(Desertasi) Transporter-Mediated Flavonoid-Drug Interactions - in Vitro and in Vivo PDF

Transporter-mediated Flavonoid-Drug
Interactions: In vitro and In vivo
By
Xiaodong Wang
August 2007
A dissertation submitted to the Faculty of the Graduate School of

State University of New York at Buffalo
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Department of Pharmaceutical Sciences
UMI Number: 3277779
UMI Microform 3277779

Copyright 2007 by ProQuest Information and Learning Company.
All rights reserved. This microform edition is protected against
unauthorized copying under Title 17, United States Code.
ProQuest Information and Learning Company

300 North Zeeb Road
P.O. Box 1346
Ann Arbor, MI 48106-1346
TABLE OF CONTENTS
Acknowledgements
iii
Abstract
iv
Chapter 1
Introduction
Chapter 2
Flavonoids as a Novel Class of Human Organic Anion Transporting 32

Polypeptide OATP1B1 (OATP-C) Modulators.
Chapter 3
Monocarboxylate Transporter 1 (MCT1)-Mediated
68
Pharmacokinetic Interaction for the Flavonoid Luteolin: Model

Fitting and Simulation.
Chapter 4
Effects of the Flavonoid Chrysin on Nitrofurantoin
110
Pharmacokinetics in Rats: Potential Involvement of ABCG2.

Chapter 5
Pharmacokinetics and Bioavailability of 7,8-Benzoflavone in
143
Rats.
Chapter 6
ABCG2-Mediated Transport of the Flavonoids Biochanin A and
175
Kaempferol.
Chapter 7
Lack of Interactions between the Flavonoid Biochanin A and P-
204
glycoprotein Substrates Doxorubicin and Cyclosporine A in Rats.
Chapter 8
Conclusions
234
Appendix
Diet/nutrient Interactions with Drug Transporters.
243
ii
ACKNOWLEDGEMENTS
I would never have been able to complete my study without the help and support from
many people to whom I would like to express my gratitude from the bottom of my heart.
My deepest thanks go to Dr. Marilyn Morris, my advisor, for her continued guidance,
encouragement, support and patience throughout the years of my graduate study. Her
devotion, enthusiasm, and passion for science inspired me and helped me mature as an
independent scientist and will have an invaluable impact on my future career.
Special thanks go to Dr. Murali Ramanathan and Dr. Joseph Balthasar for serving as my
committee members, critically reviewing my thesis, and providing insightful suggestions
and comments. I thank Dr. Philip C. Smith, Division of Molecular Pharmaceutics,
School of Pharmacy, University of North Carolina at Chapel Hill, for being my outside
reader and providing thoughtful comments and valuable suggestions to this dissertation.
I am grateful to my colleagues in Dr. Morris group for their helpful discussion and
always support. I would also like to thank the faculty, staff, and students in the
Department of Pharmaceutical Sciences for providing a friendly, challenging, and joyful
atmosphere during my Ph.D. study.
Finally, I deeply thank my parents, my wife Haiqing, and my daughter Ainsley for their
love and support. Without them, this work would have been impossible.
iii
ABSTRACT
Flavonoid-drug interactions have been increasingly observed due to the popular use of
flavonoid-containing products for disease prevention and health promotion. However,
our current understanding of the mechanisms underlying most of these interactions is
related predominantly to the modulation of drug metabolizing enzymes. In order to better
understand and predict the potential pharmacokinetic interactions of flavonoids with
conventional medicines, the interactions of flavonoids with drug transporters important in
drug disposition were investigated in this dissertation. The transporters of interest in this
dissertation include the uptake transporters, organic anion transporting polypeptide
OATP1B1 and monocarboxylate transporter 1 (MCT1), and the efflux transporters, breast
cancer resistance protein (BCRP) and P-glycoprotein.
Many of the tested flavonoids significantly inhibited the uptake of the OATP1B1
substrate [3H]dehydroepiandrosterone sulfate (DHEAS) in a concentration-dependent
manner in HeLa OATP1B1-expressing cells, with biochanin A being one of the most
potent inhibitors with an IC50 of 11.3 3.22 M. A kinetic study revealed that biochanin
A inhibited [3H]DHEAS uptake in a noncompetitive manner with a Ki of 10.2 1.89
M. This is consistent with our finding that biochanin A is not a substrate for OATP1B1.
The flavonoid luteolin has been reported to be a potent MCT1 inhibitor. In chapter 3, we
characterized the effects of luteolin on the pharmacokinetics and pharmacodynamics of
the MCT1 substrate -hydroxybutyrate (GHB) in rats. Luteolin significantly decreased
iv
the plasma concentration and AUC, and increased the total and renal clearances of GHB
in a dose-dependent manner. Moreover, luteolin significantly shortened the duration of
GHB-induced sleep in rats. A pharmacokinetic model that incorporated capacity-limited
renal reabsorption and metabolic clearance was constructed to characterize the in vivo
interaction and revealed that luteolin significantly altered the pharmacokinetics of GHB
by uncompetitively inhibiting its MCT1-mediated transport, with an inhibition constant
of 1.1 M.
The flavonoid chrysin has been reported to be a potent inhibitor of BCRP. The
pharmacokinetic interaction between chrysin and the BCRP substrate nitrofurantoin in
rats was characterized in chapter 4. Oral and intravenous coadministration of chrysin
significantly increased the AUC of nitrofurantoin. Moreover, the cumulative
hepatobiliary excretion of nitrofurantoin was significantly decreased by approximately
75% following the coadministration of chrysin. The flavonoid 7,8-benzoflavone was
more potent than chrysin in inhibiting BCRP-mediated transport, and a 50 mg/kg oral
dose significantly increased the AUC of nitrofurantoin. The investigation of the
pharmacokinetics of 7,8-benzoflavone following the administration of oral and
intravenous doses revealed nonlinear pharmacokinetics, likely due to its capacity-limited
metabolism and BCRP-mediated elimination. The studies addressing BCRP inhibition
mechanisms indicated that flavonoids, such as biochanin A and kaempferol and/or their
metabolites, inhibited BCRP-mediated transport by acting as BCRP substrates. Different
from the BCRP-mediated flavonoid-drug interactions, the flavonoid biochanin A
inhibited P-glycoprotein-mediated efflux in vitro, but had minimal effects on the
pharmacokinetics of P-glycoprotein substrates doxorubicin and cyclosporine A,

indicating other transporters or metabolizing enzymes might be important in the in vivo
disposition of doxorubicin and cyclosporine A.
In summary, this dissertation characterized the flavonoid-drug interactions mediated by

