed



Ephedrauproar
Association with
heart attacks

Withdrawal from
market
)0


Figure 1-1. The growing interest in studying herbal supplements. A) Number of
PubMed articles citing herbal supplements or herbal medicine in the last three
decades. B) Timeline for milestone events that lead to development of
interest in studying herb-drug interactions.


1800
2 1600
o
1400
, 1200
1000 -
0 8 --
400 .I
8 600 -*----I -I -
E 400 -- -

0 .,,111ii ..ii.
ri (N M it in O r- 00 C) O H (N M m in kD r- 00 cnC O r- (N Mn t Ln O rN W0 cM


Year























































Figure 1-2. Expression of UGT enzymes in the liver and the small intestine. A) Relative
expression of hepatic UGT enzyme based on 20 human liver samples.
Adapted from Izukawa et al. (2009). B) Relative expression of UGT enzymes
in the small intestine based on 3 human intestine samples. Adapted from
Ohno et al.


A. Expression of Hepatic UGT Enzymes


* UGT1A1
* UGT1A3
r UGT1A4
* UGT1A6
* UGT1A9
* UGT2B4
* UGT2B7
* UGT2B10
N UGT2B11
* UGT2B15


B. Expression of Intestinal UGT Enzymes


UGT1A1
UGT1A3
9 UGT1A5
UGT1A6
P UGT1A7
Pl UGT1A8
SUGT1A9
UGT1A10
M UGT2B7
UGT2B15









CHAPTER 2
DETERMINATION OF MYCOPHENOLIC ACID PHENOLIC GLUCURONIDE IN
MICROSOMAL INCUBATES USING HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY1

Introduction

Mycophenolic acid (MPA) is an immunosuppressant drug that has been widely and

successfully used in transplant recipients as well as in patients with immune disorders

(Staatz and Tett, 2007; Walsh et al., 2007). MPA is administered as an ester prodrug or

a sodium salt and is extensively metabolized by UDP-glucuronsyltransferases (UGTs)

to glucuronidated metabolites. MPA-7-O-glucuronide (MPAG) is the main metabolite of

MPA (Figure 2-1). Plasma concentrations of MPAG are typically 20-100-fold higher

than MPA in patients receiving mycophenolate therapy. MPAG is approximately 82%

bound to plasma albumin and is mainly excreted in the urine as the main pathway for

MPA elimination (Staatz and Tett, 2007). Other minor MPA metabolites include the acyl

glucuronide, 7-OH glucose conjugates, and 6-O-desmethyl-MPA (Shipkova et al., 1999;

Picard et al., 2004).

Formation of MPAG is carried out by various UGT enzymes. The main UGT

enzymes involved are UGT1A7 and UGT1A9, while UGT1A8 and UGT1A10 play a

smaller role in MPAG formation (Basu et al., 2004). Plasma levels of MPA and MPAG

vary widely within and between patients, which can directly affect clinical outcomes

(Hummel et al., 2007). In vitro studies with human liver microsomes, a commonly used

approach in drug metabolism and interaction studies, may provide some clues to

understanding this variability. Previous studies have used MPA microsomal incubations


1 Reprinted with permission from Mohamed MF, Harvey SS and Frye RF (2008) Determination of
mycophenolic acid glucuronide in microsomal incubations using high performance liquid chromatography-
tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 870:251-254.









to characterize the UGT enzymes involved in its glucuronidation and study MPA

interaction potential with other drugs (Vietri et al., 2000; Shipkova et al., 2001; Vietri et

al., 2002; Bernard and Guillemette, 2004; Miles et al., 2005; Picard et al., 2005).

Various reports have described assays to measure MPAG in human plasma and

urine(Aresta et al., 2004; Bolon et al., 2004; Patel et al., 2004; Indjova et al., 2005; Yau

et al., 2007); however, validated in vitro assays are lacking. This paper describes an

HPLC-tandem mass spectrometry assay for the quantitative determination of MPAG in

human liver microsomal incubations.

Experimental

Chemicals and Reagents

Mycophenolic acid (MPA) and mycophenolic acid 3-D-glucuronide (MPA-7-O-

glucuronide; MPAG) were purchased from Toronto Research Chemicals (North York,

Ontario, Canada). Potassium phosphate dibasic, uridine diphosphate glucuronic acid,

magnesium chloride, alamethicin, phenolphthalein 3-D-glucuronide (PG; internal

standard), and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, MO,

USA). Pooled human liver microsomes were purchased from In Vitro Technologies Inc.

(Baltimore, MD, USA). Acetonitrile and methanol were purchased from EMD Chemicals

Inc. (Gibbstown, NJ, USA). All chemicals used were of the highest purity available for

analytical research. Deionized water was prepared by using a Barnstead Nanopure

Diamond UV Ultrapure Water System (Dubuque, IA, USA).

Chromatography Conditions

MPAG and the internal standard (IS) PG were chromatographed with a

ThermoFinnigan Surveyor series HPLC system consisting of a Surveyor Plus

autosampler and Surveyor MS pump (Thermo Corp., San Jose, CA, USA). Gradient









chromatography was carried out at ambient temperature on a reversed-phase

Phenomenex (Torrance, CA, USA) Synergi Fusion-RP18 column (100 x 2 mm, 4 pm).

The two mobile phases consisted of (A) 1 mM acetic acid in deionized water and (B) 1

mM acetic acid in acetonitrile. Gradient elution at a flow-rate of 0.22 mL/min was

employed with the following steps: at start of the run, 30% B for one min, then increased

to 90% B in 0.75 min, held at 90% B between 1.75 and 3.1 min, and from 3.6 to 6.5 min,

the column was re-equilibrated at 30% B. The total run time was 6.5 min. The

temperature of the autosampler was maintained at 100C and the injection volume was 5

pL. A divert valve was used to divert flow to waste from 0 to 2 min and from 4.5 to 6.5

min.

Mass Spectrometry Conditions

The LC-MS/MS analysis was carried out on a TSQ Quantum triple quadrupole

mass spectrometer (Thermo Corp., San Jose, CA, USA), equipped with an electrospray

ionization (ESI) source operated in the negative ion mode. Detection of MPAG and PG

was performed for their [M-H]- ions. Analysis was carried out in the single reaction

monitoring (SRM) mode using the mass transitions of m/z 495 319 and m/z

493 175 for MPAG and phenolphthalein (3-D-glucuronide, respectively. MPA was

also monitored at a mass transition of m/z 319 191. The mass spectrometer settings

were a capillary temperature of 3500C, spray voltage of 3.0 kV, and source collision

induced dissociation (CID) of 5 V. Nitrogen was used as the sheath and auxiliary gas

set to 35 and 15 (arbitrary units), respectively. The argon collision gas pressure was set

to 1.5 mTorr (200 mPa) and the collision energy was set to 30 eV for MPA, 22 eV for

MPAG, and 25 eV for PG. The peak full width at half maximum (FWHM) was set at

0.2 Th and 0.7 Th for Q1 and Q3, respectively, and the scan time was set to 250 ms.









Data acquisition and analysis were performed with Xcalibur software version 1.4

(Thermo Corp., San Jose, CA, USA).

Stock Solutions, Standards, and Quality Controls (QCs)

Stock standard solutions of MPAG (0.2 and 2 mM) were prepared by dissolving

the appropriate amount of MPAG in methanol. A series of MPAG standards

(concentrations: 0, 1, 2, 4, 10, 15, and 20 pM) and quality control samples

(concentrations: 2.5, 7.5, and 16 pM) were prepared by subsequent dilution of the stock

standard solutions in 0.1 M phosphate buffer, pH 7.1. Working solutions of MPA (6 mM)

and the internal standard PG (1 mM) were prepared in methanol.

Microsomal Incubation Conditions and Sample Preparation

The incubation conditions were optimized with respect to time of incubation and

microsomal protein concentration. Stock solutions of UDPGA (25 mM) and MgCI2 (5

mM) were prepared in phosphate buffer. Alamethicin (0.2 mg/mL) was prepared in

phosphate buffer containing 10% ethanol. The incubation mixture (final volume, 105

pL) consisted of 300 pM MPA (for the kinetic study, 50, 100, 300, 500, 1000, 1500,

2000, and 2500 pM MPA were used), 1 mM MgCI2, 0.1 M potassium phosphate buffer

(pH 7.1), 0.16 mg/mL microsomal proteins, and 16 pg/mL alamethicin (100 pg

alamethicin/1 mg microsomal proteins). The mixture was pre-incubated on ice for 15

minutes. The reaction was started by adding UDPGA (final concentration, 1 mM). After

the mixture was incubated for 30 min at 370C, the reaction was stopped by adding 315

pL ice-cold acetonitrile and 20 pL internal standard, vortex-mixing, and placing tubes on

ice. Tubes were centrifuged for 10 min at 20,817 x g. The supernatant was diluted in a

ratio of 1:5 with purified water and 5 pL was injected into the HPLC system.









Method Validation

The method was validated for selectivity, linearity, sensitivity, precision, accuracy,

recovery and stability according to the guidelines issued by the Food and Drug

Administration (FDA) for the validation of bioanalytical methods (FDA, 2001).

Calibration, Precision and Accuracy

Calibration curves were constructed using six different concentrations of MPAG

prepared in incubation buffer. Curves were obtained daily for 3 days by calculating

peak-area ratios of MPAG to PG. Data points were fit using linear regression and a 1/y2

weighting-scheme. The precision and accuracy of the assay was determined using

quality control (QC) samples of known MPAG amounts (2.5, 7.5, and 16 pM) prepared

in incubation buffer and processed in the same manner as standards and incubations

samples. Six replicates of each QC were analyzed on 3 days, after which the inter- and

intra-day precision values were calculated using one-way ANOVA using the day as the

grouping variable as described previously (den Brok et al., 2005). Accuracy was

calculated as the percentage of the nominal MPAG concentration. For the assay to be

considered acceptable, precision determined at each concentration level was required

to be within 15% of all days mean and accuracy within 15% of nominal concentration at

all levels of concentrations.

Extraction Recovery, Matrix Effect, and Stability

Extraction recovery, absolute matrix effect, and stability were evaluated for

MPAG samples prepared at concentrations of 2.5 and 16 pM and PG internal standard

(50 pM). Each set of samples was analyzed in triplicates. Extraction recovery was

determined by comparing peak areas of the standards extracted from spiked 0.16

mg/mL microsomal proteins in phosphate buffer to control microsomal proteins









extracted in the same manner and spiked after extraction with the same standard

concentration. Matrix effect on ionization was evaluated by comparing the MPAG peak

areas of samples spiked post-extraction with corresponding peak area ratios of

standards prepared in the injection solution. Processed stability was evaluated by re-

injecting the samples after keeping them in the autosampler at 100C for 36 hours.

Comparison of MPAG and PG peak areas before and after 36-hour storage provided a

measure of stability under normal operating conditions.

Data Analysis

To estimate precision, one-way ANOVA analysis was performed using JMP IN

5.1.2 (SAS Inc, Cary, NC, USA). Data were fit to the Michaelis-Menten equation and

the apparent kinetic parameters of Km and Vmax were determined by non-linear

regression analysis (Prism 4.0, GraphPad software, San Diego, CA, USA).

Results

Chromatographic Method

MPAG, the internal standard PG, and MPA were separated within four minutes of

the chromatographic run. The retention times for MPAG, PG, and MPA were 3.35,

3.41, and 3.90 min., respectively. Representative extracted LC-MS/MS chromatograms

of processed microsomal incubations are shown in Figure 2-2. The MPAG peak was

detected only when substrate, enzyme, and co-enzyme were added. Chromatograms

of double blank incubations, which contained all incubation constituents except MPA,

did not show any interfering peaks at the retention times of either PG or MPAG.

Calibration, Precision, and Accuracy

Standard curves for MPAG were linear over the range of 1-20 pM. The mean

correlation coefficient (r2) for the standard curve was at least 0.99. Intra- and inter-day









RSD% for MPAG QC samples were less than 10% and all calculated concentrations

were within 8% of the actual concentration (Table 2-1).

Extraction Recovery, Matrix Effect, and Stability

Table 2-2 shows the results from the assessment of extraction recovery, matrix

effect, and stability for MPAG and PG. Average extraction recovery for MPAG was

87.4%. There was no significant matrix effect as the average suppression of ionization

by matrix was 12.3%. MPAG and PG were stable in the processed incubation mixtures

as well as in reconstitution solution for at least 36 hours (<10% change in measured

concentration).

Characterization of Km and Vmax

The enzyme kinetic parameters for MPAG formation were estimated by incubating

different concentrations of MPA (50 to 2500 pM) with human liver microsomes (Figure

2-3). The apparent Km and Vmaxwere 285.7 pM and 8.6 nmol/min/mg protein,

respectively. MPAG formation was consistent with Michaelis-Menten kinetics.

Conclusion

MPA glucuronidation represents the primary pathway for MPA biotransformation in

vivo. MPA-7-O-glucuronide is the main metabolite and exhibits 20-100-fold higher

plasma concentrations than MPA (Staatz and Tett, 2007). This paper describes a

specific and sensitive HPLC-tandem mass spectrometry assay for measuring MPAG in

human liver microsomes within a run time of 6.5 minutes. Although several reports

have described assays for MPAG in plasma and urine (Aresta et al., 2004; Bolon et al.,

2004; Patel et al., 2004; Indjova et al., 2005; Yau et al., 2007), this is the first detailed

report of a validated method to determine MPAG concentrations in human liver

microsomes.









The validated assay is a precise (RSD% <10%) and accurate method for

determining MPAG in microsomal incubations over a range of 1 20 pM. The method

is reproducible and subject to minimal matrix effect (Tables 2-1 and 2-2). Previous

kinetic studies on MPAG formation in vitro reported values for Km and Vmax ranging from

95 to 351 pM and from 2.5 to 20.5 nmol/min/mg protein, respectively (Vietri et al., 2000;

Bowalgaha and Miners, 2001; Shipkova et al., 2001; Vietri et al., 2002; Bernard and

Guillemette, 2004; Miles et al., 2005; Picard et al., 2005); the values determined using

this assay are within these ranges. Thus, the assay described is suitable for in vitro

pharmacogenetic and interaction studies of MPA metabolism.









Table 2-1. Precision (R.S.D. %) and accuracy (R.E. %) for MPAG in microsomal
incubations (six replicates per day for three days).
Concentration (pM) R.S.D. (%)a R.E. (%)
Nominal Measured (Mean) Intra-day Inter-day
2.50 2.70 5.6 8.9 8.0
7.50 7.40 3.9 5.1 -1.3
16.0 16.5 6.1 6.8 3.1
aEstimated using one-way ANOVA




Table 2-2. Assessment of extraction recovery, matrix effect, and stability of MPAG
analytical assay.
Nominal MPAG
Nominal MPAG Extraction Recovery a Matrix Effect b Stability C
Concentration
(poM) (%) (SD) (%) (SD) (%) (SD)
(PM)
2.5 83.3 88.0 105.3
16 91.6 87.3 109.7
50 (PG) 103.4 96.9 107.0
a Extraction recovery was calculated using the following formula: Recovery
(%) = [(mean raw peak area)pre ext. spike/(mean raw peak area)post ext. spike] x 100.
b Matrix effect was calculated using the following formula: Matrix effect (%) = [(mean
raw peak area)post ext. spike/(mean raw peak area)neat] x 100.
C Stability was calculated using the following formula: Stability (%) = [(mean raw peak
area)after 36 hours/(mean raw peak area)initial run] x 100.
PG = phenolthalein glucuronide (internal standard)












UDPGA UDP

jl


UGT1A


CH3


CH3


MPA


MPAG


Figure 2-1. Chemical structures of analytes. A) Structure of mycophenolic acid (MPA). B) Structure of mycophenolic acid
glucuronide (MPAG). C) Structure of the internal standard phenolphthalein glucuronide (PG).










- 1000t

600A
5OO
C 250

0
0
75000


B
25000
1 O


MPAG (m/z: 495 > 319)


MPAG (mz: 495 > 319)


480000 MPAG (m/z: 495 > 319)
. 360000
240000-
S120000


'" 300000 PG (m/z: 493 > 175)

S200000
D
100000

- 01


70000]
52500
35000:
175O0
0-


RT: 3.35


RT: 3.35


RT: 3.41


0 1 2 3 4
MPA (m/z: 319 >191) RT: 3.75

E
I 0o from MPAG


30T 1 2 3 4
Time (min)


Figure 2-2. Extracted HPLC-MS/MS chromatograms of incubations and spiked MPAG
samples. A) Microsomal incubations in absence of MPA and PG, B) spiked
lowest MPAG standard (1 M), C) MPAG in microsomal incubation (estimated
concentration is 10 pM), D) representative chromatogram of PG (50 pM) as
the internal standard, and E) MPA (50 pM) in microsomal incubation. The
small peak at 3.35 min. in E (enlarged in inset) is from in-source
fragmentation (loss of glucuronide) of MPAG to MPA.


~li~i


0 1 I i 4


















2' Vma = 8.6

S Km = 285.7
0I V V -. WWI
0 500 1000 1500 2000 2500
Substrate Concentration (iM)

Figure 2-3. Determination of apparent Km and Vmax for MPAG formation in human liver microsomes.









CHAPTER 3
INHIBITION OF INTESTINAL AND HEPATIC GLUCURONIDATION OF
MYCOPHENOLIC ACID BY GINKGO BILOBA EXTRACT AND FLAVONOIDS2

Introduction

Herbal supplement use continues to increase around the globe, especially in

populations looking for natural methods to promote health and wellness. In the US,

surveys estimate that 20% of the population uses at least one herbal supplement

(Bardia et al., 2007). This growing interest in herbals is manifested by annual sales in

the US of over $4 billion dollars (NBJ, 2007). Such public interest is met by concerns

from health professionals regarding possible deleterious interactions of herbals with

conventional drugs. Herbals are considered dietary supplements; hence, they are not

routinely screened for interactions with drug metabolizing enzymes (www.fda.gov).

However, numerous in vitro, animal, and clinical studies and case reports provide

evidence that herbals can interact with conventional drugs and may lead to serious

adverse effects (Gardiner et al., 2008).

Ginkgo biloba is among the most popular herbals used in the world. Its extract is

available over the counter in the US and is commonly prescribed in European countries

for cerebral insufficiency (De Smet, 2005). Antioxidant effects as well as beneficial

effects on memory and circulation have been attributed to G. biloba extract and its

components. The primary active constituents of G. biloba are terpene lactones

(ginkgolides and bilobalide) and flavone glycosides, which are hydrolyzed in vivo to

flavone-aglycones (e.g., quercetin and kaempferol) (Figure 3-1A) (Chan et al., 2007).



2 Reprinted with permission from Mohamed M and Frye R (2010) Inhibition of intestinal and hepatic
glucuronidation of mycophenolic acid by Ginkgo biloba extract and flavonoids. Drug Metabolism and
Disposition 38:270.









Several clinical and in vitro studies have investigated the effect of G. biloba on drug

metabolizing cytochrome P450 enzymes and transporters (Izzo and Ernst, 2009). In

contrast, limited research has been conducted to investigate interactions of G. biloba

and its components with conjugation pathways. In vitro studies have shown that

quercetin and kaempferol inhibit sulfotransferase 1A1 (Eaton et al., 1996; Ghazali and

Waring, 1999); meanwhile, information is lacking regarding effects of G. biloba on drug

glucuronidation.

Glucuronidation constitutes the main pathway of conjugative metabolism for a wide

variety of compounds (Ouzzine et al., 2003); substrates for UDP-glucuronsyltransferase

enzymes (UGTs) include endogenous compounds, drugs and many phytochemicals.

Many flavonoids (e.g., quercetin and kaempferol) are substrates for UGT enzymes.

Moreover, inhibitory effects of flavonoids on UGT1A enzymes have been reported in the

literature (Williams et al., 2002; D'Andrea et al., 2005). For substrates metabolized

mainly through glucuronidation, modulation of UGT activities can lead to significant

effects on pharmacokinetics (Kiang et al., 2005).

Mycophenolic acid (MPA) is an immunosuppressive drug that acts by inhibiting the

production of guanosine nucleotides in lymphocytes, ceasing their proliferation (Allison

and Eugui, 2005). Therefore, it is used to prevent graft rejection in transplant recipients

and to delay progression of the autoimmune disorders (Heatwole and Ciafaloni, 2008).

MPA is available as either a prodrug mofetil ester (CellCept) or as an enteric-coated

sodium salt (Myfortic). Although both formulations have similar pharmacokinetic and

efficacy profiles, absolute oral bioavailability of mycophenolate sodium is 72%

compared to 94% for mycophenolate mofetil (Staatz and Tett, 2007). This difference is









attributed to higher presystemic glucuronidation of MPA from the mycophenolate

sodium formulation. Following oral absorption, MPA is metabolized by UGTs to the

major phenolic conjugate 7-O-MPA-glucuronide (MPAG) (Figure 3-1B). In the liver,

UGT1A9 is the main enzyme catalyzing the formation of MPAG, while UGT1A7,

UGT1A8, and UGT1A10 contribute to MPAG formation extra-hepatically mainly in the

kidneys and intestine (Picard et al., 2005). MPA is a narrow therapeutic index drug with

wide inter- and intra-individual variability and complex pharmacokinetics in transplant

recipients (Staatz and Tett, 2007). Therefore, an alteration in MPA glucuronidation may

cause changes in exposure to the immunosuppressive drug, and consequently,

undesired clinical outcomes. The aim of this study was to investigate the effect of

ginkgo extract and its main components on MPAG formation in human intestinal and

liver microsomes. The results demonstrate that G. biloba and its primary constituents

have the ability to inhibit MPA glucuronidation in the intestine and liver.

Materials and Methods

Chemicals and Reagents

Mycophenolic acid (MPA; 98%) and mycophenolic acid-7-O-glucuronide (MPAG;

98%) were purchased from Toronto Research Chemicals (North York, ON, Canada).

Potassium phosphate dibasic, uridine diphosphate glucuronic acid, magnesium

chloride, alamethicin, phenolphthalein-3-D-glucuronide (PG; internal standard), niflumic

acid, and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Acetonitrile, methanol, quercetin dihydrate (99% purity) and kaempferol (90%) were

purchased from Fisher Scientific (Pittsburgh, PA, USA). Ginkgolide A (95.1%),

ginkgolide B (82.8%), and bilobalide (99.7%) were purchased from ChromaDex (Irvine,









CA, USA). Pooled human liver and intestinal microsomes were purchased from BD

Biosciences Discovery Labware (Woburn, MA, USA).

Herbal Extracts

Ginkgo biloba extract was provided by Finzelberg & Co. KG (Andernach, Germany)

as dry powder. The extract was standardized by the supplier to contain 24%

flavonglycosides, 6% terpene lactones, and < 5 ppm ginkgolic acids using 60% acetone

as the extraction solvent. Unhydrolyzed and acid-hydrolyzed G. biloba working

solutions were freshly prepared by dissolving 30 mg of the powder extract in 1 mL of

either 60% acetone or 60% acetone/40% 5N HCI to prepare the unhydrolyzed and acid-

hydrolyzed working extracts, respectively. The acid treated extract was heated at 900C

for one hour and neutralized with 2N KOH. The acetone-rich extracts were serially

diluted to prepare working solutions of G. biloba with concentrations of 0.05 to 5 mg/mL

and acetone content of 10%.

Inhibition of MPA Glucuronidation Assay

The incubation conditions were optimized with respect to time of incubation and

microsomal protein concentration. A typical 100 pL incubation mixture contained HLM

or HIM (protein concentration, 0.16 mg/mL), alamethicin (100 pg/mg microsomal

protein), MgCI2 (5 mM), MPA, and different concentrations of each test extract or test

compound in 100 mM phosphate buffer, pH 7.4. Microsomes were pre-incubated on ice

with alamethicin for 15 minutes to activate UGT enzymes. The reaction was started by

adding UDPGA (1 mM) and placing incubation tubes in a water bath at 370C for 30

minutes. The reaction was stopped by adding 300 pL of ice-cold acetonitrile and 20 pL

of internal standard (0.5 mg/mL phenolphthalein glucuronide). Tubes were vortex-

mixed for two minutes and centrifuged for 10 min at 20,000 x g. The supernatant was









diluted 12-fold with purified water and 5 pL was injected into the HPLC system.

Incubations with herbal extracts and the corresponding controls contained 1% acetone.

The HLM and HIM used in all experiments were from the same lot.

Screening experiments were conducted to generate ICso values by incubating MPA

at the estimated Km value in the presence of five concentrations of G. biloba

unhydrolyzed and hydrolyzed extracts (final concentrations ranging from 5-500 pg/mL)

or G. biloba individual components (final concentrations ranging from 1-100pM). In

addition to ICso values, inhibitory potency was also expressed as the volume per dose

index, which is defined as the volume in which one dose would be dissolved in to obtain

the corresponding ICso concentration as described by Strandell et al. (2004).

Comparison of this unit to physiological volumes facilitates an assessment of inhibitory

potential.

A Ki value was determined if the ICso value was lower than 100 pM. In such cases,

MPA (60-600 pM with HLM or 30-600 pM with HIM) and a range of concentrations of

individual ginkgo components (10-100 pM with HLM or 3-20 pM with HIM) were used

for the construction of Dixon plots and estimation of Ki values.

Detection of MPA-7-O-glucuronide

MPAG was determined by LC/MS/MS on a ThermoFinnigan Surveyor series HPLC

system connected to a TSQ Quantum triple quadrupole mass spectrometer (Thermo

Corp., San Jose, CA, USA) using electrospray ionization (ESI), as described previously

(Chapter 2). Average assay within-day and between-day relative standard deviations

were 5.2% and 6.9%, respectively and accuracy expressed as relative error was within

8%. Briefly, 5 pL of each sample was injected on a reversed-phase Phenomenex

(Torrance, CA, USA) Synergi Fusion-RP18 column (100 x 2 mm, 4 pm). The two









mobile phases consisted of (A) 1 mM acetic acid in deionized water and (B) 1 mM

acetic acid in acetonitrile. Gradient elution at a flow-rate of 0.22 mL/min was employed

with the following steps: at start of the run, 30% B for one min, then increased to 90% B

in 0.75 min, held at 90% B between 1.75 and 3.1 min, and from 3.6 to 6.5 min, the

column was re-equilibrated at 30% B. Analysis was carried out in the single reaction

monitoring (SRM), negative ion mode using the mass transitions of m/z 495 319 and

m/z 493 175 for MPAG and PG, respectively. MPAG standard solutions were freshly

prepared for each experiment with concentration ranges of 100 nM-4 pM for HIM or 1-

20 pM for HLM incubations.

Enzyme Kinetics Analysis

Km and Vmax were determined by nonlinear regression analysis of the MPAG

formation data using eight different MPA concentrations (0.02 to 1 mM). Data points

were fitted to the Michaelis-Menten model using Prism 4.0 (GraphPad software, San

Diego, CA, USA).

IC50 values were similarly determined by nonlinear regression fitting of the inhibition

data to the IC50 equation (Copeland, 2005) using Prism 4.0. The Ki values were

determined by fitting competitive, noncompetitive, uncompetitive, and mixed-type

inhibition models to the MPAG formation data (Copeland, 2005). The mode of inhibition

was determined on the basis of visual inspection of the Dixon plot and the Akaike

information criterion (Akaike, 1974) using SigmaPlot v.11 Enzyme Kinetics Module 1.3

(Systat Software, Inc., Chicago, IL, USA).

Results

MPA-7-O-glucuronide formation was best explained by Michaelis-Menten kinetics.

The Km and Vmax were 103.9 19.5 pM and 2.6 0.2 nmol/min/mg protein (mean









SEM), respectively, with pooled HLM, whereas with pooled HIM, these values were 67.2

10.1 pM and 408.7 17.1 pmol/min/mg protein (mean SE), respectively. These

values are similar to values previously reported (Shipkova et al., 2001; Miles et al.,

2005; Chang et al., 2009).

Inhibition of MPA Glucuronidation by Ginkgo biloba

Both unhydrolyzed and acid-hydrolyzed G. biloba extracts inhibited MPA

glucuronidation in pooled HIM and HLM (Figure 3-2). MPA concentration was 100 pM

for HLM incubations and 70 pM for HIM incubations. Results showed that unhydrolyzed

and acid-hydrolyzed G. biloba extracts inhibited MPA glucuronidation in HLM with best

fit ICso values of 84.3 11.6 and 20.9 3.6 pg/mL, respectively. More potent inhibition

of MPA glucuronidation was observed in HIM with ICso values of 6.8 0.8 and 4.3 1.2

pg/mL for the unhydrolyzed and acid-hydrolyzed extracts, respectively (Table 3-1). The

volume/dose index values, calculated to estimate the clinical significance of the

inhibition as described previously (Strandell et al., 2004), are shown in Table 3-1.

Effect of Ginkgo Compounds on MPA Glucuronidation

Ginkgo flavonoids quercetinn and kaempferol) and terpene lactones (ginkgolides A

and B, and bilobalide) were incubated with MPA to determine whether or not these

compounds inhibit MPA glucuronidation. Ginkgo flavonoids but not terpene lactones

showed inhibition with ICso values < 100 pM (Table 3-1). Quercetin and kaempferol

inhibited MPA glucuronidation in HLM with ICso values of 19.1 1.3 and 23.1 5.5 pM,

respectively. In agreement with results from incubations with G. biloba extracts,

inhibition of MPA glucuronidation was more potent in HIM, with ICso values of 5.8 0.3

and 7.6 0.6 pM for quercetin and kaempferol, respectively.









Inhibition Kinetics Analysis

To further characterize the inhibition of MPA glucuronidation by ginkgo flavonoids,

enzyme inhibition kinetic experiments were carried out. Based on the analysis of

nonlinear regression of inhibition data and Dixon plots presented in Figure 3-2,

quercetin exhibited mixed-type inhibition against MPA glucuronidation in both HLM and

HIM. Kaempferol exhibited non-competitive inhibition in HLM and mixed-type inhibition

in HIM. In HLM, Ki values were 11.3 1.7 and 33.6 2.5 pM for quercetin and

kaempferol, respectively (Table 3-2; Figure 3-2A). Again, inhibitory potency of quercetin

and kaempferol to MPA glucuronidation in HIM was three to four-fold higher than that in

HLM with Ki values of 2.8 0.4 and 4.5 1.2 pM, respectively (Table 3-2; Figure 3-2B).

Discussion

Scientific and public interest in ginkgo has grown enormously in recent years

because of its purported beneficial effects on memory and circulation (Bardia et al.,

2007). Ginkgo supplements have been widely used with little awareness of the

potential for drug interactions with conventional drugs. Although ginkgo is considered

generally safe, clinical studies and case reports have demonstrated that it can interact

with conventional drugs and may lead to severe adverse effects (Hu et al., 2005; Kupiec

and Raj, 2005). In the current study, ginkgo extract and flavone aglycones inhibited the

UGT-mediated metabolism of mycophenolic acid in human intestinal and liver

microsomes.

In intestinal microsomes, ginkgo extracts inhibited MPAG formation with ICso values

of 4.3 and 6.8 pg/mL for acid-hydrolyzed and unhydrolyzed extracts, respectively. The

clinical significance of this interaction can be postulated based on the recommended

dose of ginkgo supplements and the fraction of MPA metabolized by intestinal enzymes.









Ginkgo extracts are usually taken at a dose of 120 mg to 240 mg per day. Therefore,

ICso-equivalent concentrations can be achieved in the intestine if a 120 mg ginkgo dose

is mixed with 18 to 28 L of fluid (i.e., 6.7 to 4.3 mg/L) or if a 240 mg dose is mixed with

35 to 56 L of fluid. Thus, based on estimates of intestinal volume that range from about

0.5 to 5 L (Helium et al., 2007), concentrations in the intestine after ingestion of a ginkgo

supplement are expected to be much higher than ICso values; accordingly, inhibition of

intestinal UGT enzymes in vivo is likely. The potential for interaction is greater with

enteric-coated mycophenolate sodium, since about 28% of the dose is eliminated

through first pass metabolism (Myfortic prescribing information:

http://www.pharma.us.novartis.com/product/pi/pdf/myfortic.pdf). Inhibiting first pass

metabolism of MPA could result in higher systemic concentrations, enhanced

immunosuppressive effect and increased potential for side effects.

Incubations with HLM also showed inhibition of MPA glucuronidation by ginkgo

extracts. In the liver, UGT1A9 selectively metabolizes MPA to MPAG (Picard et al.,

2005); therefore, MPAG formation can be used as an in vitro UGT1A9 index reaction.

An effect observed on MPAG formation is expected to reproduce with other UGT1A9

substrates like propofol. In vitro screening of ginkgo components for inhibition indicates

that the observed inhibition can be attributed to ginkgo flavonoid components, but not to

terpene lactones. Ki values for inhibition of hepatic MPA glucuronidation by quercetin

and kaempferol were 11.3 and 33.6 pM, respectively.

To understand the clinical significance of this observation, adequate knowledge of

the bioavailability and hepatic concentrations of the inhibitors is necessary. Quercetin

and kaempferol are classified as flavonols, which is a class of flavonoids ubiquitously









found in plants, beverages, and dietary supplements, e.g., tea, onions, apples, red wine,

St. John's wort, and G. biloba (Nijveldt et al., 2001). A typical diet contains about 14-16

mg/day quercetin and 4-6 mg/day kaempferol according to dietary surveys in the

Netherlands and US (Hertog et al., 1993; Sampson et al., 2002); however, the intake

can reach several hundred mg in dietary supplements and herbal products and several

grams in anticancer therapy (Lamson and Brignall, 2000).