the uptake and efflux transporters. This research is of significance due to the widespread
use of dietary supplements containing flavonoids, and provides new information on the
pharmacokinetics and pharmacodynamics of this important class of dietary compounds.
vi
CHAPTER 1
INTRODUCTION
FlavonoidsDrug Interactions: Effects of Flavonoids on
Drug Transporters
Flavonoids are a class of polyphenolic compounds widely present in fruits, vegetables,

and plant-derived beverages, such as green tea, and in many herbal products marketed as
over-the-counter dietary supplements, such as St. Johns wort and milk thistle. The
concentrations of some flavonoids such as naringin and hesperidin, abundant in fruit
juices, have been reported to be as high as 145 638 mg/L 1 and 200 450 mg/L 2,3,
respectively. The average daily intake of total flavonoids from the U.S. diet was
estimated to be 200 mg to 1 g 4-8. The structures of a number of flavonoid subclasses are
shown in Table 1. Flavonoids have long been associated with a variety of biochemical
and pharmacological properties including antioxidant, antiviral, anticarcinogenic, and
anti-inflammatory activities, with no or low toxicity 9,10. These health-promoting
activities indicate that flavonoids may provide protection from cancer and cardiovascular
diseases, as well as other age-related degenerative diseases 10-12.
In recent years, flavonoid-enriched dietary supplements (e.g., St. Johns wort, ginkgo,
garlic, ginseng) have been widely used in Western countries due to an increased public
interest in alternative medicine and disease prevention 13-15. The intake of flavonoids
after taking these herbal products is likely very high. For example, the manufacturers
recommended dose of chrysin is up to 6 capsules per day and each capsule contains 500
mg chrysin (iHerb Inc., Monrovia, CA; VitaDigest, Walnut, CA); the suggested daily
dose of quercetin is one or multiple capsules and each capsule contains 500 mg quercetin
(http://www.herbsmd.com/detail/quercetin-500-1849.htm). Upon consumption of these
dietary supplements, high concentrations of flavonoids in the intestine would be
expected, raising a potential for flavonoid-drug interactions. Recently, flavonoid-drug

interactions have been increasingly evidenced in animal and clinical studies 16-20. In
some cases, these flavonoid-drug interactions can be serious or even life-threatening.
This is true especially when flavonoids or dietary supplements are used concomitantly
with narrow therapeutic index drugs, such as digoxin 20. On the other hand,
coadministration of flavonoids with conventional medicine may have significant
beneficial effects on the pharmacokinetics of the coadministered drugs. For example, the
flavonoids quercetin and flavone have been shown to increase the bioavailability of
paclitaxel in rats in a concentration-dependent manner 18,21. Additionally, the flavonoid
morin significantly increased the bioavailability of etoposide in rats 22. In human
subjects, administration of silymarin increased the clearance of metronidazole and its
major metabolite, hydroxy-metronidazole 23. Coadministration of grapefruit juice
significantly decreased the bioavailability of fexofenadine 16. Considering this
accumulating evidence for flavonoid-drug interactions from animal and clinical studies
and the popular use of flavonoid-enriched dietary supplements, which do not require
FDA approval for their marketing in the U.S., it is important to understand the
mechanisms of flavonoid-drug interactions, as well as the potential clinical consequences
of these interactions.
The mechanisms underlying the reported flavonoid-drug interactions have been largely
ascribed to the interaction with drug metabolizing enzymes 15,24-26. Recently, emerging
evidence has suggested that flavonoids can also interact with drug transporters, a class of
membrane proteins important in drug disposition 27-30. Drug transporters can be generally
classified into two major groups efflux and uptake transporters. Most efflux
transporters belong to a superfamily of ATP-binding cassette (ABC) transporters, which
utilize energy derived from ATP hydrolysis to export substrates out of cells against a
concentration gradient. Some major members of this group of transporters include Pglycoprotein (MDR1, ABCB1), multidrug resistance-associated proteins (MRPs, ABCC)
and breast cancer resistance protein (BCRP, ABCG2) 31. In contrast, uptake transporters
mediate the translocation of drugs into cells. Included in this group of transporters are
the organic anion transporting polypeptide (OATP, SLCO) family, the organic anion
transporter (OAT, SLC22A) family, the organic cation transporter (OCT, SLC22A)
family, and monocarboxylate transporter family (MCT, SLC16A) 32-35. Transportermediated drug disposition represents the dynamic interplay between uptake and efflux
transporters expressed on the apical or basolateral membranes in the epithelial cells in the
organs 36. Many of these efflux and uptake transporters are expressed in various major
organs, such as intestine, liver, kidney, blood-brain barrier, and placenta, suggesting their
essential roles in governing the oral absorption, intestinal, hepatobiliary and renal
excretion of a variety of endogenous and exogenous compounds (Figure 1) 31,35-41. In
addition, overexpression of efflux transporters (e.g., P-gp, MRP, and BCRP) in tumor
cells limits intracellular accumulation of a broad range of structurally and functionally
unrelated anticancer agents and leads to inefficient cell killing, a phenomenon known as
multidrug resistance (MDR), which remains the primary obstacle to successful cancer
chemotherapy 41-43. Due to the important roles of these transporters in drug disposition
and cancer therapy, modulation of these drug transporters by inhibitors or inducers may
significantly alter the pharmacokinetics and therapeutic efficacy of many anticancer
drugs, resulting in beneficial or adverse drug-drug interactions. Recently, flavonoids
have been identified as a class of potent modulators of major efflux transporters, such as
P-gp, MRP, and BCRP 27-30.
P-gp-Mediated Flavonoid-Drug Interactions

P-gp is a 170 kDa membrane-bound ATP-dependent efflux transporter. It consists of two
homologous halves each containing a transmembrane domain (TMD) and a cytosolic
ATP or nucleotide-binding domain (NBD) 41. P-gp transports many clinically important
drugs including anticancer drugs (e.g., vinca alkaloids, paclitaxel), HIV protease
inhibitors (e.g., saquinavir, ritonavir), -blockers (e.g., talinol), cardiac drugs (e.g.,
digoxin, quinidine), immunosuppressants (e.g., cyclosporine A, tacrolimus), H-2 receptor
antagonists (e.g., cimetidine), antibiotics (e.g., erythromycin), and corticosteroids 36,44.
The typical inhibitors of P-gp include verapamil, cyclosporine A, PSC833, GF120918,
and LY335979 44. The overexpression of P-gp has been detected in a variety of
hematological and solid tumors, suggesting its role in conferring MDR 31. In addition to
its expression in tumors, P-gp is also expressed at the apical membranes of epithelial cells
in the organs important in drug disposition such as intestine, liver, brain, and kidney 31,36.
The effects of flavonoids on P-gp have been extensively studied during the past decade
(Table 2). It was clearly shown from these studies that many of these flavonoids
demonstrated P-gp-modulating activities. However, the effects of flavonoids, especially

for some flavonols, were cell line-dependent, concentration-dependent, and substratedependent. For example, in P-gp-expressing HCT-15 colon cells, flavonols such as
quercetin, kaempferol and galangin were shown to stimulate adriamycin efflux 45. In
contrast, in MCF-7-ADR resistant breast cancer cells, quercetin was able to restore
sensitivity to adriamycin and inhibit rhodamine-123 efflux 46. Interestingly, in mouse
brain capillary endothelial cells (MBEC4), quercetin and kaempferol exhibited biphasic
effects by activating P-gp at a low concentration (10 M) and inhibiting P-gp at a high
concentration (50 M) 47. In rat hepatocytes, the effects of quercetin, kaempferol and
galangin on rhodamine-123 and doxorubicin efflux were shown to be substrate-dependent
48
. Using a purified and reconstituted P-gp system, Shapiro and Ling reported that
quercetin inhibited P-gp-mediated Hoechst 33342 efflux and enhanced its accumulation
in resistant CHRC5 cells. This effect was, at least partly, caused by the inhibition of P-gp
ATPase activity by quercetin 49. More recently, in P-gp-overexpressing KB-C2
carcinoma cells, quercetin and kaempferol significantly inhibited P-gp activity and
increased cellular accumulation of rhodamine-123 and daunorubicin 50. The reason(s) for
these results is still unclear. One possible explanation might be the existence of multiple
drug binding sites on P-gp and different allosteric effects of flavonoids on these sites. It
has been shown that quercetin could preferentially bind to the Hoechst 33342-binding site
on P-gp and inhibited Hoechst 33342 transport, presumably via competitive inhibition.
In contrast, binding of quercetin to the Hoechst 33342 site resulted in increased binding
of rhodamine-123 to another site on P-gp and stimulated rhodamine-123 transport 51.
Alternatively, the difference in experimental conditions, such as the different cell lines,
the different substrates and their concentrations, and the concentrations of flavonoids
used, might contribute to the discrepancies as well. As shown in the MBEC4 study, low
concentrations of quercetin and kaempferol activated P-gp possibly via enhancing the
phosphorylation (and hence activity) of P-gp, whereas high concentrations of quercetin
and kaempferol directly inhibited P-gp 47. Despite these conflicting observations, the
majority of recent studies have shown that many flavonoid aglycones have an inhibitory
activity on P-gp-mediated drug transport (Table 2).
Using purified C-terminal nucleotide-binding domain (NBD2) from mouse P-gp, Conseil
et al. studied the structure-activity relationship of flavonoids interacting with P-gp based
on their binding affinity. As a suitable tool for the rapid screening of P-gp modulators,
NBD2 contains an ATP-binding site as well as a close but distinct hydrophobic steroid
RU486-binding site 27. It was shown in this study that flavones (apigenin) and flavonols
(quercetin) had higher binding affinity than flavanones (naringenin), isoflavones
(genistein), or glycosylated derivatives (rutin). Interestingly, the flavonol kaempferide
exibited bifunctional interactions with both the ATP-binding and hydrophobic steroidbinding sites 27. It was shown in further studies that, among a total of 29 flavonoids
tested, only flavonols were able to bind to the ATP-binding site but flavones did not,
suggesting the essential roles of the hydroxyl group at position 3 on ring-C in interacting
with the ATP-binding site 52. Moreover, hydrophobic substitution by prenylation at
either position 6 or 8 on the A ring significantly increased the binding affinity of
flavonoids for P-gp NBD2 and abolished the interactions of flavonols with the ATPbinding site 52. Based on these findings, a tentative mechanism for the interaction of
flavonoids with P-gp was proposed by Di Pietro et al. 52. In this model, flavonols appear
to interact with the ATP-binding site via hydroxyl groups at position 3 and 5 on the A or
C rings, whereas the rest of the molecule would interact with the hydrophobic steroidbinding region. Increasing hydrophobicity by prenylation will shift flavonol binding
from the ATP-binding site to the vicinal steroid-binding and transmembrane domain
(TMD).
Additional structure-activity relationship studies using NBD2 and cell lines suggest that
high P-gp modulating activities are associated with molecules containing a 2-3 double
bond (planar structure), 3- and 5-hydroxyl groups, and hydrophobic groups on the A or B
rings. Moreover, glycosylation would dramatically decrease flavonoid P-gp-modulating
activity, as exemplified by rutin, heperidin, and naringin (Table 2). With regards to
flavanols (e.g., catechin, catechin gallate, EGC, and EGCG), the presence of a galloyl
moiety on the C ring markedly increases their activities 50,52-54.
The pharmacokinetic interactions of flavonoids with drug transporters have been reported
in a number of studies, most of which were focused on the interactions between P-gp and
quercetin. Using paclitaxel as a P-gp substrate, Choi et al. 18 investigated the effects of
quercetin on paclitaxel pharmacokinetics in rats. It was shown in this study that the oral
administration of quercetin increased the AUC and Cmax of paclitaxel (p.o. dose, 40
mg/kg) in a dose-dependent manner. At a dose of 20 mg/kg, quercetin administration

resulted in a 3.1-fold and 2.7-fold increase in paclitaxel AUC and Cmax, respectively.
Moreover, the absolute bioavailability of paclitaxel was significantly improved from 2%
(control group) to 6.2% (20 mg/kg quercetin treatment group). The t1/2 and MRT of
paclitaxel were also greatly prolonged compared to those of the control group. Similarly,
the oral administration of 40 mg/kg quercetin to pigs significantly increased the AUC of
digoxin (p.o. dose, 0.02 mg/kg) by 170% and increased the Cmax by 413%.
Unexpectedly, increasing the dose of quercetin to 50 mg/kg resulted in sudden death of
two out of three pigs within 30 min after digoxin administration 20. The adverse
interaction observed in this study raised a serious safety concern for the concomitant use
of dietary supplements and clinically important drugs, especially those with a narrow
therapeutic index. In another study, quercetin (100 M) significantly inhibited P-gpmediated transport of moxidectin and enhanced cellular accumulation of moxidectin in
rat hepatocytes. In addition, subcutaneous administration of quercetin (10 mg/kg)
significantly increased the AUC of moxidectin (p.o. dose, 0.2 mg/kg) by 83.8% 55.
Interestingly, in a study by Hsiu et al. 56, the oral administration of quercetin (50 mg/kg)
to pigs and rats resulted in significant decreases in the AUC of cyclosporine by 56% and
43%, respectively. However, in a recent clinical study, oral administration of quercetin
(5 mg/kg, 30 min or 3 days pretreatment) significantly increased cyclosporine AUC by
36% and 47%, respectively 17. The possible explanations for the contradictory results of
cyclosporine might be due to the species differences, the methods of administration
(concomitant or separate administration), and the doses (low or high doses). All these
studies indicated that flavonoid-P-gp interactions could occur in vivo. However, the
observed pharmacokinetic interactions might result from the interactions with P-gp
and/or CYP3A or both since most of these P-gp substrates are also CYP3A substrates.
MRP-Mediated Flavonoid-Drug Interactions