In contrast to kaempferol, a relatively large number of studies concerning the

absorption of quercetin have been published. However, the extent to which quercetin

reaches the liver remains largely unknown. Most studies were not able to detect free

quercetin concentrations in plasma and absorption was estimated from the quantities of

quercetin and quercetin conjugates detected in the urine (0.3-1.4% of quercetin dose)

(Scalbert and Williamson, 2000); thus it was assumed that quercetin was poorly

absorbed. However, an early study in healthy ileostomy subjects estimated quercetin

absorption to be 17-52% of orally ingested amount (Hollman et al., 1995). The authors

reported that only 0.3% of the oral quercetin dose was recovered in urine and

concluded that it might be possible that some quercetin accumulated in tissues and was

released slowly over time. A recent study investigating tissue distribution of quercetin in

pigs following long-term dietary supplementation reported that total quercetin

concentration in liver was 5 to 6 fold higher than that in plasma (Bieger et al., 2008).

Interestingly, 93% of quercetin found in the liver was in the aglycone form. Taken

together, further studies are needed to investigate whether long-term ginkgo or

flavonoid-rich supplements may lead to accumulation of quercetin in human liver to

levels that could inhibit mycophenolic acid glucuronidation.









Incubations with intestinal microsomes exhibited 3- to 12-fold more potent inhibition

of MPAG formation than in liver microsomes by ginkgo extracts, quercetin and

kaempferol (Tables 3-1 and 3-2). This difference in inhibition potency can be explained

by differentially expressed UGT enzymes in liver and intestine (Ohno and Nakajin,

2009) and the difference in catalytic activities towards MPA glucuronidation between

liver and intestine microsomes. In this study, microsomal intrinsic clearance (Vmax/Km)

for MPAG formation was 4-fold higher by HLM as compared to HIM (25.12 vs. 6.08

pL/min/mg protein). This is in accordance with previously reported values (Bowalgaha

and Miners, 2001; Shipkova et al., 2001; Picard et al., 2005). In the intestine, UGT1A7,

1A8, 1A9 and 1A10 conjugate MPA to MPAG with different affinities, while in the liver,

MPAG is selectively formed by UGT1A9 (Picard et al., 2005). In addition, UGT1A10

exhibits a much lower catalytic activity towards MPA glucuronidation than UGT1A8 and

UGT1A9, while its expression in the intestine is 13- and 25-fold greater than UGT1A8

and UGT1A9, respectively (Picard et al., 2005; Ohno and Nakajin, 2009). Due to these

differences, interactions may not always translate from liver to intestinal microsomes

with the same magnitude. Therefore, using intestinal microsomes to screen for

interactions may be necessary for drugs metabolized by intestinal glucuronidation.

Two limitations are acknowledged for this study. First, the study does not rule out the

possibility of induction of MPA metabolism by ginkgo. A recent study showed that

ginkgo and its components induce cytochrome P450 enzymes, transporters, and

UGT1A1 (Li et al., 2009). The effect of ginkgo on MPA-metabolizing enzymes in

hepatocytes warrants further research. Second, the study did not control for the

possible inhibition of UGT activities by fatty acids released from the microsomal









membrane, which may inhibit UGT1A9 and result in underestimation of inhibition

potency (Rowland et al., 2008). Although the effect of released fatty acids on MPA

glucuronidation has not been documented, it is possible that the actual potency of

inhibition is greater than what we observed.

Based on our findings, ginkgo supplements taken concomitantly with mycophenolate

sodium could lead to increased MPA exposure secondary to inhibition of presystemic

glucuronidation. Therefore, patients should be advised to avoid ginkgo supplements

while taking enteric-coated mycophenolate sodium-the form of MPA that is more

subject to presystemic metabolism. Effect of ginkgo on MPA systemic metabolism

cannot be predicted, due to lack of information on hepatic concentrations of quercetin

and kaempferol, but will likely be weaker than the presystemic inhibition. MPA is used

in HLM as a probe of UGT1A9 activity because of selective formation of MPAG by

UGT1A9. Therefore, the observed hepatic inhibition would be expected to extrapolate

to other UGT1A9 substrates like propofol and flavopiridol. The actual in vivo effect of

this interaction should be verified in clinical studies.









Table 3-1. Inhibition of MPA-7-O-glucuronidation by Ginkgo biloba extracts. Pooled
human liver or intestine microsomes (0.16 mg/mL) were incubated with
UDPGA (1 mM) and various concentrations of ginkgo extracts, and ginkgo
compounds. ICso values and volume/dose index were determined as
described under Materials and Methods. All incubations were performed in
duplicate. Data are expressed as the best-fit ICso values standard error.
Goodness of fit r2 values for the nonlinear regression model were > 0.9 for
unhydrolyzed and acid-hydrolyzed extracts, quercetin, and kaempferol.
IC50 values Volume/Dose Index*
Mean SE (L)
Extract/Ginkgo HLM HIM HLM HIM
compound
Unhydrolyzed
Unhydrolyzed 84.3 11.6 pg/mL 6.8 0.8 pg/mL 1.4 17.6
G. biloba

Acid-hydrolyzed 27.8
G. bAcid-hydrolyzed loba 20.9 3.6 pg/mL 4.3 1.2 pg/mL 5.8 27.8
G. biloba
Quercetin 19.1 1.3 pM 5.8 0.3 pM 0.7 2.2
Kaempferol 23.1 5.5 pM 7.6 0.6 pM 0.5 1.4
Ginkgolide A > 100 pM > 100 pM < 0.01 < 0.01
Ginkgolide B > 100 pM > 100 pM < 0.06 < 0.06
Bilobalide > 100 pM > 100 pM < 0.03 < 0.03
*Volume/Dose index was calculated by dividing daily dose by the IC50 value (Strandell
et al., 2004). Daily dose was considered to be 120 mg ginkgo extract containing (%w/w)
10.75% quercetin, 8.75% kaempferol, 1.2% ginkgolide A, 0.48% ginkgolide B, and
2.94% bilobalide.









Table 3-2. Inhibition of MPA-7-O-glucuronidation by ginkgo flavonoids. Alamethicin-
activated pooled human liver or intestine microsomes (0.16 mg/mL) were
incubated with UDPGA (1 mM), various concentrations of MPA and various
concentrations of quercetin or kaempferol. Ki values were determined as
described under Materials and Methods. All incubations were performed in
duplicate. Data are expressed as the best-fit Ki standard error.
Inhibitor Ki (pM) Mode of Inhibition
Quercetin
HLM 11.3 1.7 Mixed
HIM 2.8 0.4 Mixed
Kaempferol
HLM 33.6 2.5 Non-competitive
HIM 4.5 1.2 Mixed
























Ginkgolide A: X = H
Ginkgolide B: X = OH


Bilobalide


Kaempferol: X' = H
Quercetin: X' = OH


UGT1A7, 1A8, 1A9, 1A10


MPA MPAG




Figure 3-1. Chemical structures of main ginkgo components, mycophenolic acid, and MPA-7-O-glucuronide. A) Main
bioactive ginkgo components. B) Mycophenolic acid (MPA) and MPA-7-O-glucuronide.

















-- Unhvdrolyzed ginkgo extract
-*- Acid-hydrolyzed ginkgo extract

HLMs


C 0
0S

a.1
(D


-- Unhydrolyzed ginkgo extract
-A- Acid-hydrolyzed ginkgo extract

HI Ms





25 50 75 100 450 475 500
Herbal Extract Concentration ig/ml


Figure 3-2. Effect of Ginkgo biloba extracts on mycophenolic acid 7-O-glucuronidation in vitro. Alamethicin-activated
pooled human liver (panel A) or intestinal (panel B) microsomes (0.16 mg/mL) were incubated with UDPGA (1
mM) and various concentrations of unhydrolyzed (square with solid line) and acid-hydrolyzed (triangle with
dotted line) G. biloba extracts (5, 10, 50, 100, and 500 pg/mL). Incubations were performed using 100 or 70 pM
MPA for HLM and HIM, respectively. Reactions were stopped after 30 minutes by adding 300 pL ice-cold
acetonitrile. MPAG was detected by LC-MS/MS as described under Materials and Methods. Each point
represents the mean of duplicate measurements.


5 0
U- c


D4 -
0.i.


100 200 300 400 500 600
Herbal Extract Concentration uglml











[MPA] = 60 M A
[MPA]= 150 pM ~ 3.5
[MPA] = 300 pM t 3.0
[MPA]= 600 M )
S2.5 -
HLM 2.0
I 1.5 .
g 1.0


*









Sr2 = 0.99


0 20 40 60 80 100 120
B [Quercetin] (pM)
[MPA]= 60 .M
[MPA]= 150 IM o
[MPA] = 300 gM M 3.5
[MPA]= 600 iM 3.0

2.5
0 2.0 /
HLM ,


0 10 20
[Quercetin] (pM)


[MPA] = 30 M
[MPA] = 60 .iM -
[MPA]= 150 M 2
[MPA] = 300 AM D
[MPA] = 600 AM

E

HIM
I


0 20 40 60 80 100 120 0 10 20
[Kaempferol] (pM) [Kaempferol] (pM)


Figure 3-3. Inhibition of mycophenolic acid 7-O-glucuronidation by quercetin and
kaempferol. Alamethicin-activated pooled human liver (panels A and B) or
intestinal (panels C and D) microsomes (0.16 mg/mL) were incubated with
UDPGA (1 mM), various concentrations of MPA, and various concentrations
of quercetin (panels A and C) or kaempferol (panels B and D). Data shown
are representative Dixon plots. Each point represents the mean of duplicate
measurements.









CHAPTER 4
INHIBITORY EFFECTS OF COMMONLY USED HERBAL EXTRACTS ON
UGT1A1 ENZYME ACTIVITY

Introduction

According to a recent US government survey, 38% of adults and 12% of children use

one or more forms of complementary and alternative medicine (CAM) (Barnes et al.,

2008). Among the various CAM forms, herbal supplements are the most commonly

used with sales exceeding $4 billion dollars annually (NBJ, 2007). Moreover, one in

four herbal supplement users also take prescription drugs raising the potential for herb-

drug interactions (Eisenberg et al., 1998).

There are many examples of herb-drug interactions that lead to adverse clinical

outcomes such as treatment failure or serious side effects (Gardiner et al., 2008). Such

interactions usually occur through effects of phytochemicals in herbal extracts on the

pharmacodynamics or pharmacokinetics of drugs. Most pharmacokinetic interactions

occur through modulation of drug metabolizing enzyme activity. Current evidence on

herb-drug interactions come mostly from studies on cytochrome P450 enzymes (Izzo

and Ernst, 2009). For example, St. John's wort was found to be a strong inducer of

CYP3A4, which prompted label changes in prescribing information for many CYP3A4

substrates. On the other hand, there is a paucity of studies on the effect of herbal

supplements on other drug metabolism pathways.

Conjugation with glucuronic acid constitutes a major detoxification and metabolic

pathway for numerous endogenous and exogenous compounds, including many drugs

and phytochemicals (Ouzzine et al., 2003). Glucuronidation is listed as a clearance

mechanism for 1 in 10 of the top 200 drugs (Williams et al., 2004). The UDP-

glucuronosyl transferase (UGT) enzyme superfamily is comprised of two families, UGT1









and UGT2. Among UGT1 enzymes, UGT1A1 is an important glucuronidation enzyme

that is widely expressed throughout the body, especially in the liver and intestine

(Guillemette et al., 2010). Thus, it has an essential role in both first-pass and systemic

clearance of many drugs.

Variability in UGT1A1 activity has been linked to clinical outcomes in patients taking

the anti-cancer drug irinotecan. Patients who carry a variant UGTIA 1*28 allele have

lower UGT1A1 activity, and therefore, are more prone to neutropenia and diarrhea

caused by increased exposure to SN-38, the active metabolite of irinotecan (Schulz et

al., 2009). In addition to drugs, many herbal extracts are rich in phenolic

phytochemicals that are substrates for UGT1A1 (Doerge et al., 2000; Zhang et al.,

2007). Moreover, inhibitory effects of some herbal constituents (e.g., flavonoids) on

UGT1A1 enzymes have been reported in the literature (Williams et al., 2002; D'Andrea

et al., 2005). Therefore, it is important to identify herbal supplements that may affect

UGT1A1 activity and, consequently, alter drug disposition.

The aim of this study was to screen commonly used herbal extracts for inhibition of

UGT1A1 activity using human liver microsomes. Estradiol-3-O-glucuronidation, which is

selectively catalyzed by UGT1A1 in human liver microsomes (Court, 2005), was used

as an index reaction for UGT1A1 activity. Using a screening approach based on current

drug-drug interaction guidelines (FDA, 2006), we identified green tea epigallocatechin

gallate (EGCG), milk thistle extract, saw palmetto extract, and Echinacea purpurea

extracts as inhibitors of in vitro UGT1A1 activity.









Materials and Methods


Chemicals and Reagents

3-Estradiol, 3-estradiol-3-(3-D-glucuronide) [E-3-G], potassium phosphate dibasic,

uridine diphosphate glucuronic acid, magnesium chloride, alamethicin, niflumic acid,

and epigallocatechin gallate (EGCG) were purchased from Sigma-Aldrich (St. Louis,

MO, USA). Acetonitrile, ethanol, methanol, and acetone were purchased from Fisher

Scientific (Pittsburgh, PA, USA). Herbal extracts (ginseng, Panax ginseng; echinacea,

Echinacea purpurea; black cohosh, Cimicifuga racemosa; milk thistle, Silybum

marianum; garlic, Allium sativum; valerian, Valeriana officinalis, and saw palmetto,

Serenoa repens) were generously provided by Finzelberg & Co. KG (Andernach,

Germany) as dry powder. Table 4-1 summarizes the properties of the extracts

screened. UltraPool human liver microsomes (HLM) were purchased from BD

Biosciences Discovery Labware (Woburn, MA, USA). These microsomes are pooled

from 150 donors to provide lot-to-lot consistency.

Preparation of Herbal Working Solutions

Concentrations of herbal extracts in screening incubations represent the

recommended daily intake (RDI) of each extract dissolved in 53 L, 5.3 L, and 0.53 L.

These volumes roughly represent total body fluids, and two extremes of a range of

concentrations that could appear in the small intestine, assuming 100% bioavailability,

as previously described by Helium et al. (2007). For confirmation experiments, a range

of concentrations around the rough IC50 of herbal extracts was used in incubations.

Working solutions were prepared such that the concentrations were 10-fold higher than

that required in incubations. Herbal extracts were reconstituted with the solvents

originally used for extraction and standardization by the vendor (Table 4-1). Insoluble









contents were removed by centrifugation at 20,000 x g for 5 minutes and separation of

the liquid supernatant. Solutions were serially diluted to prepare the working herbal

extracts, which contained 10% of the extracting organic solvents. This way, the organic

solvent concentration was the same in all incubations and limited to 1%. EGCG

working solutions were freshly prepared in 10% methanol and 1.5 mM ascorbic acid,

which was added to ensure EGCG stability during the experiment (Lu et al., 2003). All

solutions were freshly prepared at the time of the assays. Acid-hydrolyzed ginseng

extract was prepared by dissolving 60 mg of the powder extract in 1 mL of 60%

ethanol/40% 0.5 N HCI (Sloley et al., 2006). After 90 minutes at 370, the extract was

neutralized with 0.1 N KOH and then serially diluted to prepare working solutions

containing 10% ethanol.

In Vitro Incubations

A total of nine herbal extracts were screened for inhibition of estradiol-3-O-

glucuronidation. For each experiment, positive and negative control incubations were

performed. Niflumic acid (250 pM) was used as an inhibitor in positive control

incubations. Concentration of organic solvents and excipients were the same in each

set of herbal extract incubations and controls in order to nullify their effect on any

observed inhibition. Screening experiments using three concentrations of each herbal

extract were conducted to calculate rough IC50 values. If the rough IC50 estimate was

predicted to fall in a concentration range achievable in vivo in either intestinal or

systemic fluid volume, confirmation experiments using more inhibitor concentrations

were performed to obtain a more precise IC50 estimate. In addition to IC50 values,

inhibitory potency was also expressed as the volume per dose index (V/D), which is

defined as the volume in which the typical daily dose would be dissolved to obtain the









corresponding ICso concentration (Strandell et al., 2004). Comparison of this parameter

to physiological volumes facilitates an assessment of inhibitory potential.

Microsomal incubations were performed as described previously (Alkharfy and Frye,

2002). Briefly, a typical 250 pL incubation mixture contained HLM (protein

concentration, 0.5 mg/mL), alamethicin (30 pg/mg microsomal protein), MgCI2 (1 mM),

3-estradiol (25 pM), and different concentrations of each test extract or test compound

in 100 mM phosphate buffer, pH 7.4. Microsomes were pre-incubated with alamethicin

on ice for 5 minutes to activate UGT enzymes. The reaction was started by adding

UDPGA (6 mM) and placing incubation tubes in a water bath at 370C for 30 minutes.

The reaction was stopped by adding 25 pL of 6% perchloric acid. Tubes were vortex-

mixed for two minutes and centrifuged for 10 min at 20,000 x g. 75 pL of the

supernatant was injected into the HPLC system.

HPLC Analysis

E-3-G formation was measured using an HPLC system consisting of a Shimadzu LC-

10AD VP pump (Shimadzu Scientific Instruments, Columbia, MD, USA) connected to a

Waters 717 autosampler and Waters 2475 florescence detector (Waters Corporation,

Milford, MA, USA). The HPLC method used has been described previously (Alkharfy

and Frye, 2002). Briefly, the mobile phase consisted of 35% acetonitrile and 65% 50

mM ammonium phosphate buffer (pH 3); the flow rate was 1 mL/min delivered through

an Alltima phenyl column, 5 p, 4.6x250 mm (Grace Davison, Deerfield, IL, USA). E-3-G

was detected at an excitation wavelength of 210 nm and an emission wavelength of 300

nm. The assay was linear over the concentration range of 20 to 4000 pmol; the intra-

and inter-day coefficients of variation were <6%.









Data Analysis

Remaining enzyme activity was expressed as a percent of control and estimated

from the ratio of E-3-G peak area in herbal extract incubations relative to that in

negative control incubations. Where possible, ICso values were determined by fitting the

remaining enzyme activity and inhibitor concentration data to equation 4-1 using Prism

5.02 (GraphPad Software, San Diego, CA, USA).

100 x [I]H
Y =100 [] (4-1)
IcH + []H

(Y: Remaining UGT1A1 activity (percent of control), [I]: Concentration of herbal

extract, H: Hill coefficient)

In addition to IC50, a volume per dose index was calculated as described in equation

4-2 and was used to determine the potential for in vivo inhibition. The volume per dose

index is defined as the volume in which one dose would be dissolved to obtain the

corresponding IC50 concentration as described by Strandell et al. (2004).

RDI
Inhibition index (L) = (4-2)
IC50

(RDI: recommended daily intake)

A volume per dose index cutoff value of 2.0 L/dose was used to select extracts for more

detailed characterization of the IC50 value.

Results

Screening Experiments:

Eight herbal extracts, ginseng, acid-hydrolyzed ginseng, echinacea, black cohosh,

milk thistle, valerian, saw palmetto, and EGCG inhibited 3-0-glucuronidation of estradiol

by human liver microsomes in a concentration-dependent manner (Figure 1). EGCG









completely inhibited E-3-G formation at a concentration of 500 pg/mL. Echinacea, milk

thistle, saw palmetto, and EGCG were selected for confirmatory experiments to

determine more precise ICso values because the volume/dose index exceeded 2.0

L/dose (Table 4-2).

Confirmatory Experiments and Determination of Precise ICo0 Values:

EGCG exhibited the most potent inhibition of all extracts tested with best-fit ICso

value of 7.8 0.9 pg/mL. ICso values for inhibition of UGT1A1 activity by echinacea,

milk thistle, and saw palmetto were 211.7 43.5, 30.4 6.9, and 55.2 9.2 pg/mL,

respectively (Table 4-3, Figure 4-2). Goodness of fit r2 values were > 0.9 for all ICso

curves. When recommended daily dose of each extract was taken in perspective,

volume per dose index values were 1.9, 19.7, and 5.8 L for echinacea, milk thistle, and

saw palmetto, respectively. For EGCG, the volume per dose index was 32.1 L, which is

the highest value of all extracts tested indicating the highest potential for inhibition.

Discussion

The extracts screened in this study are among the most commonly used herbal

extracts in the US and the world. In this study, we investigated their potential for

interactions with glucuronidation by UGT1A1. All screened extracts, except garlic,

exhibited concentration-dependent inhibition towards UGT1A1 activity. Only four herbal

extracts (echinacea, milk thistle, saw palmetto, and green tea polyphenol EGCG)

exhibited inhibition with ICso values achievable in vivo, particularly in the intestine. ICso

as well as volume per dose index values indicate that echinacea and saw palmetto are

expected to be mild to moderate inhibitors compared to EGCG and milk thistle. The

latter two are the focus of this discussion.









Green tea and milk thistle are among the most commonly used herbal supplements

with 2006 sales in the US of $144 million for green tea and $93 million for milk thistle

(NBJ, 2007). Studies have shown that green tea has promising anticancer, antioxidant,

weight loss, and vascular protective benefits (Nagle et al., 2006). Green tea extract is

rich in the polyphenolic compounds named catechins. Among these, EGCG is

considered the most abundant and is suggested to be the main mediator of most of the

biological effects attributed to green tea (Moore et al., 2009). We used the green tea

catechin EGCG instead of green tea extract to avoid precipitation of microsomal

proteins by tannins present in green tea, which could yield misleading results as

described previously (Butterweck and Derendorf, 2008).

In order to interpret the potential of the observed in vitro inhibition to translate in vivo,

ICso values should be considered in the context of expected in vivo concentrations. In

pharmacokinetic studies of green tea extracts, EGCG maximum plasma concentrations

were as high as 2.5 pg/mL following ingestion of a single dose of green tea extract

(Polyphenon E) containing 800 mg EGCG (Foster et al., 2007). This concentration is

less than the observed ICso for UGT1A1 inhibition by EGCG (7.8 pg/mL); therefore,

inhibition of systemic clearance of UGT1A1 substrates seems unlikely. Conversely,

intestinal concentrations of EGCG can reach higher levels than the ICso value. Based

on an intestinal fluid volume of 0.5 to 5.0 L (Helium et al., 2007), EGCG concentrations

are expected to fall in the range of 40 to 1600 pg/mL following consumption of a green

tea extract dose containing 200 to 800 mg EGCG. Considering that the ICso for

inhibition by EGCG was 7.8 pg/mL (Table 4-3), inhibition of intestinal metabolism









appears plausible if green tea is consumed concomitantly with a UGT1A1 drug

substrate.

Zhu et al. (Zhu et al., 1998) previously reported that green tea polyphenols, including

EGCG, inhibited estradiol and estrone glucuronidation in vitro using rat liver

microsomes, which is consistent with the results of the current study using human liver

microsomes. In addition to reproducing their findings in HLM, we measured E-3-G

formation as a selective probe for UGT1A1 activity as previously reported (Court, 2005).

Milk thistle extract is used by 30 to 40% of liver disease patients for its

hepatoprotective benefits (Schrieber et al., 2008). Plasma concentration of total

flavonolignans, the major constituents in milk thistle, was 24 ng/mL following ingestion

of 600 mg milk thistle extract (Schrieber et al., 2008). Again, this concentration is much

less than the observed ICso for UGT1A1 inhibition by milk thistle (30.4 pg/mL; equivalent

to 11.5 pg/mL flavonolignans; Table 4-3). In agreement with this conclusion, van Erp et

al. (2005) reported no effect of milk thistle on the pharmacokinetics of irinotecan, an

intravenous anticancer drug and substrate for UGT1A1. For comparison, the expected

intestinal concentration of milk thistle extract is 40 to 1200 pg/mL following ingestion of

200 to 600 mg milk thistle supplement. Similar to EGCG, these concentrations are

higher than the ICso for UGT1A1 inhibition by milk thistle. Therefore, inhibition of

intestinal glucuronidation of UGT1A1 substrates by milk thistle may be possible.

In this study, we opted to use HLM rather than expressed UGT1A1 or human

intestine microsomes (HIM). Compared with HLM, expressed enzymes do not mimic

the in vivo environment in terms of availability of other UGT enzymes, which may form

heterodimers in vivo. Formation of UGT heterodimers has been described in the









literature and may affect enzyme activity (Ouzzine et al., 2003). HIM were not used

because estradiol-3-O-glucuronide formation is not selectively catalyzed by UGT1A1 in

HIM as other enzymes present in the intestine (e.g. UGT1A8 and UGT1A10) also form

this conjugate (Lepine et al., 2004). To our knowledge, no substrate selective for

UGT1A1 in HIM has been identified. Thus, utilization of HLM with estradiol-3-0-

glucuronidation as an index reaction provides better specificity for UGT1A1.

Nevertheless, V/D values for inhibition by EGCG and milk thistle suggest that UGT1A1

inhibition in the intestine may be more clinically relevant.

Intestinal UGT1A1 plays an important role in the first pass glucuronidation of drugs

such as raloxifene and ezetimibe (Fisher and Labissiere, 2007). For raloxifene,

bioavailability is only 2% and most of the oral dose is cleared by intestinal

glucuronidation, mainly by UGT1A1, 1A8, and 1A10 (Kemp et al., 2002). Inhibition of

one or more of these enzymes may enhance raloxifene bioavailability resulting in

increased exposure and increased risk for side effects such as deep vein thrombosis

and pulmonary embolism (Cummings et al., 1999). Similarly, intestinal glucuronidation

of ezetimibe plays an important role in mediating the pharmacological action of the drug,

since ezetimibe glucuronide is more potent than the parent drug in inhibiting cholesterol

absorption (Ghosal et al., 2004). Thus, inhibition of intestinal glucuronidation may

increase systemic exposure to ezetimibe and could affect the therapeutic response.

The Hill coefficients for the best-fit curve for milk thistle and echinacea were smaller

than unity (0.5 for milk thistle and 0.6 for echinacea; Table 4-3; Figure 4-2). This finding

may be explained by the atypical nature of UGT1A1 kinetics observed for estradiol,

possibly because more than one ligand molecule binds to the enzyme at the same time









(Alkharfy and Frye, 2002). Another explanation can be poor aqueous solubility of one

or more milk thistle or echinacea components that restricts inhibitor accessibility at

higher concentrations (Copeland, 2005).

Based on our findings, supplements of green tea, milk thistle, and to a lesser extent,

echinacea and saw palmetto, may inhibit glucuronidation of substrates by UGT1A1,

particularly in the intestine. These findings suggest interactions of these supplements

with UGT1A1 substrates are possible. Future clinical studies are warranted to evaluate

the in vivo pharmacokinetic relevance of these interactions.









Table 4-1. List of Herbal extracts investigated for effect on UGT1A1.
Extract Scientific Percent of Key Solvent**
Name of Origin Components
(w/w)*
Black Cohosh Cimicifuga 2 5 % Total 50% ethanol
racemosa Triterpenglycosides
Echinacea root Echinacea 2 3% Cichoric acid 60% ethanol
purpurea
Garlic bulb Allium sativum 2 3.25 % Allin 80% methanol

Ginseng root Panax ginseng 2 5% Total 60% ethanol
Ginsenosides

Milk Thistle herb Silybum 37.9% Total 80% acetone
marianum Silymarin
flavonolignans
Saw Palmetto fruit Serenoa >85% Total fatty 96%
repens acids ethanol
> 0.1 Sterols
Valerian root Valeriana 2 0.1 Valerenic 70%
officinalis acids ethanol
Epigallocatechin gallate Camellia > 97% EGCG 100%
(EGCG) sinensis methanol

*Values provided by manufacturer.
**Used by manufacturer for standardization









Table 4-2. Rough ICso and volume/dose index values for inhibition of estradiol-3-0-
glucuronidation by nine herbal extracts. Estradiol was incubated with pooled
HLM and three concentrations of each extract. Rough ICso values were
calculated by fitting ICso equation (Materials and methods) to percent of
activity remaining using non-linear regression. Values reported are best-fit
ICso standard error. All reported values had r2 values for goodness of fit of
at least 0.9. Volume/dose index values were calculated as described under
(Materials and methods). Recommended daily intake (RDI) values were
determined based on PDR for Herbal Medicine (Gale Group., 2001) and
commercially available products.


Extract RDI (mg) Rough ICso Volume/Dose
(pg/mL) Index
(L/dose)
Ginseng 550 602.5 225.6 0.9
Acid-hydrolyzed Ginseng 550 NA NA
Echinacea 400 166.6 68.3 2.4*
Black Cohosh 40 298.5 18.5 0.1
Milk Thistle 600 18.0 6.8 33.3*
Garlic 1000** **
Valerian 1000 561.9 59.0 1.8
Saw Palmetto 320 51.71 8.8 6.2*
Epigallocatechin gallate 250 7.6 0.7 32.9*
(EGCG)


NA: Data points did not fit IC50 curve
* indicates volume per dose values exceeding 2.0 L
** indicates no inhibition observed









Table 4-3. Precise ICso values for herbal extracts showing strongest inhibition of
estradiol-3-O-glucuronidation. Estradiol was incubated with pooled HLM and
a range of concentrations of each extract as described under (Materials and
methods). Data are reported as best-fit ICso values standard error.
Goodness of fit r2 values for the nonlinear regression model were > 0.9. H is
Hill coefficient describing the degree of sigmoidicity of the best-fit curve.
Recommended daily intake values (RDI) were determined based on PDR for
Herbal Medicine (Gale Group., 2001) and commercially available products.
Extract RDI (mg) IC50 (pg/mL) Volume/Do H
se Index
(L/dose)
Echinacea 400 211.7 43.5 1.9 0.6
Milk Thistle 600 30.4 6.9 19.7 0.5
Saw Palmetto 320 55.2 9.2 5.8 1.2
Epigallocatechin gallate 250 7.8 0.9 32.1 0.8
(EGCG)









M Dose in 0.53 L


S10-

0
-lfo I. 1 Iii .. .. I- f





^4 <0 ^ ^ J *k 0 1
20- VA


40-






Figure 4-1. Effect of herbal extracts on E-3-G formation as an index for UGT1A1
activity in HLM. Estradiol was incubated with pooled HLM and three
concentrations of each extract. Three concentrations were tested for each
herbal extract which represent extract daily intake in 53 L (striped bars), 5.3 L
(dotted bares), and 0.53 L (checkered bars). The dotted line represents 50%
activity of control. ND indicates no metabolite detected, which was
considered as 100% inhibition. Error bars represent SE of the mean of
duplicate incubations.













Saw Palmetto


0.1 1 10 100 1000
Concentration gg/mL


B Milk Thistle


0.1 1 10 100 1000 10000
Concentration jg/mL


0.01 0.1 1 10 100 1000
Concentration pg/mL


Echinacea


1 10 100 1000 10000
Concentration pg/mL


Figure 4-2. Inhibition of E-3-G formation by herbal extracts. Estradiol was incubated
with HLM and different concentrations of a) green tea catechin ECGC, b) milk
thistle extract, c) saw palmetto extract, and d) Echinacea purpurea root
extract. Data points represent remaining UGT1A1 activity as percent of
control incubations. Data points were fitted to non-linear regression equation
as explained under (Materials and methods). Error bars represent SE of the
mean of duplicate incubations.


EGCG









CHAPTER 5
INHIBITORY EFFECTS OF EPIGALLOCATECHIN GALLTE ON RALOXIFENE IN
VITRO CLEARANCE

Introduction

Raloxifene is a selective estrogen receptor modulator that is commonly used in

postmenopausal women to prevent and treat osteoporosis and to reduce the risk of

invasive breast cancer-a leading cause of death of women in the US (Moen and

Keating, 2008). The pharmacokinetics of raloxifene exhibits high inter- and intra-

individual variability with co-efficients of variation of 30-50% for most pharmacokinetic

parameters (Raloxifene package insert: http://pi.lilly.com/us/evista-pi.pdf). Although

60% of a raloxifene dose is absorbed, only 2% of the oral dose reaches the systemic

circulation (Moen and Keating, 2008). This poor bioavailability is attributed to extensive

first-pass glucuronidation by intestinal, and to a lesser extent, hepatic UGT enzymes

(Dalvie et al., 2008; Cubitt et al., 2009). Therefore, inhibition of raloxifene

glucuronidation in the intestine by concomitantly taken drugs or herbal supplements

may increase bioavailability several-fold, which in turn may increase risk for the rare but

serious thromboembolic events associated with raloxifene use (Cummings et al., 1999).

Green tea (Camellia sinensis) is one of the most commonly used beverages and

herbal supplements in the world, with US sales of $144 million dollars in 2006 (NBJ,

2007). In addition, consumption of green tea has been reported to possess beneficial

health effects, including improving cardiovascular function and anticancer effects

(Kohlmeier et al., 1997). Green tea extract is rich in polyphenolic compounds called

catechins. The primary green tea catechin is epigallocatechin gallate (EGCG), which

like raloxifene is a substrate for intestinal UGT enzymes (Lu et al., 2003). Since green









tea is being studied for its protective effects against cancer, there is a potential that it

might be used concomitantly in patients taking raloxifene.