The family of multidrug resistance associated proteins (MRPs) is another group of ABC
transporters, and currently consists of nine members 57. Similar to P-gp, MRPs have a
typical ABC transporter core topology consisting of two homologous segments each
containing a 6-transmembrane domain and an ATP-binding domain. In addition to the
core structure, some MRPs have an extra N-terminal segment of five transmembrane
domains linked to the core structure through an intracellular loop (L0) 41,57. MRPs
transport a wide variety of endogenous and exogenous substrates, preferentially
amphiphilic anions including unconjugated compounds (e.g., methotrexate) and
compounds conjugated with glutathione, glucuronide, and sulfate 36,38,44. The typical
inhibitors of MRPs include MK571, indomethacin, and probenecid 38,44. MRP1 is
expressed ubiquitously throughout the body and is mainly localized at the basolateral
membranes of the epithelial cells in the organs 36,44. In contrast, MRP2 has a more
restricted expression pattern and is mainly localized at the apical membranes of the
epithelial cells in liver, kidney, and intestine 36,44.
A growing number of reports have been published regarding the modulating effects of
flavonoids on MRPs. Many flavonoids have been shown to interact with MRPs and the
10
potential mechanisms of MRPs reversal might involve: 1) decreasing intracellular

glutathione (GSH) concentrations via stimulating GSH transport; 2) altering the
expression of MRPs; 3) influencing drug transport possibly via direct or indirect binding
interactions with MRPs at substrate or allosteric binding sites; and 4) affecting ATPase
activity, ATP binding, and ADP release (Table 3). Using recombinant nucleotidebinding domain (NBD1) from human MRP1, Trompier et al. 58 studied the direct
interactions of flavonoids with NBD1 and revealed the presence of multiple flavonoidbinding sites. In this study, dehydrosilybin was found to bind to the ATP binding site and
inhibit leukotriene C4 (LTC4) transport. Similar to the case of P-gp, hydrophobic Cisoprenylation of dehydrosilybin increased the binding affinity for NBD1, but shifted the
flavonoid binding outside the ATP site, and decreased the inhibition of LTC4 transport.
A number of structure-activity relationship studies indicated that flavones and flavonols

were more potent in modulating MRP1 activities than isoflavones, flavanols, flavanones,
and flavanolols. Glycosylation of flavonoids resulted in a decrease in the inhibitory
activity 59-61. The structural features necessary for high MRP1 inhibitory potency
include: 1) a planar molecular structure due to the presence of a 2-3 double bond; 2) the
presence of both 3- and 4-hydroxyl groups on the B ring; and 3) hydrophobic
substitution of 4-hydroxyl group on the B ring 60-62. In a recent study including 29
flavonoids, diosmetin (3, 5, 7-trihydroxy-4-methoxyflavone ) was identified as the most
potent MRP1 inhibitor with an IC50 value of 2.7 0.6 M 61. In contrast to the wide
variety of flavonoids that can inhibit MRP1, MRP2 displays higher selectivity for
11
flavonoid type inhibition. Among 29 flavonoids tested, only robinetin and myricetin
inhibited MRP2-mediated calcein efflux with IC50 values of 15.0 3.5 M and 22.2 3.9
M, respectively. The presence of a pyrogallol group on the B ring of flavonols was an
important structural characteristic of flavonoids for MRP2 inhibition 61.
BCRP-Mediated Flavonoid-Drug Interactions

Breast Cancer Resistance Protein (BCRP) was identified recently as a new member of the
ABC transporter family. Unlike P-gp and MRPs, BCRP is a half-transporter consisting
of only 1 nucleotide-binding domain and 1 membrane-spanning domain. Recent
evidence suggests that BCRP may function as a homodimer bridged by disulfide bonds or
possibly a higher form of oligomerization. BCRP exhibits broad substrate specificity and
is resistant to a variety of anticancer drugs, such as mitoxantrone, camptothecins,
methotrexate, and anthracyclines 63. In addition, BCRP can also transport a number of
sulfate and glucuronide conjugates 64-66. The typical inhibitors of BCRP include
GF120918, fumitremorgin C (FTC), and Ko134 63,67. The expression of BCRP has been
detected in a variety of malignant tumors, normal tissues with barrier function, and
excretory organs, indicating its important roles in clinical drug resistance and drug
disposition 41,63,67. The importance of BCRP in limiting drug oral absorption and
elimination has been evidenced recently 68. In BCRP knockout mice (bcrp1-/-), it has
been reported that the oral absorption and brain penetration of BCRP substrates are
significantly higher, while the clearance and hepatobiliary elimination are significantly
decreased when compared with the wide type mice 69-71. In recent pre-clinical and
12
clinical studies, inhibition of BCRP by GF120918 has been shown to significantly

increase the bioavailability and decrease the biliary excretion of topotecan 72,73.
Therefore, BCRP represents an important determinant of in vivo drug disposition.
Several recent studies have demonstrated that many naturally occurring flavonoids can
inhibit BCRP. Among all the subclasses of flavonoids tested, flavones seemed to be the
most potent BCRP inhibitors 74,75 (Table 4). The EC50 values of the flavones chrysin and
apigenin for BCRP inhibition (measured as the concentration of flavonoids for producing
50% of the maximal increase in mitoxantrone accumulation in BCRP-overexpressing
MCF-7 MX100 cells) were shown to be within the sub- or low micromolar range (0.39
0.13 M and 1.66 0.55 M, respectively) 75. Glycosylation dramatically decreased the
BCRP-inhibiting activities of some flavonoids 30,74. Recent structure-activity relationship
studies indicated that high BCRP inhibitory potency was associated with flavonoids with
the following characteristics: 1) a planar molecular structure due to the presence of a 2-3
double bond; 2) hydroxylation at position 5; 3) lack of hydroxylation at position 3; 4)
ring-B attached at position 2; and 4) hydrophobic substitution of 6, 7, 8, or 4-hydroxyl
group 75,76. Interestingly, it was shown in a recent study that prenylation at position 6
strongly enhanced both inhibitory potency and specificity of flavones. Compared with
chrysin (IC50 = 4.6 0.5 M), the inhibitory potency of 6-prenylchrysin was significantly
enhanced (IC50 = 0.29 0.06 M). Moreover, 6-prenylchrysin seemed to represent a
specific BCRP inhibitor since no interaction was detected with either P-gp or MRP1 76. It
should be noted that the different IC50 values of chrysin reported in two studies might be
13
due to the different experimental conditions, such as cell lines and substrate concentration
used in the accumulation study 75,76.
Physiological Relevance of Flavonoid Interactions