The effect of green tea on intestinal glucuronidation of raloxifene has not been

studied. We previously showed that EGCG potently inhibits UGT1A1, which is involved

in the glucuronidation of raloxifene. The aim of this study was to investigate the effect

of EGCG, the most abundant constituent in green tea, on raloxifene intrinsic clearance

using a substrate depletion approach. In vitro intrinsic clearance calculated using this

method has been shown to be in good correlation with in vivo intrinsic clearance (Cubitt

et al., 2009).

Materials and Methods

Chemicals and Reagents

Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCI), uridine diphosphate

glucuronic acid (UDPGA), magnesium chloride, alamethicin, and epigallocatechin

gallate (EGCG; 2 97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Acetonitrile and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA).

Raloxifene and raloxifene-d4 were purchased from Toronto Research Chemicals (North

York, ON, Canada). Human intestine microsomes (HIM), pooled from five donors, were

purchased from BD Biosciences Discovery Labware (Woburn, MA, USA).

Incubations with HIM

Raloxifene and raloxifene-d4 stock solutions were prepared in methanol. Due to its

light sensitivity, raloxifene-d4 solution was kept in dark at all times. EGCG working

solution was freshly prepared at the time of the experiment and contained 10%

methanol and 1.5 mM ascorbic acid to increase its stability in aqueous medium (Lu et

al., 2003). Raloxifene depletion assay was performed as described previously with









modifications (Cubitt et al., 2009). Incubation mixture contained raloxifene (1 pM),

EGCG at concentrations of 0, 10, 50, and 100 pM, 0.15 pM ascorbic acid, 5 mM MgCI2,

0.1 M potassium phosphate buffer (pH 7.4), HIM (0.1 mg/mL), alamethicin (50 pg/mg

protein). The mixture was incubated on ice for 15 minutes. The reaction was started by

adding UDPGA (final concentration: 5 mM) to the mixture and 100-pL sample was

immediately removed for the zero-time value. 100 pL samples were taken from the

mixture at 0, 1, 5, 10, 15, 20, and 30 minutes and mixed with 100 pL acetonitrile and 20

pL internal standard (raloxifene d4), vortex-mixed, and placed on ice in the dark. Tubes

were centrifuged at 20,000 x g for 10 minutes. The supernatant was transferred to

autosampler vials for injection. Concentration of methanol in all incubations was 1%.

HPLC-MS/MS Assay of Raloxifene

Raloxifene was assayed by LC/MS/MS on a ThermoFinnigan Surveyor series HPLC

system connected to a TSQ Quantum triple quadrupole mass spectrometer (Thermo

Corp., San Jose, CA, USA) using electrospray ionization (ESI). Briefly, 5 pL of each

sample was injected on a reversed-phase Phenomenex (Torrance, CA, USA) Synergi

Max-RP column (75 x 2 mm, 4 pm). The mobile phase consisted of (A) 1% formic acid

in deionized water and (B) 1% formic acid in acetonitrile. Gradient elution at a flow-rate

of 0.2 mL/min was employed with the following steps: at start of the run, 20% B for one

min, then increased to 80% B in 0.5 min, held at 80% B between 1.5 and 4.0 min, and

from 4.5 to 6.0 min, the column was re-equilibrated at 20% B. Analysis was carried out

in the single reaction monitoring (SRM), positive ion mode using the mass transitions of

m/z 474 112 and m/z 478 112 for raloxifene and raloxifene-d4, respectively.

Retention time for both raloxifene and internal standard was 3.4 minutes.









Estimation of Non-specific Protein Binding

The free fraction of raloxifene in the incubation (fuine) was estimated using the

Hallifax-Houston equation 5-1 (Hallifax and Houston, 2006), where C is protein

concentration in milligrams per milliliter (0.1 mg/mL). The raloxifene LogP value used

was 6.2, which was calculated using Molinspiration-lnteractive logP calculator

(http://www.molinspiration.com/services/logp.html) as explained by Zhou et al.(2010).


fu = -- 1 (5-1)
f 1 + C 100 072*(logo1P)2+0 067.logo P-1 126


Data Analysis

The fraction of raloxifene remaining in the incubation was calculated from the ratio of

raloxifene to internal standard peak areas at different time points compared to the ratio

at time zero. Elimination half life was determined by fitting the data from the mean of

two incubations to a nonlinear exponential one-phase decay using Prism 5.02

(GraphPad Software, San Diego, CA, USA). In vitro CLint, u (pL/min/mg) was calculated

using equation 5-2 (Cubitt et al., 2009).

0.693 incubation volume (p/L)
CLin, = (5-2)
in vitro t1/2 amount of microsomalprotein in incubation (mg) fuin*

Results and Discussion

Calculated fu of raloxifene was 0.08 and CLint, u was 3680 pL/min/mg protein in the

absence of EGCG; both values agree with previously reported values (Cubitt et al.,

2009). Figure 1 shows the effect of adding different concentrations of EGCG on

raloxifene CLint, u. In HIM incubations, EGCG inhibited raloxifene in vitro CLint, u by 76%,

86%, and 100% at concentrations of 10, 50, and 100 pM, respectively (Table 5-1,

Figure 5-1). To compare these concentrations to putative in vivo intestinal









concentrations, EGCG content in green tea supplements and an estimate of intestinal

fluid volume of 0.5 to 5.0 L were considered. Most green tea supplements contain

about 250 mg of EGCG. Therefore, based on estimates of intestinal fluid volume that

range from 0.5 and 5 L (Helium et al., 2007), intestinal EGCG concentrations would be

expected to be 100 to 1000 pM. Moreover, doses of EGCG reaching up to 800 mg

have been used in clinical studies, which will yield putative EGCG intestinal

concentrations of 320 to 3200 pM. These concentrations are much higher than the

concentrations used in the incubations. Therefore, based on the observed inhibition it is

likely that green tea supplements will inhibit raloxifene intestinal glucuronidation-the

primary factor limiting raloxifene bioavailability. The pharmacokinetic consequences of

this interaction warrant further studies.









Table 5-1. Effect of green tea EGCG on raloxifene in vitro intrinsic clearance using
HIM. CLint,u was determined using equation 5-2 as explained under Materials
and Methods.
EGCG Concentration CLint,u (pL/min/mg Percent of control
(pM) protein)
Control 3680 100
10 pM 899.4 24.4
50 pM 557.5 15.7
100 pM 0.0 0.0


!-. ... ..
= -e" -v "- : u...- ..----
~~~Iriir~ir


--

-+-


Control
EGCG 10 cM
EGCG 50 .pM
EGCG 100 M


Time (min)

Figure 5-1. Effect of green tea EGCG on raloxifene in vitro intrinsic clearance using
HIM. Raloxifene depletion assay was performed by incubating 1 pM
raloxifene with 0.1 mg/mL alamethicin-activated HIM and 0, 10, 50, or 100 pM
EGCG. 100 pL samples were taken at various time points to estimate the
remaining raloxifene fraction compared to time zero sample. Data points are
mean values of duplicate incubations standard error. The lines present the
best-fit curves for the exponential one phase decay model by Prism 5.02
(GraphPad Software, San Diego, CA, USA).


0
c

"E

C



.1

LL
c

.



. -
U-


p -









CHAPTER 6
INHIBITORY EFFECTS OF COMMONLY USED HERBAL EXTRACTS ON
UGT1A4, 1A6, AND 1A9 ENZYME ACTIVITIES

Introduction

Conjugation of compounds with glucuronic acid represents a major disposition

pathway for endogenous and exogenous compounds, including drugs and

phytochemicals. Human glucuronidation enzymes (UDP-glucuronosyltransferases;

UGT) are divided into two families, UGT1 and UGT2, which encompass more than 18

enzymes (Tukey and Strassburg, 2000). UGT1A4, UGT1A6, and UGT1A9 enzymes

belong to the UGT1 family and conjugate a wide spectrum of drugs and

phytochemicals. UGT enzymes are differentially expressed in tissues, with liver and

intestine being the main sites for drug glucuronidation (Tukey and Strassburg, 2000).

Substrates for UGT1 enzymes include many drugs (e.g. mycophenolic acid,

trifluoperazine, tamoxifen, lamotrigine, and acetaminophen) and phytochemicals (e.g.

quercetin, kaempferol, epigallocatechin gallate) (Oliveira and Watson, 2000; Lu et al.,

2003; Kiang et al., 2005). Since these phytochemicals share UGT1 metabolic

pathway(s) with drug substrates, there is a potential for herb-drug interaction through

modulation of this pathway. We previously reported that Ginkgo biloba extract and its

polyphenolic compounds quercetin and kaempferol inhibit UGT1A9 (Chapter 3). The

aim of this study was to identify other potential herb-UGT interactions through screening

commonly used herbal extracts for inhibitory effects on the activities of UGT1A4,

UGT1A6, and UGT1A9.

Recent surveys estimate that 38% of Americans use complementary and alternative

medicine, which includes herbal supplements (Barnes et al., 2008). However, the

physiologic and metabolic effects of herbals and phytochemicals are often poorly









understood. One of the issues of concern to clinicians is the potential for herb-drug

interactions, which may lead to poor clinical outcomes (Gardiner et al., 2008). Several

case studies have described deleterious herb-drug interactions that can lead to

morbidity or even mortality (Ruschitzka et al., 2000; Kupiec and Raj, 2005).

Consequently, much attention has been given to investigating the effects of herbal

supplements on cytochrome P-450 enzymes, the primary metabolic route for the

majority of marketed drugs (Izzo and Ernst, 2009). In contrast, research is lacking

regarding the potential of herbals to alter other metabolic routes including

glucuronidation.

Identification of selective substrates for UGT enzymes allows screening of herb-UGT

interactions using human liver microsomes. Trifluoperazine, serotonin, and

mycophenolic acid were reported to be selective in vitro probe substrates for UGT1A4,

UGT1A6, and UGT1A9, respectively (Court, 2005). In this study, formation of

trifluoperazine glucuronide, serotonin glucuronide, and mycophenolic acid phenolic

glucuronide were used as index reactions for UGT1A4, 1A6, and 1A9 enzymatic

activities, respectively.

Materials and Methods

Chemicals and Reagents

Trifluoperazine (TFP; 2 99%), serotonin (2 98%), potassium phosphate dibasic,

tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCI), uridine diphosphate

glucuronic acid (UDPGA), 3-glucuronidase, magnesium chloride, bovine serum albumin

(BSA), alamethicin, niflumic acid, and epigallocatechin gallate (EGCG; 2 97%) were

purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, ethanol, methanol,

acetone, hecogenin acetate (93%), and 1-naphthol (> 99%) were purchased from Fisher









Scientific (Pittsburgh, PA, USA). Serotonin-O-3-D-glucuronide was provided by RTI

International (Research Triangle Park, NC) through the NIMH Chemical Synthesis

Program. Mycophenolic acid (MPA; 98%), mycophenolic acid 3-D-glucuronide (MPAG;

98%), and mycophenolic Acid-d3-3-D-glucuronide (MPA-d3-G; 98%) were purchased

from Toronto Research Chemicals (North York, ON, Canada). Herbal extracts (black

cohosh, Cimicifuga racemosa; cranberry, Vaccinium marocarpon, echinacea,

Echinacea purpurea; garlic, Allium sativum; ginkgo, Ginkgo biloba; ginseng, Panax

ginseng; milk thistle, Silybum marianum; saw palmetto, Serenoa repens; and valerian,

Valeriana officinalis) were generously provided by Finzelberg & Co. KG (Andernach,

Germany) as dry powder. Table 6-1 summarizes the properties of the extracts

screened. UltraPool human liver microsomes (HLM) were purchased from BD

Biosciences Discovery Labware (Woburn, MA, USA). These microsomes were pooled

from 150 donors providing lot-to-lot consistency.

Preparation of Herbal Working Solutions

Herbal extracts were reconstituted with the solvents originally used for extraction and

standardization by the vendor (Table 6-1). In order to remove any insoluble contents,

the mixture was centrifuged at 20,000 x g for 5 minutes and the liquid supernatant was

removed. Working solutions were freshly prepared so that final herbal concentrations in

screening incubations would represent the recommended daily intake of each extract in

53 L, 5.3 L, and 0.53 L. These volumes roughly represent total body fluids, and two

extremes of a range of concentrations that could appear in the small intestine, assuming

100% bioavailability as previously described by Helium et al (2007). For confirmation

experiments, a range of concentrations around the rough IC50 of herbal extracts was

used in incubations. Concentration of organic solvents in incubations was the same in









all incubations including controls and was limited to 1%. For EGCG, working solutions

were freshly prepared in 10% methanol and 1.5 mM ascorbic acid, which was added to

ensure EGCG stability during the experiment (Lu et al., 2003). Acid-hydrolyzed ginseng

extract was prepared by dissolving 60 mg of the powder extract in 1 mL of 60%

ethanol/40% 0.5 N HCI (Sloley et al., 2006). After 90 minutes at 370, the extract was

neutralized with 0.1 N KOH and was serially diluted to prepare working solutions

containing 10% ethanol. Acid-hydrolyzed ginkgo extract was prepared by dissolving 30

mg of the powder extract in 1 mL of 60% acetone/40% 5N HCI. The acid treated extract

was heated at 900C for one hour and neutralized with 2N KOH. Working solutions were

prepared so that their concentrations were 10-fold higher than the final concentrations in

incubations.

Incubations of Herbal Extracts with TFP

TFP was used as a probe substrate for UGT1A4 in HLM. Incubations with TPF were

performed as described previously by Uchaipichat and coworkers (2006). Briefly, the

incubation mixture (final volume, 250 pL) consisted of TFP, 5 mM MgCI2, 50 mM Tris-

HCI buffer (pH 7.4), 0.1 mg/mL microsomal proteins, and alamethicin (100 pg/mg

protein). Concentration of TFP in incubations was 60 pM, which corresponds to the Km

in HLM (Uchaipichat et al., 2006). The mixture was pre-incubated on ice for 15 minutes.

The reaction was started by adding UDPGA (final concentration, 5 mM). After the

mixture was incubated for 20 min at 370C, the reaction was stopped by adding 250 pL

(4% Acetic acid/96% Methanol), vortex-mixing, and placing tubes on ice. Tubes were

centrifuged for 10 min at 20,000 x g and the supernatant was transferred to HPLC

tubes. Screening experiments were performed by adding herbal extracts at three

different concentrations to the incubation mixture. Incubations with and without









hecogenin (50 pM) were performed to serve as positive and negative controls,

respectively.

Chromatographic Analysis of TFP glucuronide (TFPG)

HPLC analysis was performed with a Shimadzu LC-10AD VP pump (Shimadzu

Scientific Instruments, Columbia, MD, USA) connected to a Waters 717 autosampler

and Waters 2475 florescence detector (Waters Corporation, Milford, MA, USA). 50 pL

of the incubation supernatant was injected on a reversed-phase Phenomenex Luna

Phenyl-Hexyl column (2 x 100 mm, 3 pm). Isocratic chromatography was carried out at

ambient temperature using a mobile phase consisting of 0.1% tri-fluoroacetic acid in

acetonitrile: deionized water (30:70) at a flow-rate of 0.2 mL/min. The total run time was

15 min. TFPG was detected at an excitation wavelength of 310 nm and emission

wavelength of 475 nm (Rele et al., 2004).

The identity of the TFPG peak was verified through enzymatic hydrolysis using 3-

glucuronidase. 60 pM TFP was incubated with HLMs as described above for 1 hour at

370C. Then, 25 pL of 100 mM potassium phosphate buffer (pH 4.0), and 2,500 units of

3-glucuronidase were added. Tubes were incubated for 16 hours at 370 C. The

reaction was stopped by adding 10 pL 70% HCIO4, vortex-mixing and centrifugation at

20,000 x g for 10 minutes. The supernatant was transferred to HPLC tubes for

injection. Control incubations were performed in the same way but did not contain 3-

glucuronidase enzyme. The TFPG peak was detected in the control incubation but not

in the hydrolyzed one.

Incubations of Serotonin with Herbal Extracts

To investigate the effect of herbals on UGT1A6 activity, incubations of herbal extracts

with HLM were performed using serotonin as a probe substrate as described by









Krishnaswamy and coworkers with modifications (2003). Briefly, the incubation mixture

(final volume, 100 pL) consisted of serotonin at a concentration around Km value in HLM

(8 mM), 5 mM MgCI2, 50 mM Tris-HCI buffer (pH 7.4), 0.5 mg/mL microsomal proteins,

and alamethicin (100 pg/mg protein). The mixture was pre-incubated on ice for 15

minutes. The reaction was started by adding UDPGA (final concentration, 5 mM). After

the mixture was incubated for 60 min at 370C, the reaction was stopped by adding 10

pL 24% perchloric acid: acetonitrile (1:1, v/v), vortex-mixing, and placing tubes on ice.

Tubes were centrifuged for 10 min at 20,000 x g and the supernatant was transferred to

HPLC tubes. 1-naphthol (50 pM) was used as a positive control inhibitor in the

screening assays.

Chromatographic Analysis of Serotonin Glucuronide

Isocratic chromatography was carried out at ambient temperature on a reversed-

phase Waters C18 Symmetry column (3.9 x 150 mm, 5 pm). The mobile phase

consisted of 5% acetonitrile / 95% 2 mM ammonium acetate (pH 2.7). Isocratic elution

at flow-rate of 1.0 mL/min was employed. The total run time was 10 min and the

injection volume was 30 pL. The HPLC system consisted of a Shimadzu LC-10AD VP

pump (Shimadzu Scientific Instruments, Columbia, MD, USA) connected to a Waters

717 autosampler and Waters 2475 florescence detector (Waters Corporation, Milford,

MA, USA). Serotonin glucuronide was detected at an excitation wavelength of 225 nm

and emission wavelength of 330 nm. To confirm the identity of serotonin-glucuronide

peak, retention time was compared to serotonin glucuronide standard. In addition,

serotonin glucuronide peak was collected from the HPLC eluate and analyzed using

MS/MS. The isolated fraction showed abundant ions with m/z 353, which matches the

m/z of serotonin-glucuronide ions in the positive mode. Upon fragmentation of the









parent ion, a product ion with m/z 177 was produced, which matches the expected

breakdown of the conjugate into glucuronic acid and free serotonin.

Incubations of MPA with Herbal Extracts

Incubations with MPA were performed as described previously with modifications

(Chapter 2). Briefly, the incubation mixture (100 pL) contained HLM (protein

concentration, 0.16 mg/mL), alamethicin (100 pg/mg protein), MgCI2 (5 mM), 2% BSA,

and 100 mM phosphate buffer, pH 7.4. MPA was used at a concentration equivalent to

the Km value in HLM (240 pM). Microsomes were pre-incubated on ice with alamethicin

for 15 minutes. The reaction was started by adding UDPGA (1 mM) and placing

incubation tubes in a water bath at 370 C for 30 minutes. The reaction was stopped by

adding 300 pL of ice-cold acetonitrile and 20 pL of internal standard (20 pg/mL MPA-d3-

G). Tubes were vortex-mixed for two minutes and centrifuged for 10 min at 20,000 x g.

The supernatant was diluted 12-fold with purified water and 5 pL was injected into the

HPLC system. Incubations of MPA with niflumic acid (70 pM) were used as positive

controls.

MPAG LC-MS/MS Assay

MPAG was determined by LC/MS/MS on a ThermoFinnigan Surveyor series HPLC

system connected to a TSQ Quantum triple quadrupole mass spectrometer (Thermo

Corp., San Jose, CA, USA) using electrospray ionization (ESI), as described previously

(Chapter 2). Briefly, 5 pL of each sample was injected on a reversed-phase

Phenomenex (Torrance, CA, USA) Synergi Fusion-RP18 column (100 x 2 mm, 4 pm).

The mobile phase consisted of (A) 1 mM acetic acid in deionized water and (B) 1 mM

acetic acid in acetonitrile. Gradient elution at a flow-rate of 0.22 mL/min was employed

with the following steps: at start of the run, 30% B for one min, then increased to 90% B









in 0.75 min, held at 90% B between 1.75 and 3.1 min, and from 3.6 to 6.5 min, the

column was re-equilibrated at 30% B. Analysis was carried out in the single reaction

monitoring (SRM), negative ion mode using the mass transitions of m/z 495 319 and

m/z 498 322 for MPAG and MPA-d3-G, respectively.

Data Analysis

Remaining enzyme activity was calculated from the peak area of the glucuronide

metabolites formed in herbal extract incubations expressed as a percent of control.

Remaining enzyme activity and herbal extract concentration data were fitted to equation

4-1 using Prism 5.02 (GraphPad Software, San Diego, CA, USA) to estimate ICso

values.

Volume per dose (V/D) index was calculated using equation 6-1 and was used as a

measure of the potential of ICso concentrations to be reached in vivo as described by

Strandell et al. (2004). The V/D index is defined as the volume in which one dose

should be dissolved in order to obtain the corresponding ICso concentration.

RDI
Volume/Dos e index (L) = (6-1)
IC50

(RDI: recommended daily intake)

Results

A total of 35 herb-UGT enzyme pairs were evaluated, each at three different

concentrations. Results from the screening experiments are summarized in Table 6-2.

Rough ICso and V/D index values were estimated based on remaining enzyme activity

data at the three concentrations of each herbal extract. V/D index was used to select

the herb-UGT interactions to investigate further. A V/D cutoff value was considered to

be 5 L for UGT1A4 interactions and 2 L for UGT1A6 and 1A9 interactions. This was









based on an expression study that showed that UGT1A6 and 1A9 are expressed in the

intestine and the liver while UGT1A4 is mainly expressed in the liver (Ohno and Nakajin,

2009). Herbal extracts that showed inhibition of a UGT enzyme with V/D values

exceeding the specific cutoff value in the screening experiments were studied further in

confirmatory assays to estimate accurate ICso and V/D values. For all reported ICso

values, goodness of fit (r2) of the nonlinear regression curve was greater than 0.9.

Effect of herbal extracts on TFPG formation

Effect of 10 herbal extracts on UGT1A4 activity was achieved through incubations of

pooled HLM with TFP and monitoring formation of TFPG as an index of UGT1A4

activity. For milk thistle and acid-hydrolyzed ginkgo extracts, evaluation of their effects

of on UGT1A4 activity was not possible due to interference of the herbal extracts with

TFPG florescence. All the tested extracts inhibited TFPG formation with different

potencies (Figure 6-1). Herbal extracts showing rough ICso values less than 100 pg/mL

were (mean SE) EGCG (34.39 4.1 pg/mL), black cohosh (69.7 4.8 pg/mL), and

saw palmetto (70.6 9.3 pg/mL) (Table 6-2). Only EGCG inhibited UGT1A4 with V/D

value exceeding 5 L. This finding was confirmed by incubating TFP with increasing

concentrations of EGCG. Best-fit IC50 was (mean SE) 33.8 3.1 pg/mL and V/D

value was 7.4 L based on daily dose of 250 mg (Table 6-3, Figure 6-2).

Effect of herbal extracts on serotonin glucuronide formation

Milk thistle, saw palmetto, EGCG, and echinacea inhibited serotonin glucuronide

formation with ICso values of (mean SE) 66.9 3.5, 131.8 21.5, and 183.6 29.8

pg/mL, respectively (Table 6-2, Figure 6-1). A V/D cutoff value of 2 L was applied to

select which extracts to study further. Only saw palmetto and milk thistle exceeded the

V/D cutoff with values of 2.4 L and 9.0, respectively (Table 6-2).









Precise ICso and V/D index values were determined for inhibition of serotonin

glucuronide formation by milk thistle and saw palmetto (Figure 6-3). Best-fit ICso values

were 59.5 3.6 and 103.5 10.7 for milk thistle and saw palmetto, respectively. V/D

values were 6.3 and 3.1 L for milk thistle, and saw palmetto, respectively (Table 6-3).

Effect of herbal extracts on MPAG formation

Black cohosh, cranberry, echinacea, ginseng, acid-hydrolyzed ginseng, and milk

thistle inhibited MPAG formation (Figure 6-1). However, only milk thistle (rough ICso =

35.9 4.3 pg/mL, V/D = 16.7 L) and cranberry (rough IC50= 260.5 33.0 pg/mL, V/D =

3.8 L) exceeded the V/D cutoff of 2L and were selected for further study (Table6-2).

Precise best-fit ICso and V/D values for milk thistle and cranberry were 33.6 3.1 pg/mL

and 17.9 L, and 230.4 32.9 pg/mL and 3.1 L, respectively )Table 6-3, Figure 6-4).

Discussion

In this study, 12 commonly used herbal extracts were screened for their effects on

the glucuronidation activity of UGT1A4, 1A6, and 1A9 in pooled HLM. UGT enzyme

activities were measured in vitro using selective substrates-TFP for UGT1A4,

serotonin for UGT1A6, and MPA for UGT1A9 (Court, 2005). Based on V/D index

values, the most potent inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6

and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. These findings

highlight the possibility of herb-drug interactions through modulation of UGT enzyme

activity. The likelihood of the observed in vitro interactions to occur in vivo depends on

characteristics of the herb, the drug substrate, the specific enzyme, and the potency of

the inhibition.

UGT1A4 is known to be the primary enzyme that catalyzes N-glucuronidation of

primary, secondary and aromatic amines, which includes TFP, lamotrigine and the









estrogen receptor modulator drug tamoxifen (Kiang et al., 2005; Rowland et al., 2006;

Zhou et al., 2010). In addition, UGT1A4 shows 0-glucuronidation activity towards

steroidal compounds (Green and Tephly, 1996). Hecogenin is a known inhibitor of

UGT1A4-mediated TFP glucuronidation with ICso values of 1.5 pM (Uchaipichat et al.,

2006). Compared to hecogenin, EGCG is a non-selective UGT1A4 inhibitor with

moderate potency. EGCG has previously been shown to inhibit estradiol-3-0-

glucuronidation, an index for UGT1A1 activity, with a lower ICso value (7.8 pg/mL)

(Chapter 4). In addition, in this study EGCG showed some weak inhibitory activities

toward UGT1A6 and UGT1A9 (Figure 6-1). Pharmacokinetic studies show that

maximum plasma concentrations of EGCG are more than 10-fold less than the

observed ICso values following consumption of high dose (800 mg) EGCG (Foster et al.,

2007). This suggests that inhibition of UGT1A4-mediated systemic glucuronidation by

EGCG is unlikely. However, based on V/D index of the inhibition of 7.4 L for 250 mg

dose, effect of EGCG on hepatic first pass metabolism of UGT1A4 substrates is

possible and will be augmented with higher EGCG doses. EGCG has been studied at

doses that reach 800 mg daily for its antioxidant and anti-cancer effects (Chow et al.,

2005). Considering higher doses of EGCG (800 mg), the V/D index will be 23.6 L,

indicating that the 800 mg dose can be diluted in up to 23.6 L and still inhibit UGT1A4

activity by up to 50%. The effect of EGCG on glucuronidation of the UGT1A4

substrates TFP, lamotrigine, tamoxifen, and imipramine warrants further investigation.

UGT1A6 is typically a low affinity enzyme that catalyzes glucuronidation of drug

substrates including acetaminophen, valproic acid, and morphine (Kiang et al., 2005).

Milk thistle and saw palmetto inhibited serotonin glucuronidation with ICso









concentrations attainable if the daily doses of milk thistle (600 mg) or saw palmetto (320

mg) are diluted with 6.3 and 3.1 L, respectively. The observed milk thistle ICso for

UGT1A6 is equivalent to a total flavonolignans concentration of 22.6 pg/mL; this is

about 1000-fold higher than observed plasma concentration following intake of 600 mg

milk thistle extract (Schrieber et al., 2008). Taken together, milk thistle extract is more

likely to inhibit UGT1A6-mediated first pass rather than systemic metabolism. On the

other hand, no pharmacokinetic data are available on saw palmetto. Based on ICso

value exceeding 100 pg/mL and V/D index of 3.1 L, saw palmetto will be expected to

have mild, if any, inhibition of UGT1A6-mediated metabolism in vivo (Table 6-2).

UGT1A9 catalyzes glucuronidation of a wide range of substrates including MPA,

propofol, raloxifene, and flavopiridol (Kiang et al., 2005). In the current study, milk

thistle and cranberry inhibited MPAG formation, which was used as an index reaction

for UGT1A9 activity in HLM (Court, 2005). For milk thistle extract, the ICso value was

33.6 pg/mL, which is equivalent to 12.7 pg/mL flavonolignans. Again, this concentration

is much higher than the expected plasma concentration of flavonolignans following milk

thistle intake (Schrieber et al., 2008). Therefore, inhibition of systemic metabolism of

UGT1A9 substrates by milk thistle extract is not likely. Conversely, based on the range

of intestinal fluid volume of 0.5 to 5 liters, a single 600-mg dose of milk thistle may result

in putative concentrations of 120 to 1200 pg/mL. Accordingly, inhibition of first pass

metabolism of UGT1A9 substrates by milk thistle extract is possible.

In this study, we screened specific UGT enzyme activities using HLM rather than

human intestine microsomes (HIM) or expressed enzymes. The difference between

expressed enzymes and HLM is that the first contain single UGT enzymes while the









latter contain all the hepatic isoforms. Therefore, HLM are closer to the in vivo

environment due to the availability of other UGT enzymes that may form heterodimers,

which has been reported for some UGT enzymes and may affect enzyme activity

(Ouzzine et al., 2003). Since our goal was to screen for interactions that may have

clinical significance, the use of HLM was more appropriate. This was made feasible by

the availability of selective substrates for different UGT enzymes in HLM (Court, 2005).

Similarly, HIM contain all the intestinal UGT enzymes. However, no selective

substrates for individual UGT enzymes have been described in HIM.

Calculation of V/D index provides a helpful tool to predict the likelihood of achieving

ICso-equivalent concentrations in the intestine or plasma in the absence of clinical data

(Strandell et al., 2004). Although this approach is sufficient for the purpose of screening

and hypothesis generation, it is limited by not considering the extent of absorption of

phytochemicals through tissue and cellular barriers. Use of V/D index assumes that the

concentration in the gastrointestinal lumen is equivalent to that in the endoplasmic

reticulum of intestinal epithelial cells where UGT enzymes are located. This assumption

may lead to overestimation of the extent of the inhibition, since many phytochemicals

are poorly absorbed through the intestinal wall. Therefore, the results need to be

confirmed in clinical studies and, where available, ICso values to be compared with

concentration data obtained experimentally. It is worth noting that using V/D index to

describe inhibition potency changes the order of significance of inhibitors. For example,

based on rough ICso values, black cohosh and saw palmetto are equipotent inhibitors of

UGT1A4 activity (Rough ICso = 69.7 pg/mL and 70.6 pg/mL; Table 6-2). However, the

daily dose of saw palmetto is eight-fold higher than that of black cohosh (320 mg versus









40 mg). Thus, ingesting 320 mg of saw palmetto is expected to result in higher extent

of UGT1A4 inhibition compared to ingesting 60 mg of black cohosh.

In summary, in this study, 12 herbal extracts were screened for inhibition of three

UGT1A enzymes-UGT1A4, UGT1A6, and UGT1A9. We report inhibition of UGT1A4

by EGCG, UGT1A6 by milk thistle and saw palmetto, and UGT1A9 by cranberry and

milk thistle extracts. Among these, EGCG inhibition of UGT1A4 and milk thistle

inhibition of UGT1A6 and UGT1A9 are likely to affect first-pass glucuronidation of

substrates. The in vivo effects of these interactions on pharmacokinetics of UGT1A4,

UGT1A6, and UGT1A9 substrates remain to be determined in clinical studies.









Table 6-1. List of herbal extracts screened for UGT1A4, UGT1A6, and UGT1A9 inhibition.
Test Compound Scientific Name of Percent of Key Solvent**
Origin Components (w/w)*

Black cohosh rhizome extract Cimicifuga 2 5 % Total 50% ethanol
racemosa Triterpenglycosides
Cranberry press juice Vaccinium > 40% Total 96% Ethanol
marocarpon Proanthocyanidins
Echinacea root extract Echinacea purpurea 2 3% Cichoric acid 60% ethanol

Garlic bulb extract Allium sativum 2 3.25 % Allin 80% methanol
Ginkgo biloba leaf extract Ginkgo biloba 2 24% Ginkgo 60% acetone
flavonglycosides
2 6% Terpene lactones

Ginseng root extract Panax ginseng 2 5% Total Ginsenosides 60% ethanol

Milk Thistle herb extract Silybum marianum 37.9% Total silymarin 80% acetone
flavonolignans
Saw Palmetto fruit extract Serenoa repens >85% Total fatty acids 96% ethanol
> 0.1 Sterols

Valerian root extract Valeriana officinalis 2 0.1 Valerenic acids 70% ethanol

Epigallocatechin gallate Camellia sinensis > 97% EGCG 100% methanol
(EGCG)
*Values provided by manufacturer.
**Used by manufacturer for standardization


100









Table 6-2. Effect of commonly used herbal extracts on UGT1A4, UGT1A6, and UGT1A9 activity. Each herbal extract was
co-incubated at three concentrations with TFP (for UGT1A4), serotonin (for UGT1A6), and mycophenolic acid
(for UGT1A9) and HLM. Formation of TFPG, serotonin-glucuronide, and MPAG were used as index reactions
for activity of UGT1A4, UGT1A6, and UGT1A9 enzyme activities, respectively. Formation of glucuronides was
compared in incubations with herbal extract to negative control incubations. Data represent best-fit ICso
standard error. Goodness of fit r2 value was > 0.9 for all reported ICso value. Volume/Dose index was


calculated by


Extract


Black cohosh

Cranberry

Echinacea

Garlic
Ginkgo biloba

Acid-hydrolyzed
ginkgo biloba
Ginseng

Acid-hydrolyzed
ginseng
Milk thistle
Saw palmetto


dividing the daily intake of each herb by the rough ICso value.
UGT1A4 UGT1A6
RDI Rough ICso Volume/Dose Rough Volume/Dose
(mg) (pg/mL) Index ICso Index
(L/dose) (pg/mL) (L/dose)
40 69.7 4.8 0.6 NA NA


742.7
118.7
116.1 +
25.1
NA
268.2
48.9
Interf

368.4
66.6
288.0
42.8
Interf
70.6 9.3


> 1000

241.0
23.4
NA
NA

NA

NA

> 1000

66.9 3.5
131.8
21.5


<1.0

1.7

NA
NA

NA

NA

<0.6

9.0*
2.4*


UGT1A9
Rough ICso Volume/Dose
(pg/mL) Index
(L/dose)


321.6
102.2
260.5 33.0

858.3
158.7
NA
PB

PB

298.6 29.1

524.3 60.5

35.9 4.3
NA


0.1


3.8*

0.5

NA
PB

PB

1.8

1.0

16.7*
NA


1000

400

1000
240

240

550

550

600
320









Table 6-2. Continued
Valerian 1000 406.5 2.5 > 1000 < 1.0 NA NA
35.3
Epigallocatechin 250 34.39 4.1 7.3* 183.6 1.4 NA NA
gallate (EGCG) 29.8
NA, data points did not fit IC50 curve;
PB: IC5o values for inhibition of UGT1A9 by ginkgo and acid-hydrolyzed ginkgo extracts have been previously reported (Chapter 3). Ginkgo and
acid-hydrolyzed ginkgo extracts inhibited MPAG formation in HLM with IC50 values of 84.3 11.6 and 20.9 3.6 pg/mL, respectively. Considering
dose of 240 mg, this would result in V/D index of 2.9 and 11.4 L/Dose for unhydrolyzed and acid-hydrolyzed ginkgo extracts, respectively.
Interf: Addition of herb interfered with florescence detection of glucuronide
* indicates volume/dose index values that exceed the cutoff for further investigation


102









Table 6-3. Determination of inhibitory potency of selected UGT1A4, UGT1A6, and
UGT1A9 herbal inhibitors. Extracts were selected if their V/D index based on
rough IC50 values exceeded 4 L for UGT1A4, or 2 L for UGT1A6 and
UGT1A9. Several concentrations of each extract were co-incubated with
alamethicin-activated HLM and TFP (for UGT1A4), serotonin (for UGT1A6),
or MPA (for UGT1A9). Percent of remaining activity was measured as the
formation of each glucuronide in herbal incubation as a percent of negative
control. ICso values were calculated by fitting data points to ICso equation Hill
equation as described under Materials and Methods. Values reported are
best-fit ICso values standard error. Goodness of fit r2 value was 2 0.95 for
all reported ICso value. Volume/Dose index was calculated by dividing the
daily intake of each herb by the ICso value.
UGT Extract RDI ICso (pg/mL) Volume/Dose H
Enzyme (mg) Index
(L/dose)

UGT1A4 EGCG 250 33.8 3.1 7.4 1.0
UGT1A6 Milk thistle 600 59.5 3.6 6.3 1.1
Saw palmetto 320 103.5 10.7 3.1 1.4
UGT1A9 Cranberry 1000 230.4 32.9 3.1 1.0
Milk thistle 600 33.6 3.1 17.9 0.8


103









Figure 6-1. Effect of commonly used herbal extracts on UGT1A4, UGT1A6, and
UGT1A9 enzyme activities. HLM were co-incubated with herbal extracts and
A) TFP for UGT1A4 activity, B) serotonin for UGT1A6 activity, and C)
mycophenolic acid for UGT1A9 activity. Three concentrations were tested for
each herbal extract which represent extract daily intake in 53 L (small-dotted
bars), 5.3 L (checkered bars), and 0.53 L (striped bars). Formation of TFPG,
serotonin glucuronide, and MPAG were detected in respective herbal
incubations. Percent of activity was calculated as the percent of glucuronide
peak area in herbal incubations as compared to negative controls. Each
value represents mean of duplicate incubations. Error bars represent positive
standard error.


104










M Dose in 53 L
0 Dose in 5,3 L
120- E Dose in 0.53 L
S100
S80



a. 20-
.


1/
Cf 4


0


lie


4


IM Dose in 53 L
[ Dose in 5.3 L
M Dose in 0.53


100-
o 80
60-
40-

0-






C


S120-
C 100-

80.
S40"
4.


I"O
ol


i


1 Dose in 53 L
E Dose in 5.3 L
M Dose in 0.53 L


0V


ta-


0


$ $ J f

^y ot


I


0'
Ct
w










140-

120-
0


L 80-

60-
0 40


0-
a- 20

0 1W I Wiij v | wI W WWI N WW Ww
0.1 1 10 100 1000 10000
EGCG Concentration (pg/mL)

Figure 6-2. Inhibitory effect of green tea catechin EGCG on TFPG formation in HLM.
Increasing concentrations of EGCG were incubated with 60 pM TFP, 0.1
mg/mL alamethicin-activated HLM, 5 mM UDPGA, and 5 mM MgCI2 for 20
minutes at 370C. Formation of TFPG was used as an index for UGT1A4
activity in HLM incubations. Each data point represents mean of duplicate
incubations. Error bars represent two-sided standard error of the mean. Data
points were fitted to IC50 equation as described under Materials and Methods.
Goodness of fit r2 value was 0.98.


106









A B

120- 120
*
o 100- 100 O

o 80 80


e 40- 40



0 0
a. 20- 0. 20


10 100 1000 10 100 1000
Saw Palmettto Extract Concentration ( ~g/mL) Milk Thistle Extract Concentration ( Fg/mL)

Figure 6-3. Inhibition of serotonin glucuronide formation by saw palmetto and milk thistle extracts. Increasing
concentrations of A) saw palmetto and B) milk thistle extracts were incubated with 8 mM serotonin, 5 mM
MgCI2, 0.5 mg/mL alamethicin-activated HLM, and 5 mM UDPGA for 60 minutes at 370C. Serotonin
glucuronide formation was used as an index of UGT1A6 enzyme activity in HLM incubations. Each data point
represents mean of duplicate incubations. Error bars represent two-sided standard error of the mean. Data
points were fitted to ICso equation as described under Materials and Methods. Goodness of fit r2 value was 0.96
and 0.99 for saw palmetto and milk thistle, respectively.









A B
120 120

5 100 100-



1 1 1 I


2 80- 201


10 100 1000 10000 0.01 0.1 1 10 100 1000 10000

Cranberry Extract Concentration (pg/mL) Milk Thistle Extract Concentration (pg/mL)

Figure 6-4. Inhibition of MPAG formation by cranberry and milk thistle extracts. Increasing concentrations ofA) cranberry
and B) milk thistle extracts were incubated with 240 pM mycophenolic acid, 5 mM MgCI2, 2% BSA, 0.16 mg/mL
alamethicin-activated HLM, and 1 mM UDPGA. Formation of MPAG was used as an index for UGT1A9 activity
in HLM incubations. Each data point represents mean of duplicate incubations. Error bars represent two-sided
standard error of the mean. Data points were fitted to ICso equation as described under Materials and Methods.
Goodness of fit r2 value was 0.95 and 0.99 for cranberry and milk thistle, respectively.


108









CHAPTER 7
CONCLUSION AND FUTURE DIRECTIONS

In the last ten years, researchers have increasingly recognized the potential of herbal

supplements to interact with conventional drugs. Mechanisms by which herb-drug

interactions occur can be pharmacodynamic or pharmacokinetic. For the latter, the

majority of interactions occur through phytochemicals modulating the expression or

activity of drug metabolizing enzymes. Many case studies, review papers, in vitro,

animal and clinical studies have documented the effects of herbal supplements on CYP

enzymes. In contrast, little attention has been given to other metabolic routes. The

goal of this research project was to characterize the effects of herbal supplements on

glucuronidation reactions in vitro. Data generated from these studies provide

information for clinicians and the public regarding the safety of taking herbal

supplements with drugs metabolized by UGT enzymes. In addition, the results help

generate hypotheses for future studies to investigate the in vivo effects of the observed

in vitro interactions.

Initially, we hypothesized that Ginkgo biloba extract inhibits MPA glucuronidation in

vitro. The basis for this hypothesis was that quercetin and kaempferol, the main

flavonoid aglycones in ginkgo extract, are substrates for UGT1A9, the primary hepatic

enzyme that metabolizes MPA. In order to measure MPAG concentrations in

microsomal incubates, we developed a sensitive analytical method using LC-MS/MS.

Linearity, accuracy, and sensitivity parameters were determined to ensure reliability and

reproducibility of the method over the range of MPAG concentrations expected in

incubations. This method, described in Chapter 2, was then used to study the effect of

ginkgo extract and its main flavonoid and terpene lactone components on MPAG


109









formation using HLM and HIM (Chapter 3). We found that ginkgo extract, quercetin,

and kaempferol inhibit MPAG formation at ICso concentrations attainable in the human

intestine. This finding highlights the potential of ginkgo extract to inhibit first pass

glucuronidation of mycophenolate sodium, the form of MPA that is more susceptible to

first pass metabolism.

Next, we investigated the effect of commonly used herbal supplements on one of the

major UGT enzymes-UGT1A1. To achieve this, we developed a screening approach

similar to the screening procedure followed in pharmaceutical industry. Eight commonly

used herbal extracts were screened at three different concentrations for their potential

inhibitory effect on UGT1A1 activity (Chapter 4). Enzymatic activity of UGT1A1 was

estimated using estradiol-3-O-glucuronidation as an enzyme-selective index reaction in

pooled HLM. Using this approach, green tea catechin ECGC, milk thistle, saw palmetto,

and echinacea were found to inhibit UGT1A1. Inhibition potency was reported in terms

of ICso value and V/D index value. The latter estimates the volume of fluid in which the

daily dose of the extract should be diluted to get an ICso-equivalent concentration value.

This parameter provides a putative tool to predict, under commonly recommended

doses, whether inhibition of UGT1A1 by the herbal supplement is likely to occur in vivo.

Based on V/D values of the observed inhibitors, EGCG and milk thistle may inhibit first

pass glucuronidation of UGT1A1 substrates such as raloxifene and ezetimibe. Among

all screened herbals, EGCG showed the most potent inhibition of UGT1A1.

Raloxifene is a selective estrogen receptor modulator with poor bioavailability due to

its extensive intestinal first pass metabolism by UGT enzymes, including UGT1A1.

Therefore, our next goal was to investigate the effect of green tea EGCG on raloxifene

in vitro intrinsic clearance using HIM (Chapter 5). A substrate depletion assay was


110









applied by incubating raloxifene with HIM and different concentrations of EGCG and

monitoring the fraction of raloxifene remaining at different time points. Data obtained

were used to estimate raloxifene in vitro intrinsic clearance. EGCG strongly inhibited

raloxifene in vitro clearance at concentrations of EGCG attainable in the intestine.

These results highlight the potential for inhibition of raloxifene pre-systemic clearance,

which could cause a marked increase in raloxifene bioavailability. The clinical

consequences of the observed interaction have yet to be determined.

Finally, we screened nine commonly used herbal supplements for their inhibitory

effects on UGT1A4, UGT1A6, and UGT1A9 using TFPG, serotonin glucuronide, and

MPAG formation as index reactions, respectively (Chapter 6). HLM were incubated with

TFP, serotonin, or MPA and each herbal extract at three different concentrations.

Screening results indicated that potential inhibitors were EGCG for UGT1A4, milk thistle

for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9.

For these potential inhibitors, confirmatory experiments were conducted by incubating a

range of concentrations of each extract with the enzyme-selective substrate. ICso and

V/D values were calculated and compared to physiologic concentrations, if available.

Results from these experiments showed that EGCG may inhibit first pass

glucuronidation of UGT1A4 substrates, while milk thistle may inhibit first pass

glucuronidation of UGT1A6 and UGT1A9 substrates. Conversely, weaker inhibition of

UGT1A6 and UGT1A9 was observed by saw palmetto and cranberry, respectively.

Overall, this research highlights the potential for inhibition of UGT enzymes by herbal

extracts with potencies that may translate in vivo. Future clinical studies are warranted

to investigate the pharmacokinetic consequences of the observed interactions. In this

project, we screened commonly used supplements for interactions with UGT1A


111









enzymes. Further studies are needed to investigate effects of other herbal supplements

and other UGT enzymes.

The availability of selective in vitro probe substrates for hepatic UGT enzymes offers

an efficient and relatively inexpensive way to screen for drug-drug and herb-drug

interactions through glucuronidation. Our results show that herb-drug interactions

appear more likely with first pass rather than systemic glucuronidation of drugs.

However, selective probe substrates have not been identified for UGT enzymes in HIM,

which limits HIM utility in screening experiments. Future studies are needed to identify

selective in vitro probes that can be used with HIM.

In conclusion, the in vitro effects of commonly used herbal supplements on UGT

enzymes have been studied. Results indicate the possibility for inhibition of

glucuronidation of drugs by herbal supplements. Inhibitory effects of ginkgo on MPA

glucuronidation and of EGCG on raloxifene clearance has been described. Moreover,

inhibition of UGT1A1, 1A4, 1A6, and 1A9 by herbal extracts is reported. Future studies

are warranted to investigate the clinical relevance of the observed interactions.


112









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BIOGRAPHICAL SKETCH

Mohamed-Eslam Mohamed was born in Cairo, the capital of Egypt. He received his

bachelor's degree in pharmaceutical sciences in May of 2003, from Ain Shams

University, in Cairo. Soon after, he worked at Misr International University, where he

was a teaching assistant in clinical pharmacology courses. He began his graduate

studies in the Pharmacotherapy and Translational Research Department at the

University of Florida in August of 2005. There, he started investigating herb-drug

interactions under the supervision of Dr. Reginald Frye. He received his Ph.D. from the

University of Florida in the Summer of 2010.


127





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1 CHARACTERIZATION OF HERB-DRUG INTERACTIONS THROUGH GLUCURONIDATION By MOHAMED-ESLAM F. MOHAMED A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Mohamed-Eslam F. Mohamed

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3 To my parents