Flavonoids are abundant in the dietary supplements and in many herbal products. The
consumption of one capsule of chrysin (500 mg) and quercetin (500 mg) results in a
maximal intestinal concentration greater than 1000 M, assuming an intestinal fluid
volume of 1.65 liter 77. In fruit juices, the concentrations of major flavonoids such as
naringin and hesperidin have been reported to be as high as 145 638 mg/L 1 (equivalent
to 250-1100 M) and 200 450 mg/L
2,3
(equivalent to 330-740 M), respectively.
These concentrations are well above the IC50 values of flavonoids required to produce a
significant modulation of P-gp, MRP, and BCRP. Considering the high intestinal
concentrations of flavonoids after the ingestion of dietary supplements and the potent
inhibitory effects of flavonoids on P-gp, MRP, and BCRP, it is very likely that flavonoiddrug interactions might occur in vivo. As discussed above, accumulating evidence of the
in vivo flavonoid-drug interactions has been reported, most of which could be explained
by the modulation of drug-metabolizing enzymes and P-gp.
Summary
In recent years, a wealth of evidence has been generated from in vitro and in vivo studies
showing that many flavonoids interact extensively with efflux drug transporters and play
critical roles in multidrug resistance reversal and drug disposition. The concentrations of
14
many flavonoids required to produce significant modulation on activities of these