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4 ACKNOWLEDGMENTS First, I would like to express my gratitude to Dr. Reginald Frye, my advisor, for his continuous guidance, teaching, and support since I joined his lab. For the rest of my career, I will always be indebted to him for hi s m entorship. I would also like to thank Dr. Butterweck, Dr. Hochhaus, and Dr. Shuster fo r serving on my committee and for their helpful comments on my dissertation. I am deeply grateful to the person who encouraged me to seek graduate studies, prov ided advice, and brotherly support when I worked at Misr International University in EgyptDr. Sherief Khalifa. He is indeed one of the people who directed my path. I would like to thank the members of Fr ye lab who made the work in the lab enjoyable. Special thanks to Cheryl Gallo way and Melonie Stanton for creating a family environment in the lab; I appreciate their kindness and support. I would also like to thank Prajakta for her help wh en I first started the program I am grateful to the pharmacy students who helped me with this proj ect: Stephen Harvey, Daniel Gonzales, and Tiffany Tseng. Their hard work is an integral part of this project. To my colleagues at the pharmacotherapy department, I appreciate your friendship, the conversations we had, and the discussions in the journal clubs and seminars. At every little discussion, I learned something that affected t he way I approached my research. I would like to show my gratitude to the person who provided an indispensible emotional support and encour agementmy wife Ranaand to the little one whose pure smiles and giggles soothed the t oughness of graduate schoolmy daughter Malak. Without the two of them, graduat e school would have been much tougher.

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5 Lastly, I would like to thank my parents who have been continuously praying for my success and my brother and sisters for thei r invaluable support and for always being there for me.

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6 TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 9LIST OF FIGURES ........................................................................................................ 10LIST OF ABBR EVIATIONS ........................................................................................... 11ABSTRACT ................................................................................................................... 12 CHAPTE R 1 EFFECTS OF HERBAL SUPPLEMENTS ON DRUG GLUCURONIDATION. REVIEW OF CLINICAL, ANIMAL AND IN VITR O STUDIES ................................. 14Introduc tion ............................................................................................................. 14Popularity of Herbal S upplement Use in the US ..................................................... 15Potential for Herb-Drug Interactions through Drug Metabo lizing Enzymes ............. 16Glucuronidati on Enzy mes ....................................................................................... 17Glucuronidation as a Pathwa y for Drug Inte ractions ............................................... 18Search St rategy ...................................................................................................... 19Herbal Medicines Containing Substrat es or Modulators of UGT Enzymes ............. 19Noni Juice ......................................................................................................... 19Garlic ................................................................................................................ 19Mangosteen Juice ............................................................................................ 20Green T ea ........................................................................................................ 20Echinacea ......................................................................................................... 21Ginkgo .............................................................................................................. 22Ginseng ............................................................................................................ 22Milk Th istle ....................................................................................................... 23Soy ................................................................................................................... 24Cranberry ......................................................................................................... 25St. Johns Wort ................................................................................................. 26Aloe .................................................................................................................. 27Valerian ............................................................................................................ 27Conclusion and Summa ry ....................................................................................... 28Study Obje ctives ..................................................................................................... 28

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7 2 DETERMINATION OF MYCOPHENOLIC ACID PHENOLIC GLUCURONIDE IN MICROSOMAL INCUBATES USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY .................................. 35Introduc tion ............................................................................................................. 35Experiment al ........................................................................................................... 36Chemicals and Reagents ................................................................................. 36Chromatography Conditi ons ............................................................................. 36Mass Spectrometry Conditions ......................................................................... 37Stock Solutions, Standards, and Quality Contro ls (QCs) ................................. 38Microsomal Incubation Conditi ons and Sample Preparat ion ............................ 38Method Vali dation ............................................................................................. 39Calibration, Precision and Accuracy ................................................................. 39Extraction Recovery, Matrix Effect, and Stability .............................................. 39Data Anal ysis ................................................................................................... 40Results .................................................................................................................... 40Chromatographic Method ................................................................................. 40Calibration, Precis ion, and A ccuracy ................................................................ 40Extraction Recovery, Matrix Effect, and Stability .............................................. 41Characterization of Km and Vmax ....................................................................... 41Conclusi on .............................................................................................................. 413 INHIBITION OF INTESTINAL A ND HEPAT IC GLUCURONIDATION OF MYCOPHENOLIC ACID BY GINKGO BILOBA EXTRACT AND FLAVONOIDS .... 47Introduc tion ............................................................................................................. 47Materials and Methods ............................................................................................ 49Chemicals and Reagents ................................................................................. 49Herbal Extracts ................................................................................................. 50Inhibition of MPA Gl ucuronidati on Assay ......................................................... 50Detection of MPA-7Oglucuron ide ................................................................... 51Enzyme Kinetics Analysis ................................................................................. 52Results .................................................................................................................... 52Inhibition of MPA Glucuronidation by Ginkgo biloba ......................................... 53Effect of Ginkgo Compounds on MPA Gluc uronidation .................................... 53Inhibition Kinet ics Analysis ............................................................................... 54Discuss ion .............................................................................................................. 544 INHIBITORY EFFECTS OF COMMONLY USED HERBAL EXTRACTS ON UGT1A1 ENZYME ACTIVITY ................................................................................. 64Introduc tion ............................................................................................................. 64Materials and Methods ............................................................................................ 66Chemicals and Reagents ................................................................................. 66Preparation of Herbal Working So lutions .......................................................... 66In Vitro In cubations ........................................................................................... 67HPLC A nalysis ................................................................................................. 68

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8 Data Analysis .................................................................................................... 69Results .................................................................................................................... 69Screening Experiment s: ................................................................................... 69Confirmatory Experiments and Determination of Precise IC50 Values: ............. 70Discuss ion .............................................................................................................. 705 INHIBITORY EFFECTS OF EPIGALLOCA TECHIN GALLTE ON RALOXIFENE IN VITRO CL EARANCE ......................................................................................... 80Introduc tion ............................................................................................................. 80Materials and Methods ............................................................................................ 81Chemicals and Reagents ................................................................................. 81Incubations with HI M ........................................................................................ 81HPLC-MS/MS Assay of Ralo xifene .................................................................. 82Estimation of Non-specif ic Protein Binding ....................................................... 83Data Analysis .................................................................................................... 83Results and Discussion ........................................................................................... 836 INHIBITORY EFFECTS OF COMMONLY USED HERBAL EXTRACTS ON UGT1A4, 1A6, AND 1A9 ENZYME ACTI VITIES ..................................................... 86Introduc tion ............................................................................................................. 86Materials and Methods ............................................................................................ 87Chemicals and Reagents ................................................................................. 87Preparation of Herbal Working Solutions.......................................................... 88Incubations of Herbal Extracts with TFP .......................................................... 89Chromatographic Analysis of TFP glucuronide (TFPG) ................................... 90Incubations of Serotonin with Herbal Extracts .................................................. 90Chromatographic Analysis of Serotonin Gl ucuronide ....................................... 91Incubations of MPA wit h Herbal Extracts.......................................................... 92MPAG LC-MS/MS Assay .................................................................................. 92Data Anal ysis ................................................................................................... 93Results .................................................................................................................... 93Effect of herbal extracts on TFPG formation ..................................................... 94Effect of herbal extracts on serotonin glucur onide forma tion ............................ 94Effect of herbal extr acts on MPAG formation .................................................... 95Discuss ion .............................................................................................................. 957 CONCLUSION AND FUTU RE DIRE CTIONS ....................................................... 109REFERENC ES ............................................................................................................ 113BIOGRAPHICAL SKETCH .......................................................................................... 127

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9 LIST OF TABLES Table page 1-1 Top selling herbal supple ments in the US in 2006. ............................................. 301-2 Summary of studies on glucuronidati on of phytochemicals and modulation of UGT enzymes by phytochemic als and herbal extracts. ...................................... 312-1 Precision (R.S.D. %) and accuracy (R.E. %) for MPAG in microsomal incubati ons. ........................................................................................................ 432-2 Assessment of extraction recovery, matrix effect, and stability of MPAG analytical assay. ................................................................................................. 433-1 Inhibition of MPA7-O-glucuronidation by Ginkgo biloba extracts. ...................... 593-2 Inhibition of MPA-7-O-glucur onidation by gink go flavono ids. ............................. 604-1 List of Herbal extracts inve stigated for effe ct on UGT1A1. ................................. 754-2 Rough IC50 and volume/dose index values for inhibition of estradiol-3O glucuronidation by nine herbal ex tracts. ............................................................. 764-3 Precise IC50 values for herbal extracts s howing strongest inhibition of estradiol-3O -glucuronidation. ............................................................................ 775-1 Effect of green tea EGCG on raloxife ne in vitro intrinsic clearance using HIM.. ................................................................................................................... 856-1 List of herbal extracts scr eened for UGT1A4, UGT1A6, and UGT1A9 inhibiti on. .......................................................................................................... 1006-2 Effect of commonly used herbal extracts on UGT1A4, UGT1A6, and UGT1A9 activity. .............................................................................................................. 1016-3 Determination of inhibitory potency of selected UGT1A4, UGT1A6, and UGT1A9 herbal inhibito rs. ................................................................................ 103

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10 LIST OF FIGURES Figure page 1-1 The growing interest in studying herbal supplem ents ......................................... 331-2 Expression of UGT enzymes in th e liver and the small intestine. ..................... 342-1 Chemical structures of analytes. ......................................................................... 442-2 Extracted HPLC-MS/MS chromatogram s of incubations and spiked MPAG samples .............................................................................................................. 452-3 Determination of apparent Km and Vmax for MPAG formation in human liver microsom es. ....................................................................................................... 463-1 Chemical structures of main gi nkgo components, mycophenolic acid, and MPA-7-O-gluc uronide. ........................................................................................ 613-2 Effect of Ginkgo biloba extracts on mycophenolic acid 7-O-glucuronidation in vitro. .................................................................................................................... 623-3 Inhibition of mycophenolic acid 7-O-glucuronidation by quercetin and kaempfer ol. ......................................................................................................... 634-1 Effect of herbal extracts on E-3-G fo rmation as an index for UGT1A1 activity in HLM. ............................................................................................................... 784-2 Inhibition of E-3-G fo rmation by her bal extracts.................................................. 795-1 Effect of green tea EGCG on raloxifene in vitro intrinsic clearance using HIM. .. 856-1 Effect of commonly used herbal extracts on UGT1A4, UGT1A6, and UGT1A9 enzyme acti vities. ............................................................................................. 1056-2 Inhibitory effect of green tea ca techin EGCG on TFPG formation in HLM. ....... 1066-3 Inhibition of serotonin glucuronide fo rmation by saw palmetto and milk thistle extracts .......................................................................................................... 1076-4 Inhibition of MPAG formation by cranberry and milk th istle extracts. ................ 108

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11 LIST OF ABBREVIATIONS AUC Area under the plasma concentration-time curve CLint, u Unbound intrinsic clearance CYP Cytochrome P450 E-3-G Estradiol-3O-glucuronide ECG ( )-epicatechin-3-gallate EGC Epigallocatechin EGCG ( )-epigallocatechin-3-gallate HIM Human intestine microsomes HLM Human liver microsomes HPLC High-performance liquid chromatography IC50 Concentration of inhibitor that results in 50% inhibition of reaction Ki Dissociation constant for bi nding of inhibitor to enzyme Km Concentration of substrate that produces half-maximal velocity LC-MS/MS Liquid Chromatogr aphy/tandem mass spectrometry MPA Mycophenolic acid MPAG Mycophenolic acid -D-glucuronide MS Mass spectrometry TFP Trifluoperazine TFPG TrifluoperazineN -glucuronide UDPGA Uridine diphos phate glucuronic acid UGT UDP-glucuronosyltransferase V/D Volume per dose index Vmax Maximum enzyme velocity

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12 Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy CHARACTERIZATION OF HERB-DRUG INTERACTIONS THROUGH GLUCURONIDATION By Mohamed-Eslam F. Mohamed August 2010 Chair: Reginald F. Frye Major: Pharmaceutical Sciences The use of herbal supplements has conti nued to increase over the last decade. Many herbal supplement users concomitantly take prescription and non-prescription drugs, raising the potential for herb-drug in teractions. Cytochrome P450-mediated herb-drug interactions have been reported in m any studies. By contrast, the effects of herbal extracts on UDP-glucuronosyl tr ansferase (UGT) enzymes have not been adequately studied. The goal of this research project was to identify commonly used herbal extracts that have potential to inhibi t the glucuronidation pathway. First, we studied the effects of Ginkgo biloba extract and its major constituents on mycophenolic acid (MPA) glucuronidation. Ginkgo extrac t and its main flavonoid aglycones, quercetin and kaempferol, inhibited MPA gl ucuronidation in human liver and intestinal microsomal incubates. By comparing IC50 values to expected physiologic concentrations of ginkgo compounds in different body compartments, ginkgo extract is likely to inhibit MPA glucuronidation in the human in testine. The second aim was to identify herbal extracts that can potentially inhibit UGT1A1-mediated drug metabolism. A screening in human

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13 liver microsomes (HLM) was performed with commonly used herbal extracts to assess the potential for inhibition of UGT1A1 activi ty. Milk thistle extract and the green tea catechin epigallocatechin gallate (EGCG) we re found to be potential inhibitors of first pass metabolism of UGT1A1 substrates Among the extracts screened, EGCG exhibited the most potent inhibition. Therefore, we examined the effect of EGCG on intrinsic intestinal clearance of raloxifene, a substrate for intestinal glucuronidation by UGT1A1. EGCG exhibited concentrationdependent inhibition of ra loxifene in vitro intestinal clearance, suggesting that green t ea extracts may increase raloxifene oral bioavailability if taken concomitantly. Lastly, we screened commonly used herbal supplements for their effects on UGT1A4, 1A6, and 1A9 activi ties in HLM. In vitro inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. In conclusion this project shows that commonly used herbal supplements may inhi bit UGT-mediated drug metabolism. Based on observed inhibitory potency and predicted or known concentrations, glucuronidation is more likely to be affected in the intestine than the liver. The observed herb-UGT interactions warrant further res earch to investigate the pharmacokinetic consequences and clinical significance.

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14 CHAPTER 1 EFFECTS OF HERBAL SUPPLEMENTS ON DRUG GLUCURONIDATION. REVIEW OF CLINICAL, ANIMAL, AND IN VITRO STUDIES Introduction In the last decade, interest in stud ying the pharmacologic effects of herbal supplements, including their potential to in teract with drug metabolizing enzym es, has grown. The number of publications citing he rbal supplements has increased by nearly five-fold in the 2000s compared to the 1990s (Figure 1-1A). This upsurge coincided with an escalation in use of herbal supplements, which raised concern by health professionals regarding t he potential for herbs to adversely affect drugs pharmacokinetics and pharmacodynam ics (Gardiner et al., 2008). Several milestone events have lead to the deve lopment of interest in studying herbdrug interactions as summarized in Figure 1-1B. These events shaped the current widespread use of herbal supplements and hi ghlighted the kno wledge deficiency regarding their safety. In 1994, the Un ited States Congress passed the Dietary Supplement Health and Education Act. Under the provisions of this law, dietary supplements, including herbals, are exempt fr om regulations applied to drugs, including premarketing safety and efficacy studies (G urley, 2010). Concurr ently, the Internet became widely accessible and was commonly used to market herbal products, which lead to a boost in the use of herbal supplem ents in the mid to late 1990s (Morris and Avorn, 2003). In 1998, Congress establishe d the National Center for Complementary and Alternative Medicine with the goal of funding research on complementary and alternative medicine, including herbal suppl ements (www.nih.gov). Two years later, a milestone case study published in The Lancet described a possible interaction between St. Johns wort, an herbal supplement commonly used for depression, with the

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15 immunosuppressant drug cyclosporine (Rusch itzka et al., 2000). This case study sparked a wave of clinical, in vitro, and animal studies addressing St. Johns wort interactions with drug metabolizing enzymes (Shord et al., 2009). Meanwhile, reports emerged associating ephedra us e with heart attacks, which eventually lead to the withdrawal of all products containing ephedra fr om the US market (Haller and Benowitz, 2000). These events set off an alarm that research was needed to characterize the safety of herbal supplements as well as t heir potential to interact with conventional drugs. Recently, the scientific community has requested that the FDA play a more rigorous role in evaluating safety and efficacy of herbal supplements with calls for premarketing safety data and studies on interactions with drug metabolizing enzymes (Tsourounis and Bent, 2010). Several case studies, reports, and review articles have described the potential of herbal supplements and phytochemicals to m odulate cytochrome P450 (CYP) enzymes. On the other hand, the effect of herbal extracts on glucuronidation, a major conjugative metabolism pathway, has not been sufficiently st udied. The aim of this review is to summarize evidence regarding the potential of the top 20 selling her bal supplements to interact with UGT enzymes. Popularity of Herbal Supplement Use in the US The herbal supplement market has grown continuously in the last decade. Table 1-1 lists the top sellin g herbal supplements in the US in 2006 (NBJ, 2007). In 2006, Americans spent $4.6 billion dollars on herbal supplements, representing a 4% growth in sales from 2005 (NBJ, 2007). Survey studi es show that about 20% of Americans use at least one herbal supplement. Meanwhile, one in four herbal supplements users takes one or more prescription drugs, raising the potential for herbdrug interactions

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16 (Eisenberg et al., 1998; Bardia et al., 2007). In addition, patients with chronic diseases, which are likely to be treated by multiple drugs, use herbal supplements more frequently than the general population, ther eby increasing the risk for interactions (White et al., 2007; Miller et al., 2008). Potential for Herb-Drug Interacti ons through Drug Metabolizing Enzy mes Enzymatic biotransformation (i.e., metabolis m) plays a major role in disposition of endogenous and exogenous compounds including bot h drugs and herbal constituents. Biotransformation reactions are generally divided into two groups, phase I and phase II; each encompasses a wide range of enzymes and catalytic activities (Crettol et al., 2010). Phase I reactions involve hydrolysis, reduction, and oxidation and usually result in only a small increase in hydrophilicity (Parkinson, 2001). In phase I, CYP enzymes rank first in terms of clinical importance and number of substrates. On the other hand, phase II reactions include conjugation of compounds with a hydrophilic group producing a more hydrophilic and easily excreted product (except for acetylation and methylation). Phase II reactions may or may not be preceded by phase I reactions. For some substrates, such as morphine and mycophenolic acid, phase II conjugation with glucuronic acid represent the chief metabolic pathway (Parkinson, 2001). Herbal supplements contain a myriad of natural chemicals that share the same metabolic pathways with prescription drugs (Zhou et al., 2007). This may result in activation or inhibition of the metabolism of concomitantly taken drugs, over or underexposure to drugs, and consequently, treatment failu re or toxicity. At least 30 clinically proven herb-drug interactions mediated through CYP enzymes have been described (Skalli et al., 2007; Izzo and Ernst, 2009). Induction of CYP2C19, for example, by Ginkgo biloba resulted in subtherapeutic levels of anticonvulsant drugs, which

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17 precipitated fatal seizures (Kupiec and Ra j, 2005). St. Johns wort has the most documented evidence of pharmacokinetic dr ug interactions with more than 100 publications in the last 10 years on its interactions with prescription drugs (Izzo and Ernst, 2009). For example, induction of CYP3 A4 and P-glycoprotein by St. Johns wort resulted in decreased exposure to midazolam ( 44%), tacrolimus ( 59%), alprazolam ( 52%), verapamil ( 80%), and cyclosporine A (52%), respectively (Whitten et al., 2006). In contrast, interactions thr ough glucuronidation have not been adequately characterized. Glucuronidation Enzymes Conjugation with glucuronic ac id (glucuronidation) represents the main phas e II reaction and one of the most essential detox ification pathways in humans (Dutton, 1980). The UDP-glucuronosyl transferases (UGT) are a superfamily of enzymes which constitutes two families, UGT1 and UGT2, and three subfamilies, UGT1A, 2A, and 2B comprising at least 18 different enzymes (Figure 1-2) (Owens et al., 2005). UGT enzymes are widely and differentiall y expressed throughout the human body (Guillemette et al., 2010). Although the majori ty of UGT enzymes are expressed in the liver, UGT1A7, 1A8, and 1A10 are expressed ex clusively extrahepatically, mainly in the intestine (Izukawa et al., 2009; Ohno and Nakajin, 2009). UGT1A9, 2B7, and 2B11 are expressed at relatively high quantities in t he kidney. Figure 1-2 displays the difference in UGT expression between the liver and inte stine, which are the main sites for xenobiotic gluc uronidation. UGT enzymes conjugate a wide r ange of endogenous compounds, drugs, environmental compounds, and phytochemicals (Tukey and Strassburg, 2000; Ouzzine et al., 2003). Although UGT enzymes generally display broad and overlapping

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18 substrate specificities, selective probes have been identified for the main hepatic UGT enzymes, 1A1, 1A4, 1A6, 1A9, 2B7, 2B 15, and 2B17 (Burchell et al., 2005; Court, 2005). Identification of selective probes, development of analytical assays, and the commercial availability of human liver micr osomes (HLM) have facilitated the in vitro evaluation of glucuronidati on interactions (Court, 2005). Glucuronidation as a Pathway for Drug Interactions Several reports have commented on the clinical significance of interactions through UGT enzymes. The glucuron idation pathway has been frequent ly described as a low affinity pathway, with relatively small impact on substrate exposur e in vivo as a result of inhibition (Williams et al., 2004; Burchell et al., 2005). This has been observed for substrates that have alternative metabolic pa thways and relatively low affinity for UGT enzymes. However, if the substrate is me tabolized mainly through glucuronidation, inhibition can result in a significant increas e in exposure. For example, exposure to zidovudine, a substrate for UGT2B7, incr eased by 31% and 74% due to inhibition of glucuronidation by atovaquone and fluconazole, respectively (Sahai et al., 1994; Lee et al., 1996). Moreover, rash, which could be life -threatening, resulted from inhibition of Nglucuronidation of lamotrigine by valproic acid (Kiang et al., 2005). In addition to inhibition, interactions with glucuronidat ion can occur through induction of UGT enzymes. Studies have repor ted that rifampicin and lo pinavir/ritonavir induced lamotrigine glucuronidation, which required a doubling of the dose to achieve a therapeutic plasma concentration (Ebert et al., 2000; van der Lee et al., 2006). These examples show that drug-drug interactions through modulation of glucuronidation can be clinically significant. Similarly, since many phytochemicals are substrates for UGT enzymes, herb-drug interactions may occur through this pathway.

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19 Search Strategy Systematic literature sear ches were conducted in MEDLINE (through PubMed) and Google Scholar databases through March 2010. The search terms used were each of the 20 top-selling herbal supplem ents (Table 1-1) or their main secondary metabolites in combination with the terms glucu ronidation or UGT. Only articles written in English were included. No other restrictions we re imposed. The herbal supplements below are listed in the order of th eir 2006 sales (Table 1-1). Herbal Medicines Containing Substr ates or Modulators of UGT Enzy mes Noni Juice Noni juice ( Morinda citrifolia ) has a long history of being used as a medicinal plant for a wide range of indications including hyperte nsion, menstrual cramps, gastric ulcers, and many others (Potterat and Ha mburger, 2007). Noni juice contains several classes of secondary metabolites, including polysacchar ides, fatty acid glycosides, iridoids, anthraquinones, and flavonoids (Potterat and Hamburger, 2007). M any of these are phenolic compounds that may be substrates for UGT enzymes and may compete with metabolism of drugs. However, no studies were found regarding the glucuronidation of compounds in noni juice. In a study in ra ts, noni juice inhibited ex-vivo p-nitrophenol glucuronidation, which is mainly catalyzed by UGT1A enzymes, by 35% at a dose of 2.1 mg/kg and 49% at a dose of 21 mg/kg. Ho wever, there was no inhibition at a higher dose of 210 mg/kg (Mahfoudh et al., 2009). Garlic Garlic (Alium sativum ) bulbs have been used for over 4000 years as a medicinal plant for a variety of ailment s including headache, bites, intestinal worms and tumors (Corzo-Martnez et al., 2007). Garlic is rich in organo-sulphur compounds such as alliin,

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20 and -glutamylcysteines, diallyl sulphide, dia llyl disulphide, and ot hers (Corzo-Martnez et al., 2007). These compounds are not known to be substrates for glucuronidation. Gwilt et al. (1994) studied t he effect of garlic on acetaminophen metabolism in healthy subjects. Subjects were given 10 mL garlic extract daily (equivalent to six to seven cloves of garlic) for three months. Garlic consumption did not have a significant effect on acetaminophen or acetaminophen gluc uronide pharmacokinetic parameters. Mangosteen Juice Mangosteen (Garcinia ma ngostana) juice is well-known for its anti-inflammatory properties and it is traditionally used in the treatment of skin infections and wounds (Obolskiy et al., 2009). Mangosteen juice is rich in phenolic compounds called xanthones, mainly and -mangostin (Obolskiy et al., 2009). Bumrungpert et al. (2009) showed that -mangostin was conjugated by phase II enzymes in caco-2 cells. In their study, one third of -mangostin was conjugated after 4 hours of incubation with cells. Conjugation was measured by hydrolysis using snail enzyme that possesses both glucuronidase and sulfatase activity. Th erefore, it was not possible to determine the relative contribution of glucuronidation and sulfation. Green Tea Green tea ( Came llia sinensis ) has gained increased popularity as a beverage and an herbal supplement with many attributed health benefits includ ing reduction in the risk of cardiovascular disease and certain cancers (C abrera et al., 2006). Green tea extract is rich in polyphenolic compounds called catechin s. The major green tea catechins are: ( )-epigallocatechin-3gallate (EGCG), ( )-epicatechin-3-gallate (ECG), ( )epigallocatechin (EGC), ( )-epicatechin, (+)-gallocatechin, and (+)-catechin (Gupta et al., 2002). EGCG is believed to be the mo st biologically active and most abundant

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21 catechin in green tea extract (Feng, 2006). In vitro, animal, and human studies provide evidence that green tea catechins are meta bolized by methylation, sulfation and glucuronidation in (Feng, 2006) Lu et al. (2003) reported that EGCG was conjugated by UGT1A1, 1A8, and 1A9 and that glucuroni dation of EGCG was much higher than EGC. In terms of interactions, a study in rats showed that consumption of green tea extract for four weeks enhanced hepatic glucuronid ation of 2-nitrophenol, a substrate for UGT1A enzymes. However, the effect was not dose dependent (Bu-Abbas et al., 1998). Zhu et al. investigated the effect of administration of green tea extract for 18 days on hepatic glucuronidation activity in female Long-Evans rats. Green tea extract stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by 30%, 15% and 26%, respectively (Zhu et al., 1998). The same authors reported that green tea polyphenols, includ ing EGCG, inhibited estradiol and estrone glucuronidation in vitro usi ng rat liver microsomes with IC50 values of 10-20 g/mL (Zhu et al., 1998). In HLM, green tea catechins in hibited the glucuronid ation of SN-38, the active metabolite of the anticancer drug ir inotecan, in a concentration-dependent manner (Mirkov et al., 2007). However, in human hepatocytes, a significant decrease in glucuronide was observed in only 33% (E GCG), 44% (ECG), and 44% (EGC) of the hepatocyte preparations. Ther efore, the authors concluded that at pharmacologically relevant concentrations, catechins are unlik ely to inhibit the fo rmation of irinotecan inactive metabolites when administered concomitantly (Mirkov et al., 2007). Echinacea Echinacea products refer to herbs or roots of Echinac ea purpurea Echniacea angustifolia or Echinacea pallida or a combination of any of them (Gale Group., 2001).

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22 The herbs and roots of these different species have different comp osition and medicinal properties. Among the common compounds in echinacea are polyphenolic compounds including cichoric acid and echinacoside. Jia et al. (2009) studied phase II metabolites of echinacoside in rats and isolated two glucuronide metabolites for echinacoside (Jia et al., 2009). In vitro studies using HLM or expressed UGT enzymes are needed to characterize the contribution of UGT to echinacoside metabolism Ginkgo Ginkgo ( Ginkgo biloba) leaf extract is commonly used for its perpetual benefits on memory and circulation. The prim ary acti ve constituents of ginkgo are terpene lactones (ginkgolides and bilobalide) and flavone glycosides, which are hydrolyzed in vivo to flavone-aglycones (e.g., quercetin, kaempferol and isorhamnetin) (Chan et al., 2007). Ginkgo flavonoids are substrates for intestinal and hepatic UGT enzymes, primarily UGT1A9 and, to a lesser extent, UGT1A3 (Oliveira and Watson, 2000; Zhang et al., 2007; Chen et al., 2008b). There is in vitro and animal evidence that flavonoids modulate UGT enzymes. In a study using HLM, quercetin inhibited UGT1A1 activity with an IC50 value higher than 50 M (Williams et al., 2002; Moon et al., 2006) In contrast, quer cetin and kaempferol increased testosterone glucuronidation by almost 2.5and 4-fold, respectively, in a prostate cancer cell line (Sun et al., 1998). In a study done in rats, quercetin induced 4nitrophenol glucuronidation activity by 1.5to 4-fold in rat liv er and different parts of the intestine (Van der Logt et al., 2003). Ginseng Ginseng typically refers to roots of Panax ginseng or Panax quinque folium which are used as general tonics and adaptogens (Chen et al., 2008a). The most important

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23 bioactive components contained in ginseng are a group of saponins called ginsenosides (Chen et al., 2008a). No reports of ginsenosides glucuronidation were found in the literature. In a pharmacokinetic study in which ginsenoside Rd was administered intravenously to volunteers, no glucuronide conjugates were detected in plasma (Yang et al., 2007). Another in vitr o study on metabolism of ginsenos ide Rg3 using rat S9 liver fraction did not detect any glucuronidated me tabolites (Cai et al., 2003). In a pharmacokinetic interaction study, 10 healthy volunteers received 300 mg of zidovudine, a UGT2B7 substrat e, orally before and after 2 weeks of treatment with 200 mg American ginseng extract twice daily. Am erican ginseng did not si gnificantly affect the pharmacokinetic parameters of zidovudine or zidovudine glucuronide (Lee et al., 2008). Milk Thistle Milk thistle ( Silybum marianum ) is commonly used to trea t hepatotoxicity (Shord et al., 2009). Extract of milk this tle is rich in flavonolignans, primarily silybin, silydianin, and silychristine, which are collectively know n as silymarin (Dhiman and Chawla, 2005). There is evidence on glucuron idation of silymarin flavonolignans from both animal and human studies. In a study in rats, silybin A, silychristin, and silydianin were excreted as glucuronides (Miranda et al., 2008). Moreover silibinin monoand di-glucuronides were detected in human plasma following ingestion of silibinin phytosome capsules in colorectal carcinoma patient (Hoh et al., 2006). In vitro experiments using recombinant enzymes and hepatocytes showed inhibitory effects of milk thistle co mpounds on UGT enzymes. Silybin inhibited recombinant UGT1A1, 1A6, 1A9, 2B7 and 2B15 with IC50 values of 1.4, 28, 20, 92, and 75 M, respectively using 7-Hydroxy-4-(trifluoromethy l)coumarin as a substr ate for the different

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24 UGT enzymes (Sridar et al., 2004). In hepato cytes, silymarin inhibited glucuronidation of 4-methylumbelliferone, a substrate fo r UGT1A6 and 1A9, by about 80% and 90% at concentrations of 100 and 250 M, respective ly (Venkataramanan et al., 2000). In another in vitro study us ing HLM and estradiol-3O-glucuronidation as an index for UGT1A1 activity, silymarin inhibited UGT1 A1 at estradiol concentrations of 50 and 100 M, while results at lower concentrati ons showed mixed inhibition and activation (Williams et al., 2002). On the other hand, in a pharmacokinetic study in cancer patients, 4-day and 12-day adminis tration of milk thistle showed no significant effects on the pharmacokinetics of the anticancer drug irinotecan (van Erp et al., 2005). Soy There has been increasing intere st in soy isoflavones, especially genistein and daidzein, due to their wide range of potential biologica l activities (Nielsen and Williamson, 2007). In vitro and clinical studies provide evidence that soy isoflavones are substrates for UGT enzymes. Despite being structurally similar, genistein and daidzein conjugation exhibit preferences fo r different UGT enzym es. UGT1A1, 1A4, 1A6, 1A7, and 1A9 catalyzed 7and 4'-glucu ronidation of both ge nistein and daidzein, while UGT 1A10 was selective for genistein. The authors also report ed that genistein, but not daidzein, was conjugated in human colon microsomes (Doerge et al., 2000). The glucuronide was the predominant circul ating form for both genistein (69%) and daidzein (40%), with smaller amounts of t he aglycone and sulfate. This indicates that glucuronidation is the primary route of metabolism for these soy isoflavones. Pfeiffer et al. (2005) reported that dai dzein and genistein as well as several structurally related isoflavones modulated UGT1A1 activity in vitro using HLM. Daidzein (25 M) stimulated estradiol-3-glucuronidation, a marker for UGT1A1 activity, by about

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25 50%. In contrast, genistein (25 M) inhibited the 3-glucuronidation by about 80%. The 17-glucuronidation of estradiol was not affected by either compound. In another study in HLM, unhydrolyzed and hydrolyzed soy ex tracts inhibited dihydroestosterone glucuronidation, an index fo r UGT2B15 activity, with IC50 values of 4.6 and 6.1 g/mL, respectively (Anderson et al., 2003). In a study in mice, genistein and daidzein only slightly decreased UGT activities in some tissues (Froyen et al., 2009). The e ffect was sex and duratio n dependent. In this study, genistein and daidzein inhibited glucur onidation of 3-methyl-2-nitrophenol in the small intestine of male mice after five days of isoflavone admin istration by about 50% and 40%, respectively. This effect did not reproduce in the liver and the kidneys, or in female mice. Cranberry Cranberry ( Va ccinium macrocarpon ) is commonly consumed in the US to prevent urinary tract infections with potential activity as an antibacterial and anticancer (Neto, 2007). Cranberry juice contains a high c ontent of flavonoids and phenolic acids. Among the cranberry flavonoids, quercetin is the most abundant (Neto, 2007). As mentioned under ginkgo, querceti n is conjugated by UGT1A9 and, to a lesser extent, UGT1A3 (Oliveira and Wats on, 2000; Zhang et al., 2007; Chen et al., 2008b) In addition to flavonoids, cranberry juice contains resveratrol, which is also found in grapes and red wine (Wang et al., 2002). In vitro studies show that resveratrol is glucuronidated to two major gl ucuronide conjugates, resveratrol-3 -glucuronide and resveratrol-4 -glucuronide. The major enzymes that catalyze resveratrol glucuronidation are UGT1A1 and UGT1A9 (Brill et al., 2006; Iwuchukwu and Nagar, 2008). No studies on effects of cranberry juice on UG T enzyme activities were found.

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26 St. Johns Wort St. Johns wort ( Hypericum perforatum ) extract is a commonly used herbal therapy for insomnia and depression (Gaster and Holroyd, 2000). Flavonol glycos ides are the major class of compounds found in St. John s wort extract, with rutin, hyperoside, isoquercitrin, quercetrin (qercetin 3-rhamnoside), and miquelianin being the main compounds. Other component s include hypericin, pseudohypericin, and hyperforin (Butterweck and Schmidt, 2007). As mentioned for ginkgo, quercetin is known to be a substrate and modulator of UGT1A enzymes (Oliveira and Watson, 2000; Chen et al., 2008b). No studies regarding glucuronidation of other St. Johns wort components were found. In a recent study, Volak et al. (2010) r eported that hypericin inhibited UGT1A6mediated glucuronidation of acetaminophen in human colon cells and serotonin in UGT1A6-expressing insect cells with IC50 values of 7.1 and 0.59 M, respectively. The authors concluded that the me chanism of this interaction was through inhibition of UGT1A6 phosphorylation by protein kinase C, which is considered a novel mechanism of drug-drug interaction. In an animal study, effects of St. Johns wort on irinotecan pharmacokinetics were measured after 3 and 14 days of daily St. John s wort administration. Long-term (14day) exposure to St. Johns wo rt significantly decreased Cmax of irinotecan by 39.5% and SN-38 by 38.9%, but didnt significant ly affect SN-38 glucuronide plasma concentrations. On the other hand, short-term (3-day) administration of St. Johns wort did not significantly alter the pharmacokinet ics of CPT-11 and SN-38, but decreased the AUC0and the elimination t1/2 of SN-38 glucuronide by 31. 2% and 25.8%, respectively (Hu et al., 2007). In the sa me study, St. Johns wort ex tract (5g/mL) decreased SN-38

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27 glucuronidation by 45% in ra t liver microsomes, while pre-incubation of St. Johns wort extract in hepatoma cells significantly increa sed SN-38 glucuronidat ion. These results indicate that St. Johns wort may affect pharmacokinetics of SN-38. Aloe Aloe vera leaf extract is used as an herbal suppl ement due to its a ttributed biological benefits, including antiviral, antibacterial, laxa tive, and immunostimulatory effects (Ni et al., 2004). Aloe extract contains several clas ses of phytochemicals that have been thoroughly described (Dagne et al., 2000). Among the different classes, Aloe vera extract is rich in anthracene derivatives (e .g., aloe-emodin). There is evidence that glucuronidation is the primary route of metabo lism of aloe-emodin in rats (Shia et al., 2009). Characterization of aloe-emodin glucuronidation has not been performed. Valerian Valerian ( Valeriana officinalis ) extract is commonly used as an herbal supplement to treat sleeping disorders, restlessness, and anx iety (Pato ka and Jakl, 2010). Alkaloids, organic acids, terpenes, and valepotriat es are among the major classes of phytochemicals found in valerian extract. In terms of interactions with UGT enzymes, valerian methanolic extract inhibited UGT1A1 and UGT2B7 in HLM using estradiol and morphine as probe substrates, respectively. In the same study, valerenic acid, a monoterpene in valerian extrac t, inhibited glucuronidation of acetaminophen, estradiol, and morphine with both HLM and expressed UG T enzymes (Alkharfy and Frye, 2007). IC50 values for inhibition with valerenic acid were 9.24 M for acetaminophen glucuronidation, 8.79 M for estradiol-3O -glucuronidation, 2.33 M for estradiol-17O glucuronide, 4.96 M for morphine-3-glucuonide, and 47 .31 M for testosterone glucuronide. The clinical significance of this in vitro interaction is yet to be determined.

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28 Conclusion and Summary The studies reviewed provide evidence on the potential for modulation of UGTmediated drug metabolism by commonly used herbal supplement s. Flavonoid compounds were the most studied class of phytochemicals for metabolism by and interactions with UGT enzymes. Based on in vitro and animal studies, flavonoid-rich supplements may affect metabo lism of UGT drug substrates. However, this effect has not been studied in a clinical pharmacokinetics study. Overall, no studies were found for 6 out of the top 20 reviewed herbs regarding their glucuronidation or modulation of UGT enzymes. Moreover, only 3 clinical studies investigating the effect of herbal supplements on the pharmacokinetics of UGT drug substrates were published (Gwilt et al., 1994; van Erp et al., 2005; Lee et al., 2008) Taken together, there is a scarcity of information on glucuronidation of majority of phytochemicals and their potential to interact with UGT-mediated drug metabolism. The overall goal of this work was to charac terize the effects of commonly used herbal supplements on glucuronidation reactions in vitro. Study Objectives Study the effect of Ginkgo biloba leaf extract and its major flavonoid and terpene lactone components on MPA gl ucuronidation using hum an liver and intestine microsomes. This aim was construct ed based on the finding that quercetin and kaempferol, the major ginkgo falv onoi d aglycones, are metabolized through UGT1A9, the main enzyme metabolizing MPA. Characterize the effects of commonly used herbal extracts on UGT1A1 activity in HLM and determine inhibitory potency for po tential inhibitors. Our hypothesis was that herbal extracts would inhibit UGT1A1 due to the high content of polyphenolic phytochemicals, which can be substrates and inhibitors of UGT1A1. Assess the effect of green tea catechin EGCG on raloxifene intrinsic clearance using human intestine microsomes. Ou r hypothesis was that EGCG, having been identified as a UGT1A1 inhibitor, woul d inhibit raloxifene intrinsic clearance in vitro.

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29 Screen commonly used herbal extracts fo r inhibition of UGT1A4, UGT1A6, and UGT1A9 using HLM and characterize t he inhibitory potency of the potential inhibitors. Our aim was to identify potential inhibitors among herbal extracts that may interact with drugs metabolized through these enzymes.

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30 Table 1-1. Top selling herbal supplem ents in the US in 2006. Source: NBJs Supplement Business Report, October 2007. Top Herbs 2006 sales ($ millions ) 1 Noni Juice 257 2 Garlic 155 3 Mangosteen Juice 147 4 Green Tea 144 5 Saw Palmetto 134 6 Echinacea 129 7 Ginkgo Biloba 106 8 Ginseng 98 9 Milk Thistle 93 10 Psyllium 85 11 Soy 69 12 Cranberry 68 13 Maca 66 14 Goji 65 15 Green Foods 64 16 St. Johns wort 60 17 Aloe 60 18 Stevia 58 19 Black Cohosh 57 20 Valerian 55

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31 Table 1-2. Summary of studies on glucuronidation of phytochemicals and modulation of UGT enzymes by phytochemicals and herbal extracts. Interaction Studies Herb Phytochemicals studied for glucuronidation In vitro Animal Clinical References Noni Juice Noni juice: UGT1A (Mahfoudh et al., 2009) Garlic UGT1A6 (Gwilt et al., 1994) Mangosteen -mangostin (Obolskiy et al., 2009) Green Tea EGCG>> EGC Polyphenols: UGT1A Green tea: UGT1A (Bu-Abbas et al., 1998; Zhu et al., 1998; Lu et al., 2003) Saw Palmetto ----------------------------No studies report ed--------------------------Echinacea Echinacoside Ginkgo Biloba Flavonoids Flavonoids: UGT2B17 UGT1A1 Flavonoids: UGT1A6 (Sun et al., 1998; Oliveira and Watson, 2000; Williams et al., 2002; Van der Logt et al., 2003; Chen et al., 2008b) Ginseng ND UGT2B7 (Cai et al., 2003; Yang et al., 2007; Lee et al., 2008) Milk Thistle Flavonolignans UGT1A1 UGT 1A6 UGT 1A9 UGT 2B7 UGT 2B15 UGT1A1 (Venkataramanan et al., 2000; Williams et al., 2002; Sridar et al., 2004; van Erp et al., 2005; Hoh

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32 et al., 2006) Psyllium ---------------------------No studies report ed--------------------------Soy Isoflavones Genistein: UGT1A1 Daidzein: UGT1A1 Soy extract: UGT2B15 UGT1A (Doerge et al., 2000; Anderson et al., 2003; Pfeiffer et al., 2005; Froyen et al., 2009) Cranberry Flavonoids Resveratrol Quercetin: UGT1A1 Quercetin: UGT1A6 (Oliveira and Watson, 2000; Williams et al., 2002; Van der Logt et al., 2003; Brill et al., 2006) Maca ---------------------------No studies report ed--------------------------Goji ---------------------------No studi es reported-------------------------Green Foods ----------------------------Various contents----------------------------St. Johns wort Flavonoids SJW : UGT1A1 Quercetin: UGT1A1 Hypericin: UGT1A6 Quercetin: UGT1A6 (Oliveira and Watson, 2000; Williams et al., 2002; Hu et al., 2007; Chen et al., 2008b; Volak, 2010) Aloe Aloe-emodin (Shia et al., 2009) Stevia ---------------------------No studies report ed--------------------------Black Cohosh ----------------------------No studi es reported-------------------------Valerian Valerian & valerenic acid: UGT1A1 UGT1A6 UGT2B7 (Alkharfy and Frye, 2007) inhibition of UGT; activation or induction; no effect on UGT activity; ND, No glucuronides detected in metabolism studies.

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33 Figure 1-1. The growing inte rest in studying herbal supp lements. A) Number of PubMed articles citing herbal supplements or herbal medicine in the last three decades. B) Timeline for milestone ev ents that lead to development of interest in studying herb-drug interactions.

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34 Figure 1-2. Expression of UGT enzymes in the liver and the small intestine. A) Relative expression of hepatic UGT enzyme based on 20 human liver samples. Adapted from Izukawa et al. (2009). B) Relative expression of UGT enzymes in the small intestine based on 3 human intestine samples. Adapted from Ohno et al. A. B.

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35 CHAPTER 2 DETERMINATION OF MYCOPHENOLIC ACID PHENOLIC GLUCURONIDE IN MICROSOMAL INCUBATES USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY1 Introduction Mycophenolic acid (MPA) is an immunos uppressant drug that has been widely and successfully used in transplant recipients as well as in patient s with immune disorders (Staatz and Tett, 2007; Walsh et al., 2007). M PA is administered as an ester prodrug or a sodium salt and is extensively metaboliz ed by UDP-glucuronsy ltransferases (UGTs) to glucuronidated metabolites. MPA-7-O -glucuronide (MPAG) is the main metabolite of MPA (Figure 2-1). Plasma concentrations of MPAG are typically 20-fold higher than MPA in patients receivi ng mycophenolate therapy. M PAG is approximately 82% bound to plasma albumin and is mainly excreted in the urine as the main pathway for MPA elimination (Staatz and Te tt, 2007). Other minor MPA me tabolites include the acyl glucuronide, 7-OH gl ucose conjugates, and 6O -desmethyl-MPA (Shipkova et al., 1999; Picard et al., 2004). Formation of MPAG is carried out by various UGT enzymes. The main UGT enzymes involved are UGT1A7 and UGT1A9, while UGT1A8 and UGT1A10 play a smaller role in MPAG formation (Basu et al., 2004). Plasma levels of MPA and MPAG vary widely within and between patients, which can directly affect clinical outcomes (Hummel et al., 2007). In vi tro studies with human liver microsomes, a commonly used approach in drug metabolism and interaction studies, may provide some clues to understanding this variability. Previous st udies have used MPA microsomal incubations 1 Reprinted with permission from Mohamed MF, Harvey SS and Frye RF (2008) Determination of mycophenolic acid glucuronide in microsomal incu bations using high performance liquid chromatographytandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 870:251-254.

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36 to characterize the UGT enzymes involv ed in its glucuronidation and study MPA interaction potential with other drugs (Vietri et al., 2000; Shipkova et al ., 2001; Vietri et al., 2002; Bernard and Guillemette, 2004; Miles et al., 2005; Picard et al., 2005). Various reports have described assays to measure MPAG in human plasma and urine(Aresta et al., 2004; Bolon et al., 2004; Patel et al., 2004; Indjova et al., 2005; Yau et al., 2007); however, validated in vitro a ssays are lacking. This paper describes an HPLC-tandem mass spectrometry assay for the quantitative determi nation of MPAG in human liver microsomal incubations. Experimental Chemicals and Reagents Mycophenolic acid (MPA) and mycophenolic acid -D-glucuronide (MPA-7O glucuronide; MPAG ) were purchased from Toronto Research Chemicals (North York, Ontario, Canada). Potassium phosphate dibasi c, uridine diphosphate glucuronic acid, magnesium chloride, alamethicin, phenolphthalein -D-glucuronide (PG; internal standard), and glacial acetic acid were pur chased from Sigma-Aldrich (St. Louis, MO, USA). Pooled human liver microsomes were pur chased from In Vitro Technologies Inc. (Baltimore, MD, USA). Acet onitrile and methanol were pur chased from EMD Chemicals Inc. (Gibbstown, NJ, USA). All chemicals used were of the highest purity available for analytical research. Deionized water was prepared by using a Barnstead Nanopure Diamond UV Ultrapure Water System (Dubuque, IA, USA). Chromatography Conditions MPAG and the internal standard (IS) PG were chromatographed with a ThermoFinnigan Surveyor series HPLC system consisting of a Surveyor Plus autosampler and Surveyor MS pump (Thermo Corp., San Jose, CA, USA). Gradient

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37 chromatography was carried out at am bient temperature on a reversed-phase Phenomenex (Torrance, CA, USA) Synergi Fusion-RP18 column (100 2 mm, 4 m). The two mobile phases consisted of (A) 1 mM acetic acid in deionized water and (B) 1 mM acetic acid in acetonitrile. Gradient el ution at a flow-rate of 0.22 mL/min was employed with the following steps: at start of the run, 30% B for one min, then increased to 90% B in 0.75 min, held at 90% B between 1.75 and 3.1 min, and from 3.6 to 6.5 min, the column was re-equilibrated at 30% B. The total run time was 6.5 min. The temperature of the autosam pler was maintained at 10oC and the injection volume was 5 L. A divert valve was used to divert flow to waste from 0 to 2 min and from 4.5 to 6.5 min. Mass Spectrometry Conditions The LC-MS/MS analysis was carried out on a TSQ Quantum triple quadrupole mass spectrometer (Thermo Corp., San Jose, CA, USA), equipped with an electrospray ioniza tion (ESI) source operated in the negati ve ion mode. Detection of MPAG and PG was performed for their [M-H]ions. Analysis was carried out in the single reaction monitoring (SRM) mode using the mass transitions of m / z 495 319 and m / z 493 175 for MPAG and phenolphthalein -D-glucuronide, respectively. MPA was also monitored at a mass transition of m / z 319 191. The mass spectrometer settings were a capillary temperature of 350oC, spray voltage of 3.0 kV, and source collision induced dissociation (CID) of 5 V. Nitr ogen was used as the sheath and auxiliary gas set to 35 and 15 (arbitrary uni ts), respectively. The argon collision gas pressure was set to 1.5 mTorr (200 mPa) and the collision ener gy was set to 30 eV for MPA, 22 eV for MPAG, and 25 eV for PG. The peak full wid th at half maximum (FWHM) was set at 0.2 Th and 0.7 Th for Q1 and Q3, respecti vely, and the scan time was set to 250 ms.

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38 Data acquisition and analysis were performe d with Xcalibur software version 1.4 (Thermo Corp., San Jose, CA, USA). Stock Solutions, Standards, and Quality Controls (QCs) Stock standard solutions of MPAG (0.2 and 2 mM) w ere prepared by dissolving the appropriate amount of MPAG in met hanol. A series of MPAG standards (concentrations: 0, 1, 2, 4, 10, 15, and 20 M) and quality control samples (concentrations: 2.5, 7.5, and 16 M) were pr epared by subsequent dilution of the stock standard solutions in 0.1 M phosphate buffer, pH 7.1. Working solutions of MPA (6 mM) and the internal standard PG (1 mM) were prepared in methanol. Microsomal Incubation Conditions and Sample Preparation The incubation conditions were optimized wi th respect to time of incubation and microsomal protein concentration. Stock solutions of UDPGA (25 mM) and MgCl2 (5 mM) were prepared in phosphate buffer. Al amethicin (0.2 mg/mL) was prepared in phosphate buffer containing 10% ethanol. The incubation mixture (final volume, 105 L) consisted of 300 M MPA (for the ki netic study, 50, 100, 300, 500, 1000, 1500, 2000, and 2500 M MPA were used), 1 mM MgCl2, 0.1 M potassium phosphate buffer (pH 7.1), 0.16 mg/mL microsomal protei ns, and 16 g/mL alamethicin (100 g alamethicin/1 mg microsomal proteins). The mixture wa s pre-incubated on ice for 15 minutes. The reaction was started by adding UDPGA (final concentr ation, 1 mM). After the mixture was incubated for 30 min at 37oC, the reaction was stopped by adding 315 L ice-cold acetonitrile and 20 L internal standard, vortex-mixing, and placing tubes on ice. Tubes were centrifuged for 10 min at 20,817 x g The supernatant was diluted in a ratio of 1:5 with purifie d water and 5 L was injected into the HPLC system.

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39 Method Validation The method was validated for sele ctivity, linear ity, sensitivity, precision, accuracy, recovery and stability according to the guidelines issued by the Food and Drug Administration (FDA) for the validati on of bioanalytical methods (FDA, 2001). Calibration, Precision and Accuracy Calibration curves were constructed using six different concentrations of MPAG prepared in incubation buffer. Curves were obtained daily for 3 days by calculating peak-area ratios of MP AG to PG. Data point s were fit using linear regression and a 1/y2 weighting-scheme. The precision and accuracy of the assay was determined using quality control (QC) samples of known MPAG amounts (2.5, 7.5, and 16 M) prepared in incubation buffer and processed in t he same manner as standards and incubations samples. Six replicates of each QC were analyzed on 3 days, after which the interand intra-day precision values were calculat ed using one-way ANOVA using the day as the grouping variable as described previously (den Brok et al., 2005). Accuracy was calculated as the percentage of the nominal MPAG concentrati on. For the assay to be considered acceptable, precision determined at each concentration level was required to be within 15% of all days mean and accuracy within 15% of nominal concentration at all levels of concentrations. Extraction Recovery, Matrix Effect, and Stability Extraction recovery, absolute matrix e ffect, and stability were evaluated for MPAG samples prepared at concentrations of 2.5 and 16 M and PG internal standard (50 M). E ach set of samples was analyzed in triplicates. Extraction recovery was determined by comparing peak areas of t he standards extracted from spiked 0.16 mg/mL microsomal proteins in phosphate buffer to cont rol microsomal proteins

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40 extracted in the same manner and spiked after extraction with the same standard concentration. Matrix effect on ionization was evaluated by comparing the MPAG peak areas of samples spiked post-extraction with corresponding peak area ratios of standards prepared in the injection solution. Processed stability was evaluated by reinjecting the samples after keeping them in the autosampler at 10oC for 36 hours. Comparison of MPAG and PG peak areas before and after 36-hour storage provided a measure of stability under no rmal operating conditions. Data Analysis To estimate precision, one-way ANO VA analysis was performed using JMP IN 5.1.2 (SAS Inc, Cary, NC, USA). Data we re fit to the Mi chaelisMenten equation and the apparent kinetic parameters of Km and Vmax were determined by non-linear regression analysis (Prism 4.0, GraphP ad software, San Diego, CA, USA). Results Chromatographic Method MPAG, the internal standard PG, and MPA we re separated within four minutes of the chromatographic run. The retention times for MPAG, PG, and MPA were 3.35, 3.41, and 3.90 min., respectively. Repres entative extracted LC-MS/MS chromatograms of processed microsomal incubat ions are shown in Figure 2-2. The MPAG peak was detected only when substrate, enzyme, and co-enzyme were added. Chromatograms of double blank incubations, which contained all incubation consti tuents except MPA, did not show any interfering peaks at the retention times of either PG or MPAG. Calibration, Precision, and Accuracy Standard curves for MPAG were linear over the range of 1-20 M. The mean correlation coefficient ( r2) for the standard curve was at l east 0.99. Intraand inter-day

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41 RSD% for MPAG QC samples were less t han 10% and all calculated concentrations were within 8% of the actual concentration (Table 2-1). Extraction Recovery, Matrix Effect, and Stability Table 2-2 shows the results from the assessment of extraction recovery, matrix effect, and stability for MPAG and PG. Av erage extraction recovery for MPAG was 87.4%. There was no significant matrix effe ct as the average supp ression of ionization by matrix was 12.3%. MPAG and PG were st able in the processed incubation mixtures as well as in reconstitution solution for at least 36 hours (<10% change in measured concentration). Characterization of Km and Vmax The enzyme kinetic parameters for MPAG formation were estimated by incubating different concentrations of MPA (50 to 2500 M) with human liver microsomes (Figure 2-3). The apparent Km and Vmax were 285.7 M and 8.6 nmol/min/mg protein, respectively. MPAG formation was consistent with Michaelis-Menten kinetics. Conclusion MPA glucuronidation represents the primar y pathway for MPA biotransformation in vivo MPA7O-glucuronide is the main metabolit e and exhibits 20-100-fold higher plasma concentrations than MPA (Staatz and Tett, 2007). This paper describes a specific and sensitive HPLC-tandem mass s pectrometry assay for measuring MPAG in human liver microsomes within a run time of 6.5 minutes. Although several reports have described assays for MPAG in plasma a nd urine (Aresta et al., 2004; Bolon et al., 2004; Patel et al., 2004; Indjov a et al., 2005; Yau et al., 2007) this is the first detailed report of a validated method to determi ne MPAG concentrations in human liver microsomes.

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42 The validated assay is a precise (RSD% <10%) and accurate method for determining MPAG in microsomal incubatio ns over a range of 1 20 M. The method is reproducible and subject to minimal matr ix effect (Tables 2-1 and 2-2). Previous kinetic studies on MPAG formati on in vitro reported values for Km and Vmax ranging from 95 to 351 M and from 2.5 to 20.5 nmol/min/mg protei n, respectively (Vietri et al., 2000; Bowalgaha and Miners, 2001; Shipkova et al ., 2001; Vietri et al., 2002; Bernard and Guillemette, 2004; Miles et al., 2005; Picard et al., 2005); the values determined using this assay are within these ranges. Thus, the assay described is suitable for in vitro pharmacogenetic and interaction studies of MPA metabolism.

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43 Table 2-1. Precision (R.S.D. %) and accu racy (R.E. %) for M PAG in microsomal incubations (six replicates per day for three days). Concentration (M) R.S.D. (%)a R.E. (%) Nominal Measured (Mean)Intra-day Inter-day 2.50 2.70 5.6 8.9 8.0 7.50 7.40 3.9 5.1 -1.3 16.0 16.5 6.1 6.8 3.1 aEstimated using one-way ANOVA Table 2-2. Assessment of extraction reco very, matrix effect, and stability of MPAG analytical assay. Nominal MPAG Concentration (M) Extraction Recovery a (%) (SD) Matrix Effect b (%) (SD) Stability c (%) (SD) 2.5 83.3 88.0 105.3 16 91.6 87.3 109.7 50 (PG) 103.4 96.9 107.0 a Extraction recovery was calculated using the following formula: Recovery (%) = [(mean raw peak area)pre ext. spike/(mean raw peak area)post ext. spike] 100. b Matrix effect was calculated using the fo llowing formula: Matrix effect (%) = [(mean raw peak area)post ext. spike/(mean raw peak area)neat] 100. c Stability was calculated using the followi ng formula: Stability (%) = [(mean raw peak area)after 36 hours/(mean raw peak area)initial run] 100. PG = phenolthalein glucuronide (internal standard)

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44 Figure 2-1. Chemical structur es of analytes. A) Structure of mycophenolic acid (MPA). B) St ructure of mycophenolic acid glucuronide (MPAG). C) Structure of the internal standard phenolphthalein glucuronide (PG). A B C

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45 Figure 2-2. Extracted HPLC-MS/MS chroma tograms of incubati ons and spiked MPAG samples. A) Microsomal incubations in absence of MPA and PG, B) spiked lowest MPAG standard (1 M), C) MPAG in microsom al incubation (estimated concentration is 10 M), D) representative chromatogram of PG (50 M) as the internal standar d, and E) MPA (50 M) in microsomal incubation. The small peak at 3.35 min. in E (enlarged in inset) is from in-source fragmentation (loss of glucur onide) of M PAG to MPA. A B C D E

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46 Figure 2-3. Determination of apparent Km and Vmax for MPAG formation in human liver microsomes.

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47 CHAPTER 3 INHIBITION OF INTESTINAL A ND HEPAT IC GLUCURONIDATION OF MYCOPHENOLIC ACID BY GINKGO BILOBA EXTRACT AND FLAVONOIDS2 Introduction Herbal supplement use continues to increase around the globe, especially in populations looking for natural methods to promote health and well ness. In the US, surveys estimate that 20% of the populat ion uses at least one herbal supplement (Bardia et al., 2007). This growing interest in herbals is manifested by annual sales in the US of over $4 billion dollars (NBJ, 2007). Such public interest is met by concerns from health professionals regarding possible del eterious interactions of herbals with conventional drugs. Herbals are considered dietary supplements; hence, they are not routinely screened for interactions with drug metabolizing enzymes ( www.fda.gov ). Howeve r, numerous in vitro, animal, and clinical studies and case reports provide evidence that herbals can interact with conventional drugs and may lead to serious adverse effects (Gardi ner et al., 2008). Ginkgo biloba is among the most popular herbals used in the world. Its extract is available over the counter in the US and is commonly prescribed in European countries for cerebral insufficiency (De Smet, 2005). Antioxidant effe cts as well as beneficial effects on memory and circul ation have been attributed to G. biloba extract and its components. The primary active constituents of G. biloba are terpene lactones (ginkgolides and bilobalide) and flavone glycosides, which are hydrolyzed in vivo to flavone-aglycones (e.g., quercetin and kaempfer ol) (Figure 3-1A) (Chan et al., 2007). 2 Reprinted with permission from Mohamed M and Frye R (2010) Inhibition of intestinal and hepatic glucuronidation of mycophenolic acid by Ginkgo biloba extract and flavonoids. Drug Metabolism and Disposition 38:270.

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48 Several clinical and in vitro studies have investigated the effect of G. biloba on drug metabolizing cytochrome P450 enzymes and transporters (Izzo and Ernst, 2009). In contrast, limited research has been conducted to investigate interactions of G. biloba and its components with conjugation pathways. In vitro studies have shown that quercetin and kaempferol inhibi t sulfotransferase 1A1 (Eat on et al., 1996; Ghazali and Waring, 1999); meanwhile, informati on is lacking regarding effects of G. biloba on drug glucuronidation. Glucuronidation constitutes the main pathw ay of conjugative metabolism for a wide variety of compounds (Ouzzine et al., 2003); s ubstrates for UDP-glucuronsyltransferase enzymes (UGTs) include endogenous compounds, drugs and many phytochemicals. Many flavonoids (e.g., quercetin and kaempf erol) are substrates for UGT enzymes. Moreover, inhibitory effect s of flavonoids on UGT1A enzym es have been reported in the literature (Williams et al., 2002; D'Andrea et al., 2005). For substrates metabolized mainly through glucuronidation, modulation of UGT activities can lead to significant effects on pharmacokineti cs (Kiang et al., 2005). Mycophenolic acid (MPA) is an immunosuppre ssive drug that acts by inhibiting the production of guanosine nucleotides in lymphocytes, ceasing their proliferation (Allison and Eugui, 2005). Therefore, it is used to prevent graft reject ion in transplant recipients and to delay progression of the autoimmune di sorders (Heatwole and Ciafaloni, 2008). MPA is available as either a prodrug mofetil ester (CellCept) or as an enteric-coated sodium salt (Myfortic). Although both formulations have similar pharmacokinetic and efficacy profiles, absolute oral bioava ilability of mycophenolat e sodium is 72% compared to 94% for mycophenolate mofetil (S taatz and Tett, 2007). This difference is

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49 attributed to higher presystemic glucuron idation of MPA from the mycophenolate sodium formulation. Following oral absorpti on, MPA is metabolized by UGTs to the major phenolic conjugate 7O -MPA-glucuronide (MPAG) (Fi gure 3-1B). In the liver, UGT1A9 is the main enzyme catalyzing the formation of MPAG, while UGT1A7, UGT1A8, and UGT1A10 contribute to MPAG formation extra-hep atically mainly in the kidneys and intestine (Picard et al., 2005). MPA is a narrow therapeutic index drug with wide interand intra-individual variability and complex pha rmacokinetics in transplant recipients (Staatz and Tett, 2007). Therefore, an alteration in MPA glucuronidation may cause changes in exposure to the immunosuppressive drug, and consequently, undesired clinical outcomes. The aim of this study was to investigate the effect of ginkgo extract and its main components on MPAG formation in human intestinal and liver microsomes. The results demonstrate that G. biloba and its primary constituents have the ability to inhibit MPA glucuronidation in the intestine and liver. Materials and Methods Chemicals and Reagents Mycophenolic acid (MPA; 98%) and mycophenolic acid-7 O -glucuronide (MPAG; 98%) were purchased from Toronto Research Chemicals (North York, ON, Canada). Potassium phosphate dibasic, uridine diphosphate glucuronic acid, magnesium chloride, alamethicin, phenolphthalein-D-glucuronide (PG; intern al standard), niflumic acid, and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, methanol, quercetin dihydrate ( 99% purity) and kaempferol (90%) were purchased from Fisher Scientific (Pitts burgh, PA, USA). Gi nkgolide A (95.1%), ginkgolide B (82.8%), and bilobalide (99.7%) were purc hased from ChromaDex (Irvine,

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50 CA, USA). Pooled human liver and intestin al microsomes were purchased from BD Biosciences Discovery Labware (Woburn, MA, USA). Herbal Extracts Ginkgo biloba extract was provided by Finzel berg & Co. KG (Andernach, Germany) as dry powder. The extract was standardi z ed by the supplier to contain 24% flavonglycosides, 6% terpene lactones, and < 5 ppm ginkgolic acids using 60% acetone as the extraction solvent. Unhydrolyzed and acid-hydrolyzed G. biloba working solutions were freshly prepared by dissolving 30 mg of the powder extract in 1 mL of either 60% acetone or 60% acetone/40% 5N HCl to prepare the unhydrolyzed and acidhydrolyzed working extracts, respectively. The acid treated extract was heated at 90oC for one hour and neutralized with 2N KOH. The acetone-rich extracts were serially diluted to prepare working solutions of G. biloba with concentrations of 0.05 to 5 mg/mL and acetone content of 10%. Inhibition of MPA Glu curonidation Assay The incubation conditions were optimized wi th respect to time of incubation and microsomal protein concentration. A ty pical 100 L inc ubation mixture contained HLM or HIM (protein concentration, 0.16 mg /mL), alamethicin ( 100 g/mg microsomal protein), MgCl2 (5 mM), MPA, and different concentrati ons of each test extract or test compound in 100 mM phosphate buffer, pH 7.4. Microsomes we re pre-incubated on ice with alamethicin for 15 minutes to activate UGT enzymes. The reaction was started by adding UDPGA (1 mM) and placing incubation tubes in a water bath at 37oC for 30 minutes. The reaction was stopped by adding 300 L of ice-cold acetonitrile and 20 L of internal standard (0.5 mg /mL phenolphthalein glucuroni de). Tubes were vortexmixed for two minutes and centrifuged for 10 min at 20,000 x g The supernatant was

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51 diluted 12-fold with purified water and 5 L was injected into the HPLC system. Incubations with herbal extracts and the corr esponding controls contained 1% acetone. The HLM and HIM used in all experim ents were from the same lot. Screening experiments were conducted to generate IC50 values by incubating MPA at the estimated Km value in the presence of five concentrations of G. biloba unhydrolyzed and hydrolyzed extracts (final concentrations ranging from 5 g/mL) or G. biloba individual components (final conc entrations ranging from 1M). In addition to IC50 values, inhibitory potency was also expressed as the volume per dose index, which is defined as the volume in which one dose would be dissolved in to obtain the corresponding IC50 concentration as described by Strandell et al. (2004). Comparison of this unit to physiological volu mes facilitates an assessment of inhibitory potential. A Ki value was determined if the IC50 value was lower than 100 M. In such cases, MPA (60 M with HLM or 30 M with HI M) and a range of concentrations of individual ginkgo components (10 M wit h HLM or 3 M with HIM) were used for the construction of Dixon plots and estimation of Ki values. Detection of MPA-7O-glucuronide MPAG was determined by LC/MS/MS on a T hermoFinnigan Surveyor series HPLC system connected to a TSQ Quan tum trip le quadrupole mass spectrometer (Thermo Corp., San Jose, CA, USA) using electrospray ionization (ESI), as described previously (Chapter 2). Average assay within-day and between-day relative standard deviations were 5.2% and 6.9%, respectively and accura cy expressed as relative error was within 8%. Briefly, 5 L of each sample was injected on a reversed-phase Phenomenex (Torrance, CA, USA) Syner gi Fusion-RP18 column (100 2 mm, 4 m). The two

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52 mobile phases consisted of (A) 1 mM acet ic acid in deionized water and (B) 1 mM acetic acid in acetonitrile. Gradient eluti on at a flow-rate of 0.22 mL/min was employed with the following steps: at start of the run, 30% B for one mi n, then increased to 90% B in 0.75 min, held at 90% B between 1.75 and 3.1 min, and from 3. 6 to 6.5 min, the column was re-equilibrated at 30% B. A nalysis was carried out in the single reaction monitoring (SRM), negative ion m ode using the mass transitions of m / z 495 319 and m / z 493 175 for MPAG and PG, respectively. MPAG standard solutions were freshly prepared for each experiment with concentrati on ranges of 100 nM M for HIM or 1 20 M for HLM incubations. Enzyme Kinetics Analysis Km and Vmax were determined by nonlinear r egression analysis of the MPAG formation data using eight different MPA conc entrations (0.02 to 1 mM). Data points were fitted to the Michaelis-Menten model using Prism 4.0 (GraphPad software, San Diego, CA, USA). IC50 values were similarly determined by nonlin ear regression fitting of the inhibition data to the IC50 equation (Copeland, 2005) us ing Prism 4.0. The Ki values were determined by fitting competitive, noncom petitive, uncompetitive, and mixed-type inhibition models to the MPAG formation data (Copeland, 2005). The mode of inhibition was determined on the basis of visual insp ection of the Dixon plot and the Akaike information criterion (Akaike, 1974) using SigmaPlot v.11 Enzyme Kinetics Module 1.3 (Systat Software, Inc., Chicago, IL, USA). Results MPA-7O -glucuronide formation wa s best explained by MichaelisMenten kinetics. The Km and Vmax were 103.9 19.5 M and 2.6 0.2 nmol/min/mg protein (mean

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53 SEM), respectively, with pooled HLM, whereas with pooled HIM, these values were 67.2 10.1 M and 408.7 17.1 pmol /min/mg protein (mean SE), respectively. These values are similar to values previously reported (Shipkova et al., 2001; Miles et al., 2005; Chang et al., 2009). Inhibition of MPA Glucuronidation by Ginkgo biloba Both unhydrolyzed and acid-hydrolyzed G. biloba extrac ts inhibited MPA glucuronidation in pooled HIM and HLM (Figure 3-2). MPA conc entration was 100 M for HLM incubations and 70 M for HIM incu bations. Results showed that unhydrolyzed and acid-hydrolyzed G. biloba extracts inhibited MPA glucuronidation in HLM with best fit IC50 values of 84.3 11.6 and 20. 9 3.6 g/mL, respectively. More potent inhibition of MPA glucuronidation wa s observed in HIM with IC50 values of 6.8 0.8 and 4.3 1.2 g/mL for the unhydrolyzed and acid-hydrolyzed ex tracts, respectively (Table 3-1). The volume/dose index values, calculated to es timate the clinical significance of the inhibition as described previously (Strande ll et al., 2004), are shown in Table 3-1. Effect of Ginkgo Compounds on MPA Glucuronidation Ginkgo flavonoids (quercetin and kaempferol) and terpene lactones (ginkgolides A and B, and bilobalide) were incubated with MPA to determine whether or not these compounds inhibit MPA glucuronidation. Ginkgo flavonoids but not terpene lactones showed inhibition with IC50 values < 100 M (Table 3-1). Quercetin and kaempferol inhibited MPA glucuronidation in HLM with IC50 values of 19.1 1.3 and 23.1 5.5 M, respectively. In agreement with results from incubations with G. biloba extracts, inhibition of MPA glucuronidation was more potent in HIM, with IC50 values of 5.8 0.3 and 7.6 0.6 M for quercetin and kaempferol, respectively.

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54 Inhibition Kinetics Analysis To further characterize the inhibition of MPA glucuronidation by ginkgo flavonoids, enzyme inhibition kinetic ex periments were carried out. Based on the analysis of nonlinear regression of inhi bition data and Dixon plots presented in Figure 3-2, quercetin exhibited mixed-type inhibition aga inst MPA glucuronidation in both HLM and HIM. Kaempferol exhibited non-competitive inhibition in HLM and mixed-type inhibition in HIM. In HLM, Ki values were 11.3 1.7 and 33.6 2.5 M for quercetin and kaempferol, respectively (Table 3-2; Figure 3-2A ). Again, inhibitory potency of quercetin and kaempferol to MPA glucuronidation in HIM wa s three to four-fold higher than that in HLM with Ki values of 2.8 0.4 and 4. 5 1.2 M, respectively (Table 3-2; Figure 3-2B). Discussion Scientific and public interest in gink go has grown enormously in recent years because of its purported beneficial effects on memory and circulation (Bardia et al., 2007). Ginkgo supplements have been widely used with little awareness of the potential for drug interactions wit h conventio nal drugs. Although ginkgo is considered generally safe, clinical studies and case reports have demonstrated that it can interact with conventional drugs and may lead to severe adverse effects (Hu et al., 2005; Kupiec and Raj, 2005). In the current study, ginkgo extract and flavone aglycones inhibited the UGT-mediated metabolism of mycophenolic acid in human intestinal and liver microsomes. In intestinal microsomes, ginkgo extr acts inhibited MPAG formation with IC50 values of 4.3 and 6.8 g/mL for acid-hydrolyzed an d unhydrolyzed extracts, respectively. The clinical significance of this interaction can be postulated based on the recommended dose of ginkgo supplements and the fraction of MPA metabolized by intestinal enzymes.