transporters appear to be, in general, within the micromolar range, which is achievable in
the intestine after intake of food and, especially dietary supplements. Therefore, altered
disposition of drug transporter substrates following ingestion of a regular diet is likely to
occur. However, most in vivo interaction studies reported to date are primarily focused
on the interactions between the flavonoids and P-gp. The in vivo flavonoid-drug
interactions mediated by other efflux transporter such as BCRP are largely unknown.
Recently, the significant roles of uptake transporters such as OATP transporters
(especially OATP1B1) and MCT transporters (especially MCT 1) in the hepatic and renal
drug disposition have also been demonstrated 35,78-80. However, the in vitro and in vivo
interactions of flavonoids with these uptake transporters have not been well
characterized.
The objectives of this thesis were therefore to investigate the in vitro and in vivo
interactions of flavonoids with efflux transporter P-glycoprotein and BCRP and uptake
transporters OATP1B1 and MCT1. The information obtained from these studies will
help us better understand and predict the potential in vivo flavonoid-drug interactions
mediated by these drug transporters. The altered drug disposition due to these
pharmacokinetic interactions may result in clinically relevant changes in drug ADME
properties and therefore drug efficacy, or, as demonstrated in our studies with hydroxybutyrate, in designing detoxification strategies.
15
References:
1. Ross SA, Ziska DS, Zhao K, ElSohly MA 2000. Variance of common flavonoids by
brand of grapefruit juice. Fitoterapia 71(2):154-161.
2.Erlund I, Meririnne E, Alfthan G, Aro A 2001. Plasma kinetics and urinary excretion of
the flavanones naringenin and hesperetin in humans after ingestion of orange juice and
grapefruit juice. J Nutr 131(2):235-241.
3. Manach C, Morand C, Gil-Izquierdo A, Bouteloup-Demange C, Remesy C 2003.
Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of
two doses of orange juice. Eur J Clin Nutr 57(2):235-242.
4. Kuhnau J 1976. The flavonoids. A class of semi-essential food components: their role
in human nutrition. World Rev Nutr Diet 24:117-191.
5.
Harborne JB, Williams CA 2000. Advances in flavonoid research since 1992.
Phytochemistry 55(6):481-504.
6. Scalbert A, Williamson G 2000. Dietary intake and bioavailability of polyphenols. J
Nutr 130(8S Suppl):2073S-2085S.
7. Vinson JA, Hao Y, Su X, Zubik L 1998. Phenol antioxidant quantity and quality in
foods: vegetables. J Agric Food Chem 46(9):3630-3634.
8. Vinson JA, Su X, Zubik L, Bose P 2001. Phenol antioxidant quantity and quality in
foods: fruits. J Agric Food Chem 49(11):5315-5321.
9.Middleton E, Jr., Kandaswami C, Theoharides TC 2000. The effects of plant flavonoids
on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol
Rev 52(4):673-751.
10. Havsteen BH 2002. The biochemistry and medical significance of the flavonoids.
Pharmacol Ther 96(2-3):67-202.
11. Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D 1993. Dietary
antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study.
Lancet 342(8878):1007-1011.
12. Kohno H, Tanaka T, Kawabata K, Hirose Y, Sugie S, Tsuda H, Mori H 2002.
Silymarin, a naturally occurring polyphenolic antioxidant flavonoid, inhibits
azoxymethane-induced colon carcinogenesis in male F344 rats. Int J Cancer 101(5):461468.
13.Ohnishi N, Yokoyama T 2004. Interactions between medicines and functional foods
or dietary supplements. Keio J Med 53(3):137-150.
14.Izzo AA 2005. Herb-drug interactions: an overview of the clinical evidence. Fundam
Clin Pharmacol 19(1):1-16.
15.Sparreboom A, Cox MC, Acharya MR, Figg WD 2004. Herbal remedies in the United
States: potential adverse interactions with anticancer agents. J Clin Oncol 22(12):24892503.
16.Dresser GK, Bailey DG, Leake BF, Schwarz UI, Dawson PA, Freeman DJ, Kim RB
2002. Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to
decrease the oral availability of fexofenadine. Clin Pharmacol Ther 71(1):11-20.
17.Choi JS, Choi BC, Choi KE 2004. Effect of quercetin on the pharmacokinetics of oral
cyclosporine. Am J Health Syst Pharm 61(22):2406-2409.
16
18. Choi JS, Jo BW, Kim YC 2004. Enhanced paclitaxel bioavailability after oral
administration of paclitaxel or prodrug to rats pretreated with quercetin. Eur J Pharm
Biopharm 57(2):313-318.
19.Wang Z, Hamman MA, Huang SM, Lesko LJ, Hall SD 2002. Effect of St John's wort
on the pharmacokinetics of fexofenadine. Clin Pharmacol Ther 71(6):414-420.
20. Wang YH, Chao PD, Hsiu SL, Wen KC, Hou YC 2004. Lethal quercetin-digoxin
interaction in pigs. Life Sci 74(10):1191-1197.
21. Choi JS, Choi HK, Shin SC 2004. Enhanced bioavailability of paclitaxel after oral
coadministration with flavone in rats. Int J Pharm 275(1-2):165-170.
22.Li X, Yun JK, Choi JS 2007. Effects of morin on the pharmacokinetics of etoposide in
rats. Biopharm Drug Dispos 28(3):151-156.
23.Rajnarayana K, Reddy MS, Vidyasagar J, Krishna DR 2004. Study on the influence of
silymarin pretreatment on metabolism and disposition of metronidazole.
Arzneimittelforschung 54(2):109-113.
24. Harris RZ, Jang GR, Tsunoda S 2003. Dietary effects on drug metabolism and
transport. Clin Pharmacokinet 42(13):1071-1088.
25. Ioannides C 2002. Pharmacokinetic interactions between herbal remedies and
medicinal drugs. Xenobiotica 32(6):451-478.
26. Moon YJ, Wang X, Morris ME 2006. Dietary flavonoids: effects on xenobiotic and
carcinogen metabolism. Toxicol In Vitro 20(2):187-210.
27. Conseil G, Baubichon-Cortay H, Dayan G, Jault JM, Barron D, Di Pietro A 1998.
Flavonoids: a class of modulators with bifunctional interactions at vicinal ATP- and
steroid-binding sites on mouse P-glycoprotein. Proc Natl Acad Sci U S A 95(17):98319836.
28.Cooray HC, Janvilisri T, van Veen HW, Hladky SB, Barrand MA 2004. Interaction of
the breast cancer resistance protein with plant polyphenols. Biochem Biophys Res
Commun 317(1):269-275.
29. Nguyen H, Zhang S, Morris ME 2003. Effect of flavonoids on MRP1-mediated
transport in Panc-1 cells. J Pharm Sci 92(2):250-257.
30. Zhang S, Yang X, Morris ME 2004. Flavonoids are inhibitors of breast cancer
resistance protein (ABCG2)-mediated transport. Mol Pharmacol 65(5):1208-1216.
31. Leslie EM, Deeley RG, Cole SP 2005. Multidrug resistance proteins: role of Pglycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicol Appl
Pharmacol 204(3):216-237.
32.Hagenbuch B, Meier PJ 2004. Organic anion transporting polypeptides of the OATP/
SLC21 family: phylogenetic classification as OATP/ SLCO superfamily, new
nomenclature and molecular/functional properties. Pflugers Arch 447(5):653-665.
33. You G 2004. Towards an understanding of organic anion transporters: structurefunction relationships. Med Res Rev 24(6):762-774.
34.Koepsell H, Schmitt BM, Gorboulev V 2003. Organic cation transporters. Rev Physiol
Biochem Pharmacol 150:36-90.
35. Enerson BE, Drewes LR 2003. Molecular features, regulation, and function of
monocarboxylate transporters: implications for drug delivery. J Pharm Sci 92(8):15311544.
17
36. Ho RH, Kim RB 2005. Transporters and drug therapy: implications for drug
disposition and disease. Clin Pharmacol Ther 78(3):260-277.
37. Allen JD, Schinkel AH 2002. Multidrug resistance and pharmacological protection
mediated by the breast cancer resistance protein (BCRP/ABCG2). Mol Cancer Ther
1(6):427-434.
38.Beringer PM, Slaughter RL 2005. Transporters and their impact on drug disposition.
Ann Pharmacother 39(6):1097-1108.
39. Borst P, Evers R, Kool M, Wijnholds J 2000. A family of drug transporters: the
multidrug resistance-associated proteins. J Natl Cancer Inst 92(16):1295-1302.
40. Konig J, Nies AT, Cui Y, Leier I, Keppler D 1999. Conjugate export pumps of the
multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2mediated drug resistance. Biochim Biophys Acta 1461(2):377-394.
41.
Litman T, Druley TE, Stein WD, Bates SE 2001. From MDR to MXR: new
understanding of multidrug resistance systems, their properties and clinical significance.
Cell Mol Life Sci 58(7):931-959.
42.Gottesman MM, Pastan I 1993. Biochemistry of multidrug resistance mediated by the
multidrug transporter. Annu Rev Biochem 62:385-427.
43.Leonard GD, Fojo T, Bates SE 2003. The role of ABC transporters in clinical practice.
Oncologist 8(5):411-424.
44. Endres CJ, Hsiao P, Chung FS, Unadkat JD 2006. The role of transporters in drug
interactions. Eur J Pharm Sci 27(5):501-517.
45. Critchfield JW, Welsh CJ, Phang JM, Yeh GC 1994. Modulation of adriamycin
accumulation and efflux by flavonoids in HCT-15 colon cells. Activation of Pglycoprotein as a putative mechanism. Biochem Pharmacol 48(7):1437-1445.
46. Scambia G, Ranelletti FO, Panici PB, De Vincenzo R, Bonanno G, Ferrandina G,
Piantelli M, Bussa S, Rumi C, Cianfriglia M, et al. 1994. Quercetin potentiates the effect
of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: Pglycoprotein as a possible target. Cancer Chemother Pharmacol 34(6):459-464.
47.Mitsunaga Y, Takanaga H, Matsuo H, Naito M, Tsuruo T, Ohtani H, Sawada Y 2000.
Effect of bioflavonoids on vincristine transport across blood-brain barrier. Eur J
Pharmacol 395(3):193-201.
48.
Chieli E, Romiti N, Cervelli F, Tongiani R 1995. Effects of flavonols on Pglycoprotein activity in cultured rat hepatocytes. Life Sci 57(19):1741-1751.
49.Shapiro AB, Ling V 1997. Effect of quercetin on Hoechst 33342 transport by purified
and reconstituted P-glycoprotein. Biochem Pharmacol 53(4):587-596.
50.Kitagawa S, Nabekura T, Takahashi T, Nakamura Y, Sakamoto H, Tano H, Hirai M,
Tsukahara G 2005. Structure-activity relationships of the inhibitory effects of flavonoids
on P-glycoprotein-mediated transport in KB-C2 cells. Biol Pharm Bull 28(12):22742278.
51. Shapiro AB, Ling V 1997. Positively cooperative sites for drug transport by Pglycoprotein with distinct drug specificities. Eur J Biochem 250(1):130-137.
52.Di Pietro A, Conseil G, Perez-Victoria JM, Dayan G, Baubichon-Cortay H, Trompier
D, Steinfels E, Jault JM, de Wet H, Maitrejean M, Comte G, Boumendjel A, Mariotte
AM, Dumontet C, McIntosh DB, Goffeau A, Castanys S, Gamarro F, Barron D 2002.
18
Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and

related ABC transporters. Cell Mol Life Sci 59(2):307-322.
53. Boumendjel A, Di Pietro A, Dumontet C, Barron D 2002. Recent advances in the
discovery of flavonoids and analogs with high-affinity binding to P-glycoprotein
responsible for cancer cell multidrug resistance. Med Res Rev 22(5):512-529.
54. Kitagawa S 2006. Inhibitory effects of polyphenols on p-glycoprotein-mediated
transport. Biol Pharm Bull 29(1):1-6.
55. Dupuy J, Larrieu G, Sutra JF, Lespine A, Alvinerie M 2003. Enhancement of
moxidectin bioavailability in lamb by a natural flavonoid: quercetin. Vet Parasitol
112(4):337-347.
56.
Hsiu SL, Hou YC, Wang YH, Tsao CW, Su SF, Chao PD 2002. Quercetin
significantly decreased cyclosporin oral bioavailability in pigs and rats. Life Sci
72(3):227-235.
57. Kruh GD, Belinsky MG 2003. The MRP family of drug efflux pumps. Oncogene
22(47):7537-7552.
58.Trompier D, Baubichon-Cortay H, Chang XB, Maitrejean M, Barron D, Riordon JR,
Di Pietro A 2003. Multiple flavonoid-binding sites within multidrug resistance protein
MRP1. Cell Mol Life Sci 60(10):2164-2177.
59.Leslie EM, Mao Q, Oleschuk CJ, Deeley RG, Cole SP 2001. Modulation of multidrug
resistance protein 1 (MRP1/ABCC1) transport and atpase activities by interaction with
dietary flavonoids. Mol Pharmacol 59(5):1171-1180.
60. van Zanden JJ, Geraets L, Wortelboer HM, van Bladeren PJ, Rietjens IM, Cnubben
NH 2004. Structural requirements for the flavonoid-mediated modulation of glutathione
S-transferase P1-1 and GS-X pump activity in MCF7 breast cancer cells. Biochem
Pharmacol 67(8):1607-1617.
61.van Zanden JJ, Wortelboer HM, Bijlsma S, Punt A, Usta M, Bladeren PJ, Rietjens IM,
Cnubben NH 2005. Quantitative structure activity relationship studies on the flavonoid
mediated inhibition of multidrug resistance proteins 1 and 2. Biochem Pharmacol
69(4):699-708.
62.Lania-Pietrzak B, Michalak K, Hendrich AB, Mosiadz D, Grynkiewicz G, Motohashi
N, Shirataki Y 2005. Modulation of MRP1 protein transport by plant, and synthetically
modified flavonoids. Life Sci 77(15):1879-1891.
63. Mao Q, Unadkat JD 2005. Role of the breast cancer resistance protein (ABCG2) in
drug transport. Aaps J 7(1):E118-133.
64. Suzuki M, Suzuki H, Sugimoto Y, Sugiyama Y 2003. ABCG2 transports sulfated
conjugates of steroids and xenobiotics. J Biol Chem 278(25):22644-22649.
65.Adachi Y, Suzuki H, Schinkel AH, Sugiyama Y 2005. Role of breast cancer resistance
protein (Bcrp1/Abcg2) in the extrusion of glucuronide and sulfate conjugates from
enterocytes to intestinal lumen. Mol Pharmacol 67(3):923-928.
66. Imai Y, Asada S, Tsukahara S, Ishikawa E, Tsuruo T, Sugimoto Y 2003. Breast
cancer resistance protein exports sulfated estrogens but not free estrogens. Mol
Pharmacol 64(3):610-618.
67. Doyle LA, Ross DD 2003. Multidrug resistance mediated by the breast cancer
resistance protein BCRP (ABCG2). Oncogene 22(47):7340-7358.
19
68. van Herwaarden AE, Schinkel AH 2006. The function of breast cancer resistance
protein in epithelial barriers, stem cells and milk secretion of drugs and xenotoxins.
Trends Pharmacol Sci 27(1):10-16.
69. Merino G, Jonker JW, Wagenaar E, van Herwaarden AE, Schinkel AH 2005. The
breast cancer resistance protein (BCRP/ABCG2) affects pharmacokinetics, hepatobiliary
excretion, and milk secretion of the antibiotic nitrofurantoin. Mol Pharmacol 67(5):17581764.
70. Breedveld P, Pluim D, Cipriani G, Wielinga P, van Tellingen O, Schinkel AH,
Schellens JH 2005. The effect of Bcrp1 (Abcg2) on the in vivo pharmacokinetics and
brain penetration of imatinib mesylate (Gleevec): implications for the use of breast cancer
resistance protein and P-glycoprotein inhibitors to enable the brain penetration of
imatinib in patients. Cancer Res 65(7):2577-2582.
71.van Herwaarden AE, Jonker JW, Wagenaar E, Brinkhuis RF, Schellens JH, Beijnen
JH, Schinkel AH 2003. The breast cancer resistance protein (Bcrp1/Abcg2) restricts
exposure to the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
Cancer Res 63(19):6447-6452.
72. Jonker JW, Smit JW, Brinkhuis RF, Maliepaard M, Beijnen JH, Schellens JH,
Schinkel AH 2000. Role of breast cancer resistance protein in the bioavailability and fetal
penetration of topotecan. J Natl Cancer Inst 92(20):1651-1656.
73.Kruijtzer CM, Beijnen JH, Rosing H, ten Bokkel Huinink WW, Schot M, Jewell RC,
Paul EM, Schellens JH 2002. Increased oral bioavailability of topotecan in combination
with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918. J Clin
Oncol 20(13):2943-2950.
74.Imai Y, Tsukahara S, Asada S, Sugimoto Y 2004. Phytoestrogens/flavonoids reverse
breast cancer resistance protein/ABCG2-mediated multidrug resistance. Cancer Res
64(12):4346-4352.
75.Zhang S, Yang X, Coburn RA, Morris ME 2005. Structure activity relationships and
quantitative structure activity relationships for the flavonoid-mediated inhibition of breast
cancer resistance protein. Biochem Pharmacol 70(4):627-639.
76.Ahmed-Belkacem A, Pozza A, Munoz-Martinez F, Bates SE, Castanys S, Gamarro F,
Di Pietro A, Perez-Victoria JM 2005. Flavonoid structure-activity studies identify 6prenylchrysin and tectochrysin as potent and specific inhibitors of breast cancer
resistance protein ABCG2. Cancer Res 65(11):4852-4860.
77.Davies B, Morris T 1993. Physiological parameters in laboratory animals and humans.
Pharm Res 10(7):1093-1095.
78.Kim RB 2003. Organic anion-transporting polypeptide (OATP) transporter family and
drug disposition. Eur J Clin Invest 33 Suppl 2:1-5.
79. Halestrap AP, Meredith D 2004. The SLC16 gene family-from monocarboxylate
transporters (MCTs) to aromatic amino acid transporters and beyond. Pflugers Arch
447(5):619-628.
80.Wang Q, Darling IM, Morris ME 2006. Transport of gamma-hydroxybutyrate in rat
kidney membrane vesicles: Role of monocarboxylate transporters. J Pharmacol Exp Ther
318(2):751-761.
20
81.Tseng E, Liang W, Wallen C, Morris ME 2001. Effect of flavonoids on P-gp mediated