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55 Ginkgo extracts are usually taken at a dose of 120 mg to 240 mg per day. Therefore, IC50equivalent concentrations can be achieved in the intestine if a 120 mg ginkgo dose is mixed with 18 to 28 L of fluid (i.e., 6.7 to 4.3 mg/L) or if a 240 mg dose is mixed with 35 to 56 L of fluid. Thus, based on estimates of intestinal volume that range from about 0.5 to 5 L (Hellum et al., 2007), concentrations in the intestine after ingestion of a ginkgo supplement are expected to be much higher than IC50 values; accordingly, inhibition of intestinal UGT enzymes in vivo is likely. The potential for interaction is greater with enteric-coated mycophenolate sodium, since about 28% of the dose is eliminated through first pass metabolism (Myfortic prescribing information: http://www.pharma.us.novartis.com/product/pi/pdf/myfortic.pdf ). Inhibiting first pass metabolism of MPA could result in higher systemic concentrations, enhanced immunosuppressive effect and increased potential for side effects. Incubations with HLM also showed inhibi tion of MPA glucuronidation by ginkgo extracts. In the liver, UGT1A9 selectively metabolizes MPA to MPAG (Picard et al., 2005); therefore, MPAG formation can be used as an in vitro UGT1A9 index reaction. An effect observed on MPAG formation is expected to reproduce with other UGT1A9 substrates like propofol. In vitro screening of ginkgo components for inhibition indicates that the observed inhibition can be attribut ed to ginkgo flavonoid components, but not to terpene lactones. Ki values for inhibition of hepatic MPA glucuronidation by quercetin and kaempferol were 11.3 and 33.6 M, respectively. To understand the clinical significance of this observation, adequate knowledge of the bioavailability and hepatic concentrations of the inhibitors is necessary. Quercetin and kaempferol are classified as flavonols, which is a class of flavonoids ubiquitously

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56 found in plants, beverages, and dietary supplements, e.g., tea, onions, apples, red wine, St. John's wort, and G. biloba (Nijveldt et al., 2001). A typica l diet contains about 14-16 mg/day quercetin and 4-6 mg/day kaempferol according to dietary surveys in the Netherlands and US (Hertog et al., 1993; Sampson et al., 2002); however, the intake can reach several hundred mg in dietary supplements and herbal products and several grams in anticancer therapy (Lamson and Bri gnall, 2000). In contrast to kaempferol, a relatively large number of studies concerning the absorption of quercetin have bee n published. However, the extent to which quercetin reaches the liver remains largely unknown. Most studies were not able to detect free quercetin concentrations in plasma and absorption was estimated from the quantities of quercetin and quercetin conjugates detected in the urine (0. 3.4% of quercetin dose) (Scalbert and Williamson, 2000); thus it was assumed that quercetin was poorly absorbed. However, an early study in heal thy ileostomy subjects estimated quercetin absorption to be 17% of orally ingested amount (Hollman et al., 1995). The authors reported that only 0.3% of the oral quercetin dose wa s recovered in urine and concluded that it might be possible that some quercetin accumulated in tissues and was released slowly over time. A recent study in vestigating tissue distribution of quercetin in pigs following long-term dietary supplementation reported that total quercetin concentration in liver was 5 to 6 fold higher than that in plasma (Bieger et al., 2008). Interestingly, 93% of quercetin found in t he liver was in the aglycone form. Taken together, further studies are needed to investigate whether long-term ginkgo or flavonoid-rich supplements may lead to accu mulation of quercetin in human liver to levels that could inhibit m ycophenolic acid glucuronidation.

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57 Incubations with intestinal microsomes exhibi ted 3to 12-fold more potent inhibition of MPAG formation than in liver microsom es by ginkgo extracts, quercetin and kaempferol (Tables 3-1 and 3-2). This diffe rence in inhibition potency can be explained by differentially expressed UGT enzymes in liver and intestine (Ohno and Nakajin, 2009) and the difference in catalytic activiti es towards MPA glucuronidation between liver and intestine microsomes. In this study, microsomal intrinsic clearance ( Vmax/Km) for MPAG formation was 4-fold higher by HLM as compared to HIM (25.12 vs. 6.08 L/min/mg protein). This is in accordance with previously reported values (Bowalgaha and Miners, 2001; Shipkova et al., 2001; Picard et al., 2005). In th e intestine, UGT1A7, 1A8, 1A9 and 1A10 conjugate MPA to MPAG with different affinities, while in the liver, MPAG is selectively formed by UGT1A9 (Picard et al., 2005). In addition, UGT1A10 exhibits a much lower cata lytic activity towards MPA glucuronidation than UGT1A8 and UGT1A9, while its expression in the intesti ne is 13and 25-fold gr eater than UGT1A8 and UGT1A9, respectively (Picard et al., 2005; Ohno and Nakajin, 2009). Due to these differences, interactions may not always transla te from liver to intestinal microsomes with the same magnitude. Therefore, us ing intestinal micr osomes to screen for interactions may be necessary for drugs me tabolized by intestinal glucuronidation. Two limitations are ack nowledged for this study. First, the study does not rule out the possibility of induction of MPA metabolism by ginkgo. A recent study showed that ginkgo and its components induce cytochrome P450 enzymes, transporters, and UGT1A1 (Li et al., 2009). The effect of ginkgo on MPA-metabolizing enzymes in hepatocytes warrants further re search. Second, the stud y did not control for the possible inhibition of UGT activities by fatty acids released fr om the microsomal

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58 membrane, which may inhibit UGT1A9 and re sult in underestimation of inhibition potency (Rowland et al., 2008). Although the effect of released fatty acids on MPA glucuronidation has not been doc umented, it is possible that the actual potency of inhibition is greater than what we observed. Based on our findings, ginkgo supplements taken concomitantly with mycophenolate sodium could lead to increased MPA exposure secondary to inhibition of presystemic glucuronidation. Therefor e, patients should be advised to avoid ginkgo supplements while taking enteric-coated mycophenolate sodiumthe form of MPA that is more subject to presystemic metabolism. Effect of ginkgo on MPA systemic metabolism cannot be predicted, due to lack of informa tion on hepatic concentrations of quercetin and kaempferol, but will likely be weaker than t he presystemic inhibition. MPA is used in HLM as a probe of UGT1A9 activity bec ause of selective formation of MPAG by UGT1A9. Therefore, the obs erved hepatic inhibition would be expected to extrapolate to other UGT1A9 substrates like propofol and fl avopiridol. The actual in vivo effect of this interaction should be verified in clinical studies.

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59 Table 3-1. Inhibition of MPA-7-O-glucuronidation by Ginkgo biloba extracts. Pooled human liver or intestine microsomes (0.16 mg/mL) were incubated with UDPGA (1 mM) and various concentrati ons of ginkgo extracts, and ginkgo compounds. IC50 values and volume/dose index were determined as described under Materials and Methods. A ll incubations were performed in duplicate. Data are expressed as the best-fit IC50 values standard error. Goodness of fit r2 values for the nonlinear regr ession model were > 0.9 for unhydrolyzed and acid-hydrolyzed extracts, quercetin, and kaempferol. IC50 values Mean SE Volume/Dose Index* (L) Extract/ Ginkgo compound HLM HIM HLM HIM Unhydrolyzed G. biloba 84.3 11.6 g/mL 6.8 0.8 g/mL 1.4 17.6 Acid-hydrolyzed G. biloba 20.9 3.6 g/mL 4.3 1.2 g/mL 5.8 27.8 Quercetin 19.1 1.3 M 5.8 0.3 M 0.7 2.2 Kaempferol 23.1 5.5 M 7.6 0.6 M 0.5 1.4 Ginkgolide A > 100 M > 100 M < 0.01 < 0.01 Ginkgolide B > 100 M > 100 M < 0.06 < 0.06 Bilobalide > 100 M > 100 M < 0.03 < 0.03 *Volume/Dose index was calculated by dividing daily dose by the IC50 value (Strandell et al., 2004). Daily dose was considered to be 120 mg ginkgo extract containing (%w/w) 10.75% quercetin, 8.75% kaempferol, 1.2% ginkgolide A, 0.48% ginkgolide B, and 2.94% bilobalide.

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60 Table 3-2. Inhibition of MPA-7-O-glucuron idation by ginkgo flavonoids. Alamethicinactivated pooled human liver or intesti ne microsomes (0.16 mg/mL) were incubated with UDPGA (1 mM), various concentrations of MPA and various concentrations of quercetin or kaempferol. Ki values were determined as described under Materials and Methods. A ll incubations were performed in duplicate. Data are expressed as the best-fit Ki standard error. Inhibitor Ki (M) Mode of Inhibition Quercetin HLM 11.3 1.7 Mixed HIM 2.8 0.4 Mixed Kaempferol HLM 33.6 2.5 Non-competitive HIM 4.5 1.2 Mixed

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61 Figure 3-1. Chemical structures of main ginkgo component s, mycophenolic acid, and MPA-7-O-glucuronide. A) Main bioactive ginkgo components. B) Mycophenolic acid (MPA) and MPA-7-O-glucuronide. B A

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62 Figure 3-2. Effect of Ginkgo biloba extracts on mycophenolic acid 7-O-glucuroni dation in vitro. Alamethicin-activated pooled human liver (panel A) or intestinal (panel B) microsomes (0.16 mg/mL) were incubated with UDPGA (1 mM) and various concentrations of unhydrolyzed (square with solid line) and acid-h ydrolyzed (triangle with dotted line) G. biloba extracts (5, 10, 50, 100, and 500 g/mL). Incu bations were performed using 100 or 70 M MPA for HLM and HIM, respectively. Reactions were stopped after 30 minutes by adding 300 L ice-cold acetonitrile. MPAG was detected by LC-MS/MS as described under Mate rials and Methods. Each point represents the mean of dup licate measurements. B A

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63 Figure 3-3. Inhibition of mycophenolic ac id 7-O-glucuronidation by quercetin and kaempferol. Alamethicin-activated pooled human liver (panels A and B) or intestinal (panels C and D) microsom es (0.16 mg/mL) were incubated with UDPGA (1 mM), various concentrations of MPA, and various concentrations of quercetin (panels A and C) or kaem pferol (panels B and D). Data shown are representative Dixon plots. Each point represents the mean of duplicate measurements. A B C D

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64 CHAPTER 4 INHIBITORY EFFECTS OF COMMONLY USED HERBAL EXTRACTS ON UGT1A1 ENZYME ACTIVITY Introduction According to a recent US government survey, 38% of adults and 12% of children use one or more forms of complementary and al ternative medicine (C AM) (Barnes et al., 2008). Among the various CAM forms, herba l supplements are the most commonly used with sales exceeding $4 billion dollars annually (NBJ, 2007). Moreover, one in four herbal supplement users also take pres cription drugs raising the potential for herbdrug interactions (Eisenberg et al., 1998). There are many examples of herb-drug inte ractions that lead to adverse clinical outcomes such as treatment failu re or serious side effects (Gardiner et al., 2008). Such interactions usually occur through effects of phytochemicals in herbal extracts on the pharmacodynamics or pharmacokinetics of drugs Most pharmacokinetic interactions occur through modulation of drug metabolizin g enzyme activity. Current evidence on herb-drug interactions come mostly from studies on cytochrome P450 enzymes (Izzo and Ernst, 2009). For example, St. Johns wo rt was found to be a strong inducer of CYP3A4, which prompted label changes in pr escribing information for many CYP3A4 substrates. On the other hand, there is a paucity of studies on the effect of herbal supplements on other drug metabolism pathways. Conjugation with glucuronic ac id constitutes a major detoxification and metabolic pathway for numerous endogenous and exogenous compounds, including many drugs and phytochemicals (Ouzzine et al., 2003). Glucuronidation is listed as a clearance mechanism for 1 in 10 of the top 200 drugs (Williams et al., 2004). The UDPglucuronosyl transferase (UGT) enzyme superfam ily is comprised of two families, UGT1

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65 and UGT2. Among UGT1 enzymes, UGT1A1 is an important glucuronidation enzyme that is widely expressed throughout the body, especially in the liver and intestine (Guillemette et al., 2010). Thus, it has an essential role in both first-pass and systemic clearance of many drugs. Variability in UGT1A1 activity has been linked to clinical outcomes in patients taking the anti-cancer drug irinotecan. Patients who carry a variant UGT1A1*28 allele have lower UGT1A1 activity, and therefore, are more prone to neutropenia and diarrhea caused by increased exposure to SN-38, the ac tive metabolite of irinotecan (Schulz et al., 2009). In addition to drugs, many herbal extracts are rich in phenolic phytochemicals that are substrates for UGT1A1 (Doerge et al., 2000; Zhang et al., 2007). Moreover, inhibitory effects of so me herbal constituents (e.g., flavonoids) on UGT1A1 enzymes have been reported in the lit erature (Williams et al., 2002; D'Andrea et al., 2005). Therefore, it is important to identify herbal supplements that may affect UGT1A1 activity and, consequently, alter drug disposition. The aim of this study was to screen commonl y used herbal extracts for inhibition of UGT1A1 activity using human liver microsomes. Estradiol-3O-glucuronidation, which is selectively catalyzed by UGT1A1 in human liver microsomes (Court, 2005), was used as an index reaction for UGT1A1 activity Using a screening approach based on current drug-drug interaction guidelines (FDA, 2006), we identified green tea epigallocatechin gallate (EGCG), milk thistle extract, saw palmetto extract, and Echinacea purpurea extracts as inhibitors of in vitro UGT1A1 activity.

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66 Materials and Methods Chemicals and Reagents -Estradiol, -estradiol-3-( -D-glucuronide) [E-3-G], potassium phosphate dibasic, uridine diphosphate glucuronic acid, magnesium chloride, alamethicin, niflumic acid, and epigallocatechin gallate (EGCG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, ethanol, methanol, and acetone were purchased from Fisher Scientific (Pittsburgh, PA, USA) Herbal extracts (ginseng, Panax ginseng ; echinacea, Echinacea purpurea ; black cohos h, Cimicifuga racemosa; milk thistle, Silybum marianum ; garlic, Allium sativum ; valerian, Valeriana officinalis and saw palmetto, Serenoa repens ) were generously provided by Finzelberg & Co. KG (Andernach, Germany) as dry powder. Table 4-1 summa rizes the properties of the extracts screened. UltraPool human liver microsomes (HLM) were purchased from BD Biosciences Discovery Labware (Woburn, MA USA). These microsomes are pooled from 150 donors to provide lot-to-lot consistency. Preparation of Herbal Working Solutions Concentrations of herbal extracts in screening incubations represent the recommended daily intake (RDI) of each extract dissolved in 53 L, 5.3 L, and 0.53 L. These volumes roughly represent total body fluids, and two extremes of a range of concentrations that could appear in the small intestine, assuming 100% bioavailability, as previously described by Hellum et al. (2 007). For confirmation experiments, a range of concentrations around the rough IC50 of herbal extracts was used in incubations. Working solutions were prepared such that the concentrations were 10-fold higher than that required in incubations. Herbal extr acts were reconstituted with the solvents originally used for extraction and standardization by the vendor (Table 4-1). Insoluble

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67 contents were removed by centrifugation at 20,000 x g for 5 minutes and separation of the liquid supernatant. Solutions were serially diluted to prepare the working herbal extracts, which contained 10% of the extracti ng organic solvents. This way, the organic solvent concentration was the same in a ll incubations and limited to 1%. EGCG working solutions were freshly prepared in 10% methanol and 1.5 mM ascorbic acid, which was added to ensure EGCG stability during the experiment (Lu et al., 2003). All solutions were freshly prepared at the time of the assays. Acid-hydrolyzed ginseng extract was prepared by dissolving 60 mg of the powder extract in 1 mL of 60% ethanol/40% 0.5 N HCl (Sloley et al ., 2006). After 90 minutes at 37o, the extract was neutralized with 0.1 N KOH and then serially diluted to prepare working solutions containing 10% ethanol. In Vitro Incubations A total of nine herbal extracts were screened for inhibition of estradio l-3O glucuronidation. For each experiment, positive and negative control incubations were performed. Niflumic acid (250 M) was used as an inhibitor in positive control incubations. Concentration of organic solv ents and excipients were the same in each set of herbal extract incubati ons and controls in order to nullify their effect on any observed inhibition. Screening experiments us ing three concentrations of each herbal extract were conducted to calculate rough IC50 values. If the rough IC50 estimate was predicted to fall in a concentration range achiev able in vivo in ei ther intestinal or systemic fluid volume, confirmation experim ents using more inhibitor concentrations were performed to obtain a more precise IC50 estimate. In addition to IC50 values, inhibitory potency was also expressed as t he volume per dose index (V/D), which is defined as the volume in which the typical daily dose would be dissolved to obtain the

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68 corresponding IC50 concentration (Strandell et al., 2004) Comparison of this parameter to physiological volumes facilitates an assessment of inhibitory potential. Microsomal incubations were performed as described previously (Alkharfy and Frye, 2002). Briefly, a typical 250 L incubat ion mixture contained HLM (protein concentration, 0.5 mg/mL), alamethicin (30 g/mg microsomal protein), MgCl2 (1 mM), -estradiol (25 M), and different concentrati ons of each test extract or test compound in 100 mM phosphate buffer, pH 7. 4. Microsomes were preincubated with alamethicin on ice for 5 minutes to activate UGT enzym es. The reaction was started by adding UDPGA (6 mM) and placing incubati on tubes in a water bath at 37oC for 30 minutes. The reaction was stopped by adding 25 L of 6% perchloric acid. Tubes were vortexmixed for two minutes and centrifuged for 10 min at 20,000 x g 75 L of the supernatant was injected into the HPLC system. HPLC Analysis E-3-G formation was measured using an HP LC system consisting of a Shimadzu LC10AD VP pump (Shimadzu Scientific Instrum ents, Columbia, MD, USA) conn ected to a Waters 717 autosampler and Waters 2475 florescence detector (Waters Corporation, Milford, MA, USA). The HPLC method us ed has been described previously (Alkharfy and Frye, 2002). Briefly, the mobile phase consisted of 35% acetonitrile and 65% 50 mM ammonium phosphate buffer (p H 3); the flow rate was 1 mL/min delivered through an Alltima phenyl column, 5 4.6 mm (Grace Davison, D eerfield, IL, USA). E-3-G was detected at an excitation wavelength of 210 nm and an emission wavelength of 300 nm. The assay was linear over the concentration range of 20 to 4000 pmol; the intraand inter-day coefficients of variation were <6%.

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69 Data Analysis Remaining enzyme activity was expressed as a percent of control and estimated from the ratio of E-3-G peak area in herbal extract incubations relative to that in negative control incubations. Where possible, IC50 values were determined by fitting the remaining enzyme activity and inhibitor c oncentration data to equation 4-1 using Prism 5.02 (GraphPad Software, San Diego, CA, USA). H H HIIC I Y 50100 100 (4-1) ( Y : Remaining UGT1A1 activity (percent of control), [ I ]: Concentration of herbal extract, H : Hill coefficient) In addition to IC50, a volume per dose index was ca lculated as described in equation 4-2 and was used to determine the potential for in vivo inhibition. The volume per dose index is defined as the volume in which one dose would be dissolved to obtain the corresponding IC50 concentration as described by Strandell et al. (2004). 50 (L)index Inhibition IC RDI (4-2) ( RDI: recommended daily intake ) A volume per dose index cutoff value of 2.0 L/ dose was used to select extracts for more detailed characterization of the IC50 value. Results Screening Experiments: Eight herbal extracts, ginseng, acid-hydro lyzed ginseng, echinacea, black cohosh, milk thistle, valerian, saw palmetto, and EGCG inhibited 3Oglucuronidation of estradiol by human liver microsomes in a concentra tion-dependent manner (Figure 1). EGCG

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70 completely inhibited E-3-G formation at a concentration of 500 g/mL. Echinacea, milk thistle, saw palmetto, and EGCG were se lected for confirmatory experiments to determine more precise IC50 values because the volume/dose index exceeded 2.0 L/dose (Table 4-2). Confirmatory Experiments and Determination of Precise IC50 Values: EGCG exhibited the most potent inhibiti on of all extracts tested with best-fit IC50 value of 7.8 0.9 g/mL. IC50 values for inhibition of UGT1 A1 activity by echinacea, milk thistle, and saw palmetto were 211.7 43.5, 30.4 6.9, and 55.2 9.2 g/mL, respectively (Table 4-3, Figure 4-2). Goodness of fit r2 values were > 0.9 for all IC50 curves. When recommended daily dose of eac h extract was taken in perspective, volume per dose index values were 1.9, 19.7, and 5.8 L for echinacea, milk thistle, and saw palmetto, respectively. For EGCG, the volu me per dose index was 32.1 L, which is the highest value of all extrac ts tested indicating the highes t potential for inhibition. Discussion The extracts screened in this study ar e among the most commonly used herbal extracts in the US and the world. In this study, we investigated their potential for interactions with glucuronidation by UGT1 A1. All screened extracts, except garlic, exhibited concentration-dependent inhibition towards UGT1A1 ac tivity. Only four herbal extracts (echinacea, milk thistle, saw palmetto, and green tea polyphenol EGCG) exhibited inhibition with IC50 values achievable in vivo, par ticularly in the intestine. IC50 as well as volume per dose index values in dicate that echinacea and saw palmetto are expected to be mild to moderate inhibitors compared to EGCG and milk thistle. The latter two are the focus of this discussion.

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71 Green tea and milk thistle are among the most commonly used herbal supplements with 2006 sales in the US of $144 million for green tea and $93 million for milk thistle (NBJ, 2007). Studies have show n that green tea has promising anticancer, antioxidant, weight loss, and vascular protective benefits (Nagle et al., 2006). Green tea extract is rich in the polyphenolic compounds named catechins. Among these, EGCG is considered the most abundant and is suggested to be the main mediator of most of the biological effects attributed to green tea (Moore et al ., 2009). We used the green tea catechin EGCG instead of green tea extract to avoid precipitation of microsomal proteins by tannins present in green tea, which could yield misleading results as described previously (Butterweck and Derendorf, 2008). In order to interpret the potent ial of the observed in vitro inhibition to translate in vivo, IC50 values should be considered in the contex t of expected in vivo concentrations. In pharmacokinetic studies of green tea extrac ts, EGCG maximum plasma concentrations were as high as 2.5 g/mL following inges tion of a single dose of green tea extract (Polyphenon E) containing 800 mg EGCG (Fos ter et al., 2007). This concentration is less than the observed IC50 for UGT1A1 inhibition by EG CG (7.8 g/mL); therefore, inhibition of systemic clearance of UGT1A1 substrates seems unlikely. Conversely, intestinal concentrations of EGCG can reach higher levels than the IC50 value. Based on an intestinal fluid volume of 0.5 to 5.0 L (Hellum et al., 2007), EGCG concentrations are expected to fall in the range of 40 to 1600 g/mL following consumption of a green tea extract dose containing 200 to 800 mg EGCG. Considering that the IC50 for inhibition by EGCG was 7.8 g/mL (Table 4-3), inhibition of intestinal metabolism

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72 appears plausible if green tea is consumed concomitantly with a UGT1A1 drug substrate. Zhu et al. (Zhu et al., 1998) previously r eported that green tea polyphenols, including EGCG, inhibited estradiol and estrone gl ucuronidation in vitro using rat liver microsomes, which is consistent with the resu lts of the current st udy using human liver microsomes. In addition to reproducing their findings in HLM, we measured E-3-G formation as a selective probe for UGT1A1 acti vity as previously reported (Court, 2005). Milk thistle extract is used by 30 to 40% of liver disease patients for its hepatoprotective benefits (Schrieber et al., 2008). Plasma concentration of total flavonolignans, the major constituents in milk thistle, was 24 ng/mL following ingestion of 600 mg milk thistle extract (Schrieber et al ., 2008). Again, this concentration is much less than the observed IC50 for UGT1A1 inhibition by milk thistle (30.4 g/mL; equivalent to 11.5 g/mL flavonolignans; Table 4-3). In agreement with this conclusion, van Erp et al. (2005) reported no effect of milk this tle on the pharmacokinetics of irinotecan, an intravenous anticancer drug and substrate for UGT1A1. Fo r comparison, the expected intestinal concentration of milk thistle ex tract is 40 to 1200 g/mL following ingestion of 200 to 600 mg milk thistle supplement. Sim ilar to EGCG, these concentrations are higher than the IC50 for UGT1A1 inhibition by milk this tle. Therefore, inhibition of intestinal glucuronidation of UGT1A1 substr ates by milk thistle may be possible. In this study, we opted to use HLM rather than expressed UGT1A1 or human intestine microsomes (HIM). Compared with HLM, expressed enzymes do not mimic the in vivo environment in terms of availab ility of other UGT enzymes, which may form heterodimers in vivo. Formation of UGT heterodimers has been described in the

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73 literature and may affect enzyme activity (Ouzzine et al., 2003). HIM were not used because estradiol-3Oglucuronide formation is not sele ctively catalyzed by UGT1A1 in HIM as other enzymes present in the intest ine (e.g. UGT1A8 and UGT1A10) also form this conjugate (Lepine et al., 2004). To our knowledge, no substrate selective for UGT1A1 in HIM has been identified. T hus, utilization of HL M with estradiol-3Oglucuronidation as an inde x reaction provides better specificity for UGT1A1. Nevertheless, V/D values for inhibition by EGCG and milk thistle suggest that UGT1A1 inhibition in the intestine may be more clinically relevant. Intestinal UGT1A1 plays an im portant role in the first pa ss glucuronidation of drugs such as raloxifene and ezetimibe (Fis her and Labissiere, 2007). For raloxifene, bioavailability is only 2% and most of the oral dose is cleared by intestinal glucuronidation, mainly by UGT1A1, 1A8, and 1A10 (Kemp et al., 2002). Inhibition of one or more of these enzymes may enhance ra loxifene bioavailability resulting in increased exposure and increased risk for side effects such as deep vein thrombosis and pulmonary embolism (Cummings et al., 1999). Similarly, intestinal glucuronidation of ezetimibe plays an important role in mediating the pharmacological action of the drug, since ezetimibe glucuronide is more potent t han the parent drug in inhibiting cholesterol absorption (Ghosal et al., 2004) Thus, inhibition of inte stinal glucuronidation may increase systemic exposure to ezetimibe an d could affect the therapeutic response. The Hill coefficients for the best-fit curve for milk thistle and echinacea were smaller than unity (0.5 for milk thistle and 0.6 for echi nacea; Table 4-3; Figure 4-2). This finding may be explained by the atypical nature of UGT1A1 kinetics observed for estradiol, possibly because more than one ligand molecule binds to the enzyme at the same time

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74 (Alkharfy and Frye, 2002). Another expla nation can be poor aqueous solubility of one or more milk thistle or echinacea components that restricts inhibitor accessibility at higher concentrations (Copeland, 2005). Based on our findings, supplements of green t ea, milk thistle, and to a lesser extent, echinacea and saw palmetto, may inhibit glucuronidation of substrates by UGT1A1, particularly in the intestine. These findings suggest interactions of these supplements with UGT1A1 substrates are possible. Future clinical studies are warranted to evaluate the in vivo pharmacokinetic relevance of these interactions.

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75 Table 4-1. List of Herbal extracts investigated for effect on UGT1A1. Extract Scientific Name of Origin Percent of Key Components (w/w)* Solvent** Black Cohosh Cimicifuga racemosa 5 % Total Triterpenglycosides 50% ethanol Echinacea root Echinacea purpurea 3% Cichoric acid 60% ethanol Garlic bulb Allium sativum 3.25 % Allin 80% methanol Ginseng root Panax ginseng 5% Total Ginsenosides 60% ethanol Milk Thistle herb Silybum marianum 37.9% Total Silymarin flavonolignans 80% acetone Saw Palmetto fruit Serenoa repens >85% Total fatty acids > 0.1 Sterols 96% ethanol Valerian root Valeriana officinalis 0.1 Valerenic acids 70% ethanol Epigallocatechin gallate (EGCG) Camellia sinensis > 97% EGCG 100% methanol *Values provided by manufacturer. **Used by manufacturer for standardization

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76 Table 4-2. Rough IC50 and volume/dose index values for inhibition of estradiol-3O glucuronidation by nine herbal extracts Estradiol was incubated with pooled HLM and three concentrations of each extract. Rough IC50 values were calculated by fitting IC50 equation (Materials and me thods) to percent of activity remaining using non-linear regre ssion. Values reported are best-fit IC50 standard error. A ll reported values had r2 values for goodness of fit of at least 0.9. Volume/dose index val ues were calculated as described under (Materials and methods). Recommended daily intake (RDI) values were determined based on PDR for Herbal Medicine (Gale Group., 2001) and commercially available products. Extract RDI (mg) Rough IC50 (g/mL) Volume/Dose Index (L/dose) Ginseng 550 602.5 225.6 0.9 Acid-hydrolyzed Ginseng 550 NA NA Echinacea 400 166.6 68.3 2.4* Black Cohosh 40 298.5 18.5 0.1 Milk Thistle 600 18.0 6.8 33.3* Garlic 1000 -** -** Valerian 1000 561.9 59.0 1.8 Saw Palmetto 320 51.71 8.8 6.2* Epigallocatechin gallate (EGCG) 250 7.6 0.7 32.9* NA: Data points did not fit IC50 curve indicates volume per dose values exceeding 2.0 L ** indicates no inhibition observed

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77 Table 4-3. Precise IC50 values for herbal extracts showing strongest inhibition of estradiol-3O -glucuronidation. Estradiol was incubated with pooled HLM and a range of concentrations of each extr act as described under (Materials and methods). Data are reported as best-fit IC50 values standard error. Goodness of fit r2 values for the nonlinear r egression model were > 0.9. H is Hill coefficient describing the degree of sigmoidicity of the best-fit curve. Recommended daily intake values (RDI ) were determined based on PDR for Herbal Medicine (Gale Group., 2001) and commercially available products. Extract RDI (mg) IC50 (g/mL) Volume/Do se Index (L/dose) H Echinacea 400 211.7 43.5 1.9 0.6 Milk Thistle 600 30.4 6.9 19.7 0.5 Saw Palmetto 320 55.2 9.2 5.8 1.2 Epigallocatechin gallate (EGCG) 250 7.8 0.9 32.1 0.8

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78 Figure 4-1. Effect of herbal extracts on E-3-G formation as an index for UGT1A1 activity in HLM. Estradiol was incubated with pooled HLM and three concentrations of each extract. Three concentrations were tested for each herbal extract which represent extract daily intake in 53 L (striped bars), 5.3 L (dotted bares), and 0.53 L (checkered bar s). The dotted line represents 50% activity of control. ND indicate s no metabolite detected, which was considered as 100% inhibition. E rror bars represent SE of the mean of duplicate incubations.

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79 Figure 4-2. Inhibition of E3-G formation by herbal extrac ts. Estradiol was incubated with HLM and different concentrations of a) green tea catechin ECGC, b) milk thistle extract, c) saw palmetto ex tract, and d) Echinacea purpurea root extract. Data points repr esent remaining UGT1A1 ac tivity as percent of control incubations. Data points were fitted to non-linear regression equation as explained under (Materials and methods). Error bars represent SE of the mean of duplicate incubations. A B C D

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80 CHAPTER 5 INHIBITORY EFFECTS OF EPIGALLOCATE CHIN GALLTE ON RALOXIFENE IN VITRO CLEARANCE Introduction Raloxifene is a selective es trogen receptor modulator that is commonly used in postmenopausal women to prevent and treat osteoporosis and to reduce the risk of invasiv e breast cancera leading cause of death of women in the US (Moen and Keating, 2008). The pharmacokinetics of raloxifene exhibits high interand intraindividual variability with co-efficients of variation of 30% for most pharmacokinetic parameters (Raloxifene package insert: http://pi.lilly.com/us/evista-pi.pdf ). Although 60% of a raloxifene dose is absorbed, only 2% of the oral dose reaches the systemic circulation ( Moen and Keating, 2008). This poor bioavailability is attributed to extensive first-pass glucuronidation by intestinal, and to a lesser extent, hepatic UGT enzymes (Dalvie et al., 2008; Cubitt et al., 2009). Therefore, inhibition of raloxifene glucuronidation in the intest ine by concomitantly taken drugs or herbal supplements may increase bioavailability several-fold, whic h in turn may increase risk for the rare but serious thromboembolic events associated with raloxifene use (Cummings et al., 1999). Green tea ( Camellia sinensis) is one of the most commonly used beverages and herbal supplements in the world, with US sales of $144 million dollars in 2006 (NBJ, 2007). In addition, consumption of green tea has been reported to possess beneficial health effects, including improving card iovascular function and anticancer effects (Kohlmeier et al., 1997). Green tea extract is rich in polyphenolic compounds called catechins. The primary green tea catechin is epigallocatechin gallate (EGCG), which like raloxifene is a substrate for intestinal UGT enzymes (Lu et al., 2003). Since green

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81 tea is being studied for its protective effects against cancer, there is a potential that it might be used concomitantly in patients taking raloxifene. The effect of green tea on intestinal glucuronidation of ra loxifene has not been studied. We previously showed that EGCG potently inhibits UGT1A1, which is involved in the glucuronidation of raloxifene. The aim of this study was to investigate the effect of EGCG, the most abundant constituent in gr een tea, on raloxifene intrinsic clearance using a substrate depletion approach. In vitr o intrinsic clearance calculated using this method has been shown to be in good correlation with in vivo intrinsic clearance (Cubitt et al., 2009). Materials and Methods Chemicals and Reagents Tris(hydroxymethyl)aminomethane hydrochl oride (Tris-HCl), uridin e diphosphate glucuronic acid (UDPGA), magnesium chloride, alamethi cin, and epigallocatechin gallate (EGCG; 97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were purchased from Fi sher Scientific (Pittsburgh, PA, USA). Raloxifene and raloxifene-d4 were purchased from Toronto Research Chemicals (North York, ON, Canada). Human inte stine microsomes (HIM), pool ed from five donors, were purchased from BD Biosciences Di scovery Labware (Woburn, MA, USA). Incubations with HIM Raloxifene and raloxifene-d4 st ock solutions were prepared in methanol. Due to its light sensitivity, raloxif ene-d4 solution was k ept in dark at all times. EGCG working solution was freshly prepared at the time of the experiment and contained 10% methanol and 1.5 mM ascorbic acid to increase its stability in aqueous medium (Lu et al., 2003). Raloxifene depleti on assay was performed as de scribed previously with

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82 modifications (Cubitt et al., 2009). Incubation mixture contained raloxifene (1M), EGCG at concentrations of 0, 10, 50, and 100 M, 0.15 M ascorbic acid, 5 mM MgCl2, 0.1 M potassium phosphate buffer (pH 7.4), HI M (0.1 mg/mL), alamethicin (50 g/mg protein). The mixture was incubated on ice fo r 15 minutes. The reaction was started by adding UDPGA (final concentration: 5 mM ) to the mixture and 100-L sample was immediately removed for the zero-time val ue. 100 L samples were taken from the mixture at 0, 1, 5, 10, 15, 20, and 30 minutes and mixe d with 100 L acetonitrile and 20 L internal standard (raloxifene d4), vortex-mixed, and placed on ice in the dark. Tubes were centrifuged at 20,000 x g for 10 minutes. The supernatant was transferred to autosampler vials for injection. Concentrati on of methanol in all incubations was 1%. HPLC-MS/MS Assay of Raloxifene Raloxifene was assayed by LC/MS/MS on a ThermoFin nigan Surveyor series HPLC system connected to a TSQ Quantum trip le quadrupole mass spectrometer (Thermo Corp., San Jose, CA, USA) using electrospray ionization (ESI). Briefly, 5 L of each sample was injected on a reversed-phase Phenomenex (Torrance, CA, USA) Synergi Max-RP column (75 2 mm, 4 m). The mobile phase consis ted of (A) 1% formic acid in deionized water and (B) 1% fo rmic acid in acetonitrile. Gr adient elution at a flow-rate of 0.2 mL/min was employed wi th the following steps: at star t of the run, 20% B for one min, then increased to 80% B in 0.5 min, he ld at 80% B between 1.5 and 4.0 min, and from 4.5 to 6.0 min, the co lumn was re-equilibrated at 20% B. Analysis was carried out in the single reaction monitoring (SRM), posit ive ion mode using the mass transitions of m / z 474 112 and m / z 478 112 for raloxifene and raloxifene-d4, respectively. Retention time for both raloxifene and internal standard was 3.4 minutes.

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83 Estimation of Non-sp ecific Protein Binding The free fraction of raloxi fene in the incubation (fuinc) was estimated using the Hallifax-Houston equation 5-1 (Hal lifax and Houston, 2006), where C is protein concentration in milligrams per milliliter (0.1 mg/mL). The raloxi fene LogP value used was 6.2, which was calculated using Molin spiration-Interactive logP calculator ( http://www.molinspiration.com/services/logp.html ) as explained by Zhou et al.(2010). 126.1log067.0)(log072.0 inc10 2 10101 1 fu P PC (5-1) Data Analysis The fraction of raloxifene remaining in the incubation was calculat ed from the ratio of raloxifene to internal standard peak areas at different time points compared to the ratio at time zero. Elimination half life was det ermined by fitting the data from the mean of two incubat ions to a nonlinear exponential one-phase decay using Prism 5.02 (GraphPad Software, San Diego, CA, USA). In vitro CLint, u (L/min/mg) was calculated using equation 5-2 (Cubitt et al., 2009). inc 1/2 int,fu (mg) incubation in protein microsomal ofamount L)( volume incubation t vitro 0.693 in CLu (5-2) Results and Discussion Calculated fu of raloxifene was 0.08 and CLint, u was 3680 L/min/mg protein in the absence of EGCG; both values agree with prev iously reported values (Cubitt et al., 2009). Figure 1 shows the effect of addi ng different concentra tions of EGCG on raloxifene CLint, u. In HIM incubations, EGCG i nhibited raloxifene in vitro CLint, u by 76%, 86%, and 100% at concentrations of 10, 50, and 100 M, respectively (Table 5-1, Figure 5-1). To compare these concentra tions to putative in vivo intestinal

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84 concentrations, EGCG content in green tea supplements and an estimate of intestinal fluid volume of 0.5 to 5.0 L were consi dered. Most green tea supplements contain about 250 mg of EGCG. Therefore, based on esti mates of intestinal fluid volume that range from 0.5 and 5 L (Hellum et al., 2007), intestinal EGCG concentrations would be expected to be 100 to 1000 M. Moreover, doses of EGCG reaching up to 800 mg have been used in clinical studies, which will yield putative EGCG intestinal concentrations of 320 to 3200 M. These concentrations are much higher than the concentrations used in the incubations. Ther efore, based on the observed inhibition it is likely that green tea supplements will inhibit ra loxifene intestinal glucuronidationthe primary factor limiting raloxi fene bioavailability. The pha rmacokinetic consequences of this interaction warrant further studies.

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85 Table 5-1. Effect of green tea EGCG on ra loxifene in vitro intrinsic clearance using HIM. CLint,u was determined using equation 5-2 as explained under Materials and Methods. EGCG Concentration (M) CLint,u (L/min/mg protein) Percent of control Control 3680 100 10 M 899.4 24.4 50 M 557.5 15.7 100 M 0.0 0.0 Figure 5-1. Effect of green tea EGCG on ra loxifene in vitro intrinsic clearance using HIM. Raloxifene depletion assay was performed by incubating 1 M raloxifene with 0.1 mg/mL alamethicin-acti vated HIM and 0, 10, 50, or 100 M EGCG. 100 L samples were taken at various time points to estimate the remaining raloxifene fraction compared to time zero sample. Data points are mean values of duplicate incubations st andard error. The lines present the best-fit curves for the exponential one phase decay model by Prism 5.02 (GraphPad Software, San Diego, CA, USA).

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86 CHAPTER 6 INHIBITORY EFFECTS OF COMMONLY USED HERBAL EXTRACTS ON UGT1A4, 1A6, AND 1A9 ENZYME ACTIVITIES Introduction Conjugation of compounds with glucuronic acid represents a major disposition pathway for endogenous and exogenous compounds, including drugs and phytochemicals. Human glucuronidation enzymes (UDP-glucuronosyltransferases; UGT) are divided into two families, UG T1 and UGT2, which encompass more than 18 enzymes (Tukey and Strassburg, 2000). UGT1A4, UGT1A6, and UGT1A9 enzymes belong to the UGT1 family and conj ugate a wide spectrum of drugs and phytochemicals. UGT enzymes are differentially expressed in tissues, with liver and intestine being the main sites for drug gl ucuronidation (Tukey and Strassburg, 2000). Substrates for UGT1 enzymes include many drugs (e.g. mycophenolic acid, trifluoperazine, tamoxifen, lamotrigine, and acetaminophen) and phytochemicals (e.g. quercetin, kaempferol, epigallocatechin gallate) (Oliveira and Watson, 2000; Lu et al., 2003; Kiang et al., 2005). Since these phytochemicals share UGT1 metabolic pathway(s) with drug substrates, there is a potential for herb-dr ug interaction through modulation of this pathway. We previously reported that Ginkgo biloba extract and its polyphenolic compounds quercetin and kaempferol inhibit UGT1A9 (Chapter 3). The aim of this study was to identify other pot ential herb-UGT interactions through screening commonly used herbal extracts for inhibitory effects on the activities of UGT1A4, UGT1A6, and UGT1A9. Recent surveys estimate that 38% of Am ericans use complementary and alternative medicine, which includes herbal supplemen ts (Barnes et al., 2008). However, the physiologic and metabolic effects of herbal s and phytochemicals are often poorly

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87 understood. One of the issues of concern to clinicians is the potential for herb-drug interactions, which may lead to poor clinical outcomes (Gardiner et al., 2008). Several case studies have described deleterious herb-drug interactions that can lead to morbidity or even mortality (Ruschitzka et al., 2000; Kupiec and Raj, 2005). Consequently, much attention has been given to investigating the effects of herbal supplements on cytochrome P-450 enzymes, the primary metabolic route for the majority of marketed drugs (Izzo and Ernst, 2009). In contrast, research is lacking regarding the potential of herbals to alter other metabolic routes including glucuronidation. Identification of selective substrates for UGT enzymes allows scr eening of herb-UGT interactions using human liver microsom es. Trifluoperazine, serotonin, and mycophenolic acid were reported to be select ive in vitro probe substrates for UGT1A4, UGT1A6, and UGT1A9, respectively (Cour t, 2005). In this study, formation of trifluoperazine glucuronide, serotonin gluc uronide, and mycophenolic acid phenolic glucuronide were used as index reactions for UGT1A4, 1A6, and 1A9 enzymatic activities, respectively. Materials and Methods Chemicals and Reagents Trifluoperazine (TFP; 99%), serotonin ( 98%), potassium phosphate dibasic, tris(hydroxymethyl)aminomethane hydrochl oride (Tris-HCl), uridine diphosphate glucuronic acid (UDPGA), -glucuronidase, magnesium chlo ride, bovine serum albumin (BSA), alamethicin, niflumic acid, and epigallocatechin gallate (EGCG; 97%) were purchased from Sigma-Aldrich (St. Louis, MO USA). Acetonitrile, ethanol, methanol, acetone, hecogenin acetate (93% ), and 1-naphthol (> 99%) were purchased from Fisher

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88 Scientific (Pittsburgh, PA, USA). SerotoninO -D-glucuronide was provided by RTI International (Research Triangle Park, NC) through the NIMH Chemical Synthesis Program. Mycophenolic acid (M PA; 98%), mycophenolic acid -D-glucuronide (MPAG; 98%), and mycophenolic Acid-d3-D-glucuronide (MPA-d3-G; 98%) were purchased from Toronto Research Chemicals (North York, ON, Canada). Her bal extracts (black cohosh, Cimicifuga racemosa; cranberry, Vaccinium marocarpon echinacea, Echinacea purpurea ; garlic, Allium sativum ; ginkgo, Ginkgo biloba ; ginseng, Panax ginseng; milk thistle, Silybum marianum ; saw palmetto, Serenoa repens ; and valerian, Valeriana officinalis ) were generously provided by Fi nzelberg & Co. KG (Andernach, Germany) as dry powder. Table 6-1 summa rizes the properties of the extracts screened. UltraPool human liver microsomes (HLM) were purchased from BD Biosciences Discovery Labware (Woburn, MA, USA). These microsomes were pooled from 150 donors providing lo t-to-lot consistency. Preparation of Herbal Working Solutions Herbal extracts were reconstituted with t he solvents originally used for extraction and standardiz ation by the vendor (Table 6-1). In order to remove any insoluble contents, the mixture was centrifuged at 20,000 x g for 5 minutes and the liquid supernatant was removed. Working solutions were freshly pr epared so that final herbal concentrations in screening incubations would repr esent the recommended daily intake of each extract in 53 L, 5.3 L, and 0.53 L. These volumes roughly represent total body fluids, and two extremes of a range of concentrations that c ould appear in the small intestine, assuming 100% bioavailability as previously described by Hellum et al (2007). For confirmation experiments, a range of conc entrations around the rough IC50 of herbal extracts was used in incubations. Concentration of organic solvents in incubations was the same in

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89 all incubations including controls and was li mited to 1%. For EGCG, working solutions were freshly prepared in 10% methanol and 1.5 mM ascorbic acid, which was added to ensure EGCG stability during the experiment (Lu et al., 2003) Acid-hydrolyzed ginseng extract was prepared by dissolving 60 mg of the powder extract in 1 mL of 60% ethanol/40% 0.5 N HCl (Sloley et al ., 2006). After 90 minutes at 37o, the extract was neutralized with 0.1 N KOH and was serially diluted to prepare working solutions containing 10% ethanol. Acid-hydrolyzed ginkgo extract was prepared by dissolving 30 mg of the powder extract in 1 mL of 60% ac etone/40% 5N HCl. The acid treated extract was heated at 90oC for one hour and neutralized with 2N KOH. Working solutions were prepared so that their concentrations were 10-fo ld higher than the final concentrations in incubations. Incubations of Herbal Extracts with TFP TFP was used as a probe substrate for UGT1A4 in HLM. Incubations with TP F were performed as described previously by Uchai pichat and coworkers (2006). Briefly, the incubation mixture (final volume, 250 L) consisted of TFP, 5 mM MgCl2, 50 mM TrisHCl buffer (pH 7.4), 0.1 mg/mL microsomal proteins, and alamethicin (100 g/mg protein). Concentration of TFP in inc ubations was 60 M, which corresponds to the Km in HLM (Uchaipichat et al., 2006). The mixtur e was pre-incubated on ice for 15 minutes. The reaction was started by adding UDPGA (final concentration, 5 mM). After the mixture was incubated for 20 min at 37oC, the reaction was stopped by adding 250 L (4% Acetic acid/96% Methanol), vortex-mixi ng, and placing tubes on ice. Tubes were centrifuged for 10 min at 20,000 x g and the supernatant was transferred to HPLC tubes. Screening experiments were perfo rmed by adding herbal extracts at three different concentrations to the incubation mixture. In cubations with and without

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90 hecogenin (50 M) were performed to se rve as positive and negative controls, respectively. Chromatographic Analysis of TFP glucuronide (TFPG) HPLC analysis was performed wi th a Shimadzu LC-10AD VP pump (Shimadzu Scientific Instruments, Co lumbia, MD, USA) connected to a Waters 717 autosampler and Waters 2475 florescence detector (Waters Corporation, Milfor d, MA, USA). 50 L of the incubation supernatant was in jected on a reversed-phase Phenomenex Luna Phenyl-Hexyl column (2 x 100 mm, 3 m). Is ocratic chromatography was carried out at ambient temperature using a m obile phase consisting of 0.1% tri-fluoroacetic acid in acetonitrile: deionized water (30:70) at a flow-ra te of 0.2 mL/min. T he total run time was 15 min. TFPG was detected at an exci tation wavelength of 310 nm and emission wavelength of 475 nm (Rele et al., 2004). The identity of the TFPG peak was veri fied through enzymatic hydrolysis using glucuronidase. 60 M TFP was incubated with HLMs as described above for 1 hour at 37oC. Then, 25 L of 100 mM potassium phosphat e buffer (pH 4.0), and 2,500 units of -glucuronidase were added. Tubes were incubated for 16 hours at 37o C. The reaction was stopped by adding 10 L 70% HClO4, vortex-mixing and centrifugation at 20,000 x g for 10 minutes. The supernatant was transferred to HPLC tubes for injection. Control incubations were perfo rmed in the same way but did not contain glucuronidase enzyme. The TFPG peak was detected in the control incubation but not in the hydrolyzed one. Incubations of Serotonin with Herbal Extracts To investigate the effect of herbals on UGT1A6 activity, incubations of herbal extracts with HLM were performed using serotonin as a probe substrate as described by

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91 Krishnaswamy and coworkers with modifications (2003). Briefly, t he incubation mixture (final volume, 100 L) consisted of serotonin at a concentration around Km value in HLM (8 mM), 5 mM MgCl2, 50 mM Tris-HCl buffer (pH 7.4), 0.5 mg/mL microsomal proteins, and alamethicin (100 g/mg protein). The mixture was pre-incubated on ice for 15 minutes. The reaction was started by adding UDPGA (final concentr ation, 5 mM). After the mixture was incubated for 60 min at 37oC, the reaction was stopped by adding 10 L 24% perchloric acid: acetonitrile (1:1, v/v), vortex-mixing, and placing tubes on ice. Tubes were centrifuged for 10 min at 20,000 x g and the supernatant was transferred to HPLC tubes. 1-naphthol (50 M) was used as a positive control inhibitor in the screening assays. Chromatographic Analysis of Serotonin Glucuronide Isocratic chromatography was carried out at ambient temperature on a reversedphase Waters C18 Symmetry column (3.9 x 150 mm, 5 m). The mobile phase consisted of 5% acetonitrile / 95% 2 mM a mmonium acetate (pH 2.7) Isocratic elution at flow-rate of 1.0 mL/min was employed. The total run time was 10 min and the injection volume was 30 L. The HPLC system consisted of a Shimadzu LC-10AD VP pump (Shimadzu Scientific In struments, Columbia, MD, USA) connected to a Waters 717 autosampler and Waters 2475 florescence det ector (Waters Corpor ation, Milford, MA, USA). Serotonin glucur onide was detected at an excitation wavelength of 225 nm and emission wavelength of 330 nm. To conf irm the identity of serotonin-glucuronide peak, retention time was compared to sero tonin glucuronide standard. In addition, serotonin glucuronide peak was collected from the HPLC eluate and analyzed using MS/MS. The isolated fraction showed abundant ions with m / z 353, which matches the m / z of serotonin-glucuronide ions in the pos itive mode. Upon fragmentation of the

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92 parent ion, a product ion with m / z 177 was produced, which matches the expected breakdown of the conjugate into gluc uronic acid and free serotonin. Incubations of MPA with Herbal Extracts Incubations with MPA were performed as de scribed previously with modifications (Chapter 2). Briefly, the incubation mixture (100 L) contained HLM (protein concentration, 0.16 mg/mL), alamet hicin (100 g/mg protein), MgCl2 (5 mM), 2% BSA, and 100 mM phosphate buffer, pH 7.4. MPA was used at a concentration equivalent to the Km value in HLM (240 M). Microsomes were pre-inc ubated on ice with alamethicin for 15 minutes. The reaction was started by adding UDPGA (1 mM) and placing incubation tubes in a water bath at 37o C for 30 minutes. The reaction was stopped by adding 300 L of ice-cold acetonitrile and 20 L of internal stan dard (20 g/mL MPA-d3G). Tubes were vortex-mixed for two minutes and centrifuged for 10 min at 20,000 x g The supernatant was diluted 12-fold with pur ified water and 5 L was injected into the HPLC system. Incubations of MPA with niflum ic acid (70 M) were used as positive controls. MPAG LC-MS/MS Assay MPAG was determined by LC/MS/MS on a T hermoFinnigan Surveyor series HPLC system connected to a TSQ Quan tum trip le quadrupole mass spectrometer (Thermo Corp., San Jose, CA, USA) using electrospray ionization (ESI), as described previously (Chapter 2). Briefly, 5 L of each sa mple was injected on a reversed-phase Phenomenex (Torrance, CA, USA) Synergi Fusion-RP18 column (100 2 mm, 4 m). The mobile phase consisted of (A) 1 mM acetic acid in deionized water and (B) 1 mM acetic acid in acetonitrile. Gradient eluti on at a flow-rate of 0.22 mL/min was employed with the following steps: at start of the run, 30% B for one mi n, then increased to 90% B

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93 in 0.75 min, held at 90% B between 1.75 and 3.1 min, and from 3. 6 to 6.5 min, the column was re-equilibrated at 30% B. A nalysis was carried out in the single reaction monitoring (SRM), negative ion m ode using the mass transitions of m / z 495 319 and m / z 498 322 for MPAG and MPA-d3-G, respectively. Data Analysis Remaining enzyme activity was calculated from the peak area of the glucuronide metabolites formed in herbal extract incubati ons expres sed as a percent of control. Remaining enzyme activity and herbal extract concentration data were fitted to equation 4-1 using Prism 5.02 (GraphPad Software, San Diego, CA, USA) to estimate IC50 values. Volume per dose (V/D) index was calculat ed using equation 6-1 and was used as a measure of the potential of IC50 concentrations to be reached in vivo as described by Strandell et al. (2004). The V/D index is defined as the volume in which one dose should be dissolved in order to obtain the corresponding IC50 concentration. 50 (L)index e Volume/Dos IC RDI (6-1) ( RDI: recommended daily intake ) Results A total of 35 herb-UGT enzyme pairs were evaluated, each at three different concentrations. Results from the screening experiments are su mmarized in Table 6-2. Rough IC50 and V/D index values were estimat ed based on remaining enzyme activity data at the three concentrations of each herbal extract. V/D index was used to select the herb-UGT interactions to investigate furt her. A V/D cutoff value was considered to be 5 L for UGT1A4 interactions and 2 L for UGT1A6 and 1A9 interactions. This was

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94 based on an expression study that showed t hat UGT1A6 and 1A9 are expressed in the intestine and the liver while UGT1A4 is mainly expressed in the liver (Ohno and Nakajin, 2009). Herbal extracts that showed inhi bition of a UGT enzyme with V/D values exceeding the specific cutoff value in the screening experiments were studied further in confirmatory assays to estimate accurate IC50 and V/D values. For all reported IC50 values, goodness of fit (r2) of the nonlinear regression curve was greater than 0.9. Effect of herbal extracts on TFPG formation Effect of 10 herbal extracts on UGT1A4 ac tivity was achieved through incubations of pooled HLM with TFP and monitoring formation of TFPG as an index of UGT1A4 activity. For milk thistle and acidhydrolyz ed ginkgo extracts, evaluation of their effects of on UGT1A4 activity was not possible due to interference of the herbal extracts with TFPG florescence. All the tested extracts inhibited TFPG formation with different potencies (Figure 6-1). Herbal extracts showing rough IC50 values less than 100 g/mL were (mean SE) EGCG (34. 39 4.1 g/mL), black cohosh (69.7 4.8 g/mL), and saw palmetto (70.6 9.3 g/mL) (Table 6-2). Only EGCG inhibit ed UGT1A4 with V/D value exceeding 5 L. This finding was c onfirmed by incubating TFP with increasing concentrations of EGCG. Best-fit IC50 was (mean SE) 33.8 3.1 g/mL and V/D value was 7.4 L based on daily dose of 250 mg (Table 6-3, Figure 6-2). Effect of herbal extracts on serotonin glucuronide formation Milk thistle, saw palmetto, EGCG, and echi nacea inhibited serotonin glucuronide formation with IC50 values of (mean SE) 66.9 3.5, 131.8 21.5, and 183.6 29.8 g/mL, respectively (Table 6-2, Figure 6-1). A V/D cutoff value of 2 L was applied to select which extracts to study further. On ly saw palmetto and milk thistle exceeded the V/D cutoff with values of 2.4 L and 9.0, respectively (Table 6-2).

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95 Precise IC50 and V/D index values were determined for inhibition of serotonin glucuronide formation by milk thistle and saw palmetto (Figure 6-3). Best-fit IC50 values were 59.5 3.6 and 103.5 10.7 for milk th istle and saw palmetto, respectively. V/D values were 6.3 and 3.1 L for milk thistle, and saw palmetto, respectively (Table 6-3). Effect of herbal extracts on MPAG formation Black cohosh, cranberry, echinacea, ginsen g, acid-hydrolyzed ginseng, and milk thistle inhibited MPAG formation (Figure 61). However, only milk thistle (rough IC50 = 35.9 4.3 g/mL, V/D = 16. 7 L) and cranberry (rough IC50 = 260.5 33.0 g/mL, V/D = 3.8 L) exceeded the V/D cutoff of 2L and were selected for further study (Table6-2). Precise best-fit IC50 and V/D values for milk thistl e and cranberry were 33.6 3.1 g/mL and 17.9 L, and 230.4 32.9 g/mL and 3.1 L, respectively )Table 6-3, Figure 6-4). Discussion In this study, 12 commonly used herbal ex tracts were screened for their effects on the glucuronidation activity of UGT1A4, 1A6, and 1A9 in pooled HLM. UGT enzyme activities were measured in vitro usi ng selec tive substratesTFP for UGT1A4, serotonin for UGT1A6, and MPA for UGT1 A9 (Court, 2005). Based on V/D index values, the most potent inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. These findings highlight the possibility of herb-drug inte ractions through modul ation of UGT enzyme activity. The likelihood of the observed in vitro interactions to occur in vivo depends on characteristics of the herb, t he drug substrate, the specif ic enzyme, and the potency of the inhibition. UGT1A4 is known to be the pr imary enzyme that catalyzes N -glucuronidation of primary, secondary and arom atic amines, which include s TFP, lamotrigine and the

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96 estrogen receptor modulator drug tamoxifen (Kiang et al., 2005; Rowland et al., 2006; Zhou et al., 2010). In addition, UGT1A4 shows O -glucuronidation activity towards steroidal compounds (G reen and Tephly, 1996). Hecogenin is a known inhibitor of UGT1A4-mediated TFP gl ucuronidation with IC50 values of 1.5 M (Uchaipichat et al., 2006). Compared to hecogenin EGCG is a non-selective UGT1A4 inhibitor with moderate potency. EGCG has previously been shown to inhibit estradiol-3Oglucuronidation, an index for UGT1A1 activity, with a lower IC50 value (7.8 g/mL) (Chapter 4). In addition, in this study EGCG showed some weak inhibitory activities toward UGT1A6 and UGT1A9 (Figure 6-1). Pharmacokinetic studies show that maximum plasma concentrations of EGCG are more than 10-fold less than the observed IC50 values following consumption of high dose (800 mg) EGCG (Foster et al., 2007). This suggests that inhibition of UG T1A4-mediated systemic glucuronidation by EGCG is unlikely. However, based on V/D i ndex of the inhibition of 7.4 L for 250 mg dose, effect of EGCG on hepatic first pa ss metabolism of UGT1A4 substrates is possible and will be augmented with higher EGCG doses. EGCG has been studied at doses that reach 800 mg daily for its antioxidant and anti-cancer e ffects (Chow et al., 2005). Considering higher doses of EGCG ( 800 mg), the V/D inde x will be 23.6 L, indicating that the 800 mg dose can be diluted in up to 23.6 L and still inhibit UGT1A4 activity by up to 50%. The effect of EGCG on glucuronidation of the UGT1A4 substrates TFP, lamotrigine, tamoxifen, and imipramine warrants furt her investigation. UGT1A6 is typically a low affinity enzym e that catalyzes glucuronidation of drug substrates including acetaminophen, valpro ic acid, and morphine (Kiang et al., 2005). Milk thistle and saw palmetto inhibi ted serotonin glucuronidation with IC50

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97 concentrations attainable if the daily doses of milk thistle (600 mg) or saw palmetto (320 mg) are diluted with 6.3 and 3.1 L, respec tively. The observed milk thistle IC50 for UGT1A6 is equivalent to a total flavonoli gnans concentration of 22.6 g/mL; this is about 1000-fold higher than observed plasma concentration following intake of 600 mg milk thistle extract (Schrieber et al., 2008). Taken together, milk thistle extract is more likely to inhibit UGT1A6-mediated first pass rather than systemic metabolism. On the other hand, no pharmacokinetic data are available on saw palmetto. Based on IC50 value exceeding 100 g/mL and V/D index of 3.1 L, saw palmetto will be expected to have mild, if any, inhibition of UGT1A6-mediated metabolism in vivo (Table 6-2). UGT1A9 catalyzes glucuronidation of a wide range of substrates including MPA, propofol, raloxifene, and flavopiridol (Kiang et al., 2005). In the current study, milk thistle and cranberry inhibited MPAG formati on, which was used as an index reaction for UGT1A9 activity in HLM (Court, 2005). For milk thistle extract, the IC50 value was 33.6 g/mL, which is equivalent to 12.7 g/mL flavonolignans. Again, this concentration is much higher than the expected plasma c oncentration of flavono lignans following milk thistle intake (Schrieber et al., 2008). Therefore, inhibition of systemic metabolism of UGT1A9 substrates by milk thistle extract is not likely. Conversely, based on the range of intestinal fluid volume of 0.5 to 5 liters, a si ngle 600-mg dose of milk thistle may result in putative concentrations of 120 to 1200 g/mL. Accordingly, inhibition of first pass metabolism of UGT1A9 substrates by milk thistle extract is possible. In this study, we screened specific UGT enzyme activities using HLM rather than human intestine microsomes (HIM) or ex pressed enzymes. The difference between expressed enzymes and HLM is that the firs t contain single UGT enzymes while the

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98 latter contain all the hepatic isoforms. T herefore, HLM are closer to the in vivo environment due to the availability of other UGT enzymes that may form heterodimers, which has been reported for some UGT en zymes and may affect enzyme activity (Ouzzine et al., 2003). Since our goal was to screen for interactions that may have clinical significance, the use of HLM was mo re appropriate. This was made feasible by the availability of selective substrates for different UGT enzymes in HLM (Court, 2005). Similarly, HIM contain all the intestinal UGT enzymes. However, no selective substrates for individual UGT enzymes have been described in HIM. Calculation of V/D index provides a helpful tool to predict the likelihood of achieving IC50-equivalent concentrations in the intestine or plasma in the absence of clinical data (Strandell et al., 2004). Although this approach is sufficient for the purpose of screening and hypothesis generation, it is limited by not considering the extent of absorption of phytochemicals through tissue and cellular barrier s. Use of V/D index assumes that the concentration in the gastrointestinal lumen is equivalent to that in the endoplasmic reticulum of intestinal epithelial cells wher e UGT enzymes are located. This assumption may lead to overestimation of the extent of the inhibition, since many phytochemicals are poorly absorbed through the intestinal wa ll. Therefore, t he results need to be confirmed in clinical studies and, where available, IC50 values to be compared with concentration data obtained experimentally. It is worth noting that using V/D index to describe inhibition potency changes the order of significance of inhibitors. For example, based on rough IC50 values, black cohosh and saw palmetto are equipotent inhibitors of UGT1A4 activity (Rough IC50 = 69.7 g/mL and 70.6 g/mL; Table 6-2). However, the daily dose of saw palmetto is eight-fold higher than that of black cohosh (320 mg versus

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99 40 mg). Thus, ingesting 320 mg of saw palmetto is expected to result in higher extent of UGT1A4 inhibition compared to ingesting 60 mg of black cohosh. In summary, in this study, 12 herbal extrac ts were screened for inhibition of three UGT1A enzymesUGT1A4, UGT1A6, and UGT1A9 We report inhibition of UGT1A4 by EGCG, UGT1A6 by milk thistle and sa w palmetto, and UGT1A9 by cranberry and milk thistle extracts. Among these, EGCG inhibition of UGT1A4 and milk thistle inhibition of UGT1A6 and UGT1A9 are likely to affect first-pass glucuronidation of substrates. The in vivo effects of these interactions on pharmacokinetics of UGT1A4, UGT1A6, and UGT1A9 substrates remain to be determined in clinical studies.

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100 Table 6-1. List of herbal extracts screened for UGT1A4, UGT1A6, and UGT1A9 inhibition. Test Compound Scientific Name of Origin Percent of Key Components (w/w)* Solvent** Black cohosh rhizome extract Cimicifuga racemosa 5 % Total Triterpenglycosides 50% ethanol Cranberry press juice Vaccinium marocarpon > 40% Total Proanthocyanidins 96% Ethanol Echinacea root extract Echinacea purpurea 3% Cichoric acid 60% ethanol Garlic bulb extract Allium sativum 3.25 % Allin 80% methanol Ginkgo biloba leaf extract Ginkgo biloba 24% Ginkgo flavonglycosides 6% Terpene lactones 60% acetone Ginseng root extract Panax ginseng 5% Total Ginsenosides 60% ethanol Milk Thistle herb extract Silybum marianum 37.9% Total silymarin flavonolignans 80% acetone Saw Palmetto fruit extract Serenoa repens >85% Total fatty acids > 0.1 Sterols 96% ethanol Valerian root extract Valeriana officinalis 0.1 Valerenic acids 70% ethanol Epigallocatechin gallate (EGCG) Camellia sinensis > 97% EGCG 100% methanol *Values provided by manufacturer. **Used by manufacturer for standardization

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101 Table 6-2. Effect of commonly used herbal extracts on UGT1A4, UGT1A6, and UGT1A9 activity. Each herbal extract was co-incubated at three concentrations wit h TFP (for UGT1A4), serotonin (for UGT1A6), and mycophenolic acid (for UGT1A9) and HLM. Formation of TFPG, serotoninglucuronide, and MPAG were used as index reactions for activity of UGT1A4, UGT1A6, and UGT1A9 enzyme activities, respective ly. Formation of glucuronides was compared in incubations with herbal extract to negative control incubati ons. Data represent best-fit IC50 standard error. Goodness of fit r2 value was > 0.9 for all reported IC50 value. Volume/Dose index was calculated by dividing the daily intake of each herb by the rough IC50 value. UGT1A4 UGT1A6 UGT1A9 Extract RDI (mg) Rough IC50 (g/mL) Volume/Dose Index (L/dose) Rough IC50 (g/mL) Volume/Dose Index (L/dose) Rough IC50 (g/mL) Volume/Dose Index (L/dose) Black cohosh 40 69.7 4. 8 0.6 NA NA 321.6 102.2 0.1 Cranberry 1000 742.7 118.7 1.3 > 1000 < 1.0 260.5 33.0 3.8* Echinacea 400 116.1 25.1 3.4 241.0 23.4 1.7 858.3 158.7 0.5 Garlic 1000 NA NA NA NA NA NA Ginkgo biloba 240 268.2 48.9 0.9 NA NA PB PB Acid-hydrolyzed ginkgo biloba 240 Interf NA NA PB PB Ginseng 550 368.4 66.6 1.5 NA NA 298.6 29.1 1.8 Acid-hydrolyzed ginseng 550 288.0 42.8 1.9 > 1000 < 0.6 524.3 60.5 1.0 Milk thistle 600 Interf 66.9 3.5 9.0* 35.9 4.3 16.7* Saw palmetto 320 70.6 9.3 4.5 131.8 21.5 2.4* NA NA

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102 Table 6-2. Continued Valerian 1000 406.5 35.3 2.5 > 1000 < 1.0 NA NA Epigallocatechin gallate (EGCG) 250 34.39 4.1 7.3* 183.6 29.8 1.4 NA NA NA, data points did not fit IC50 curve; PB: IC50 values for inhibition of UGT1A9 by ginkgo and acid-hydrolyzed ginkgo extracts have been previously reported (Chapter 3). Gink go and acid-hydrolyzed ginkgo extracts inhibited MPAG formation in HLM with IC50 values of 84.3 11.6 and 20.9 3.6 g/mL, respectiv ely. Considering dose of 240 mg, this would result in V/D index of 2.9 and 11.4 L/ Dose for unhydrolyzed and acid-hydrolyzed ginkgo extracts, res pectively. Interf: Addition of herb interfered with florescence detection of glucuronide indicates volume/dose index values that exceed the cutoff for further investigation

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103 Table 6-3. Determination of inhibitory potency of selected UGT1A4, UGT1A6, and UGT1A9 herbal inhibitors. Extracts we re selected if their V/D index based on rough IC50 values exceeded 4 L for UGT1A4, or 2 L for UGT1A6 and UGT1A9. Several concentrations of each extract were co-incubated with alamethicin-activated HLM and TFP (for UGT1A4), serotonin (for UGT1A6), or MPA (for UGT1A9). Percent of re maining activity was measured as the formation of each glucuronide in herbal incubation as a per cent of negative control. IC50 values were calculated by fitting data points to IC50 equation Hill equation as described under Materials and Methods. Values reported are best-fit IC50 values standard error. Goodness of fit r2 value was 0.95 for all reported IC50 value. Volume/Dose index was calculated by dividing the daily intake of each herb by the IC50 value. UGT Enzyme Extract RDI (mg) IC50 (g/mL) Volume/Dose Index (L/dose) H UGT1A4 EGCG 250 33. 8 3.1 7.4 1.0 UGT1A6 Milk thistle 600 59.5 3.6 6.3 1.1 Saw palmetto 320 103.5 10.7 3.1 1.4 UGT1A9 Cranberry 1000 230.4 32.9 3.1 1.0 Milk thistle 600 33. 6 3.1 17.9 0.8

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104 Figure 6-1. Effect of commonly used herbal extracts on UGT1A4, UGT1A6, and UGT1A9 enzyme activities. HLM were co-incubated with herbal extracts and A) TFP for UGT1A4 activity, B) se rotonin for UGT1A6 activity, and C) mycophenolic acid for UGT1A9 activity. Three concentrations were tested for each herbal extract which represent extract daily intake in 53 L (small-dotted bars), 5.3 L (checkered bars), and 0.53 L (striped bars). Formation of TFPG, serotonin glucuronide, and MPAG were detected in respective herbal incubations. Percent of activity was calculated as the percent of glucuronide peak area in herbal incubations as compared to negative controls. Each value represents mean of duplicate incubat ions. Error bars represent positive standard error.

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105 A B C

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106 Figure 6-2. Inhibitory effect of green t ea catechin EGCG on TFPG formation in HLM. Increasing concentrations of EGCG we re incubated with 60 M TFP, 0.1 mg/mL alamethicin-activated HL M, 5 mM UDPGA, and 5 mM MgCl2 for 20 minutes at 37oC. Formation of TFPG was used as an index for UGT1A4 activity in HLM incubations. Each data point represents mean of duplicate incubations. Error bars represent twosided standard error of the mean. Data points were fitted to IC50 equation as described under Materials and Methods. Goodness of fit r2 value was 0.98.

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107 Figure 6-3. Inhibition of serotonin gl ucuronide formation by saw palmetto and milk thistle extracts. Increasing concentrations of A) saw palmetto and B) milk this tle extracts were incubated with 8 mM serotonin, 5 mM MgCl2, 0.5 mg/mL alamethicin-activated HLM, and 5 mM UDPGA for 60 minutes at 37oC. Serotonin glucuronide formation was used as an i ndex of UGT1A6 enzyme activity in HLM incubations. Each data point represents mean of duplicate incubations Error bars represent two-sided st andard error of the mean. Data points were fitted to IC50 equation as described under Materials and Methods. Goodness of fit r2 value was 0.96 and 0.99 for saw palmetto and milk thistle, respectively. A B

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108 Figure 6-4. Inhibition of MPAG formation by cranberry and milk thistle extracts. Increasing concentrations of A) cranberry and B) milk thistle extracts were incubat ed with 240 M mycophenolic acid, 5 mM MgCl2, 2% BSA, 0.16 mg/mL alamethicin-activated HLM, and 1 mM UDPGA. Formation of MPAG was used as an index for UGT1A9 activity in HLM incubations. Each data point represents mean of duplicate incubations. Error bars represent two-sided standard error of the mean. Da ta points were fitted to IC50 equation as described under Materials and Methods. Goodness of fit r2 value was 0.95 and 0.99 for cranberry and milk thistle, respectively. A B

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109 CHAPTER 7 CONCLUSION AND FUTURE DIRECTIONS In the last ten years, res earchers have increasingly recogni zed the potential of herbal supplements to interact with conventiona l drugs. Mechanisms by which herb-drug interactions occur can be pharmacodynamic or pharmacokinetic. For the latter, the majority of interactions occur through phy tochemicals modulating the expression or activity of drug metabolizing enzymes. Many case studies, review papers, in vitro, animal and clinical studies have documented the effects of herbal supplements on CYP enzymes. In contrast, little attention has been given to other metabolic routes. The goal of this research project was to charac terize the effects of herbal supplements on glucuronidation reactions in vitro. Data generated from these studies provide information for clinicians and the public regarding the safety of taking herbal supplements with drugs metabolized by UGT enzymes. In addition, the results help generate hypotheses for future studies to invest igate the in vivo e ffects of the observed in vitro interactions. Initially, we hypothesized that Ginkgo biloba extract inhibits MPA glucuronidation in vitro. The basis for this hypothesis wa s that quercetin and k aempferol, the main flavonoid aglycones in ginkgo extract, are su bstrates for UGT1A9 the primary hepatic enzyme that metabolizes MPA. In order to measure MPAG concentrations in microsomal incubates, we developed a sensitive analytical method using LC-MS/MS. Linearity, accuracy, and sensit ivity parameters were determi ned to ensure reliability and reproducibility of the method over the range of MPAG concentrations expected in incubations. This method, described in Chapte r 2, was then used to study the effect of ginkgo extract and its main flavonoid and terpene lactone co mponents on MPAG

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110 formation using HLM and HIM (Chapter 3). We found that ginkgo extract, quercetin, and kaempferol inhibit MPAG formation at IC50 concentrations attainable in the human intestine. This finding highlights the potent ial of ginkgo extract to inhibit first pass glucuronidation of my cophenolate sodium, the form of MPA that is more susceptible to first pass metabolism. Next, we investigated the effect of co mmonly used herbal supplements on one of the major UGT enzymesUGT1A1. To achiev e this, we develo ped a screening approach similar to the screening procedure followed in pharmaceutical industry. Eight commonly used herbal extracts were scr eened at three different concent rations for their potential inhibitory effect on UGT1A1 activity (Chapter 4). Enzymatic activity of UGT1A1 was estimated using estradiol-3O-glucuronidation as an enzyme-se lective index reaction in pooled HLM. Using this approa ch, green tea catechin ECGC, m ilk thistle, saw palmetto, and echinacea were found to inhibit UGT1A1. Inhibition potency was reported in terms of IC50 value and V/D index value. The latter esti mates the volume of fluid in which the daily dose of the extract should be diluted to get an IC50-equivalent concentration value. This parameter provides a putative t ool to predict, under commonly recommended doses, whether inhibition of UGT1A1 by the herbal supplement is likely to occur in vivo. Based on V/D values of the obs erved inhibitors, EGCG and milk thistle may inhibit first pass glucuronidation of UGT1A1 substrates such as raloxi fene and ezetimibe. Among all screened herbals, EGCG showed the most potent inhibition of UGT1A1. Raloxifene is a selective estrogen receptor modulator with poor bioavailability due to its extensive intestinal first pass metabolism by UGT enzymes, including UGT1A1. Therefore, our next goal was to investigate the effect of green tea EGCG on raloxifene in vitro intrinsic clearance using HIM (C hapter 5). A substrate depletion assay was

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111 applied by incubating raloxifene with HI M and different concentrations of EGCG and monitoring the fraction of raloxifene remaini ng at different time points. Data obtained were used to estimate raloxife ne in vitro intrinsic clearan ce. EGCG strongly inhibited raloxifene in vitro clearance at concentration s of EGCG attainable in the intestine. These results highlight the potential for inhibi tion of raloxifene pre-systemic clearance, which could cause a marked increase in ra loxifene bioavailability. The clinical consequences of the observed interaction have yet to be determined. Finally, we screened nine commonly used herbal supplements for their inhibitory effects on UGT1A4, UGT1A6, and UGT1A9 using TFPG, serotonin glucuronide, and MPAG formation as index reactions, respecti vely (Chapter 6). HLM were incubated with TFP, serotonin, or MPA and each herbal extrac t at three different concentrations. Screening results indicated that potential inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. For these potential inhibitors, confirmatory experiments were conducted by incubating a range of concentrations of each extract wit h the enzyme-selectiv e substrate. IC50 and V/D values were calculated and compared to ph ysiologic concentrations, if available. Results from these experiments showed that EGCG may inhibit first pass glucuronidation of UGT1A4 substrates, while milk thistle may inhibit first pass glucuronidation of UGT1A6 and UGT1A9 substrates. Conver sely, weaker inhibition of UGT1A6 and UGT1A9 was observed by saw palmetto and cranberry, respectively. Overall, this research high lights the potential for inhibiti on of UGT enzymes by herbal extracts with potencies that may translate in vivo. Future clinical studies are warranted to investigate the pharmacokinetic consequences of the observed interactions. In this project, we screened commonly used suppl ements for interactions with UGT1A

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112 enzymes. Further studies ar e needed to investigate effects of other herbal supplements and other UGT enzymes. The availability of selective in vitro pr obe substrates for hepatic UGT enzymes offers an efficient and relatively inexpensive way to screen for dr ug-drug and herb-drug interactions through glucuronidation. Our results show that herb-drug interactions appear more likely with first pass rather than systemic glucuroni dation of drugs. However, selective probe substrates have not been identified for UGT enzymes in HIM, which limits HIM utility in screening experiments Future studies ar e needed to identify selective in vitro probes t hat can be used with HIM. In conclusion, the in vitro effects of commonly used herbal supplements on UGT enzymes have been studied. Results indicate the possibility for inhibition of glucuronidation of drugs by herbal supplements. Inhibito ry effects of ginkgo on MPA glucuronidation and of EGCG on raloxifene clearance has been described. Moreover, inhibition of UGT1A1, 1A4, 1A6, and 1A9 by her bal extracts is reported. Future studies are warranted to investigate the clinical re levance of the observed interactions.

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127 BIOGRAPHICAL SKETCH Mohamed-Eslam Mohamed was born in Cairo, t he capital of Egypt. He received his bachelors degree in pharmaceutical scienc es in May of 2003, from Ain Shams University, in Cairo. Soon after, he work ed at Misr International University, where he was a teaching assist ant in clinical pharmacology courses. He began his graduate studies in the Pharmacotherapy and Trans lational Research Department at the University of Florida in August of 2005. There, he started inve stigating herb-drug interactions under the supervision of Dr. Reginal d Frye. He received his Ph.D. from the University of Florida in the Summer of 2010.