transport in a human breast cancer cell line. AAPS PharmSci 3:S2081.
82. Zhang S, Morris ME 2003. Effects of the flavonoids biochanin A, morin, phloretin,
and silymarin on P-glycoprotein-mediated transport. J Pharmacol Exp Ther 304(3):12581267.
83. Chung SY, Sung MK, Kim NH, Jang JO, Go EJ, Lee HJ 2005. Inhibition of Pglycoprotein by natural products in human breast cancer cells. Arch Pharm Res
28(7):823-828.
84. Jodoin J, Demeule M, Beliveau R 2002. Inhibition of the multidrug resistance Pglycoprotein activity by green tea polyphenols. Biochim Biophys Acta 1542(1-3):149159.
85.Kitagawa S, Nabekura T, Kamiyama S 2004. Inhibition of P-glycoprotein function by
tea catechins in KB-C2 cells. J Pharm Pharmacol 56(8):1001-1005.
86. Qian F, Wei D, Zhang Q, Yang S 2005. Modulation of P-glycoprotein function and
reversal of multidrug resistance by (-)-epigallocatechin gallate in human cancer cells.
Biomed Pharmacother 59(3):64-69.
87.Zhang S, Morris ME 2003. Effect of the flavonoids biochanin A and silymarin on the
P-glycoprotein-mediated transport of digoxin and vinblastine in human intestinal Caco-2
cells. Pharm Res 20(8):1184-1191.
88.Castro AF, Altenberg GA 1997. Inhibition of drug transport by genistein in multidrugresistant cells expressing P-glycoprotein. Biochem Pharmacol 53(1):89-93.
89.Versantvoort CH, Schuurhuis GJ, Pinedo HM, Eekman CA, Kuiper CM, Lankelma J,
Broxterman HJ 1993. Genistein modulates the decreased drug accumulation in non-Pglycoprotein mediated multidrug resistant tumour cells. Br J Cancer 68(5):939-946.
90. Hooijberg JH, Broxterman HJ, Scheffer GL, Vrasdonk C, Heijn M, de Jong MC,
Scheper RJ, Lankelma J, Pinedo HM 1999. Potent interaction of flavopiridol with MRP1.
Br J Cancer 81(2):269-276.
91. Leslie EM, Deeley RG, Cole SP 2003. Bioflavonoid stimulation of glutathione
transport by the 190-kDa multidrug resistance protein 1 (MRP1). Drug Metab Dispos
31(1):11-15.
92. Hooijberg JH, Broxterman HJ, Heijn M, Fles DL, Lankelma J, Pinedo HM 1997.
Modulation by (iso)flavonoids of the ATPase activity of the multidrug resistance protein.
FEBS Lett 413(2):344-348.
93. van Zanden JJ, de Mul A, Wortelboer HM, Usta M, van Bladeren PJ, Rietjens IM,
Cnubben NH 2005. Reversal of in vitro cellular MRP1 and MRP2 mediated vincristine
resistance by the flavonoid myricetin. Biochem Pharmacol 69(11):1657-1665.
94. Kauffmann HM, Pfannschmidt S, Zoller H, Benz A, Vorderstemann B, Webster JI,
Schrenk D 2002. Influence of redox-active compounds and PXR-activators on human
MRP1 and MRP2 gene expression. Toxicology 171(2-3):137-146.
95. Wu CP, Calcagno AM, Hladky SB, Ambudkar SV, Barrand MA 2005. Modulatory
effects of plant phenols on human multidrug-resistance proteins 1, 4 and 5 (ABCC1, 4
and 5). Febs J 272(18):4725-4740.
96.
Versantvoort CH, Broxterman HJ, Lankelma J, Feller N, Pinedo HM 1994.
Competitive inhibition by genistein and ATP dependence of daunorubicin transport in
21
intact MRP overexpressing human small cell lung cancer cells. Biochem Pharmacol
48(6):1129-1136.
97. Takeda Y, Nishio K, Niitani H, Saijo N 1994. Reversal of multidrug resistance by
tyrosine-kinase inhibitors in a non-P-glycoprotein-mediated multidrug-resistant cell line.
Int J Cancer 57(2):229-239.
22
Table 1. Chemical structures of subclasses of flavonoids

Structural
formula
Representative
flavonoids
Substitutions
5
Apigenin
Chrysin
Luteolin
Diosmetin
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
OH
OH
OH
H
OH
O-Me
H
H
H
H
Fisetin
Galangin
Kaempferol
Morin
Myricetin
Quercetin
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
OH
H
H
OH
H
H
H
OH
OH
OH
H
OH
OH
OH
OH
H
H
H
H
OH
H
Hesperitin
Naringenin
OH
OH
OH
OH
H
H
OH
H
O-Me
OH
H
H
Epicatechin
Epigallocatechin
OH
OH
OH
OH
H
H
OH
OH
OH
OH
H
OH
Silibinin
OH
OH
Selane
Biochanin A
Genistein
Daidzein
OH
OH
H
OH
OH
OH
H
H
H
H
H
H
O-Me
OH
OH
H
H
H
Flavones
3'
4'
2'
O
7
6
5'
5
O
Flavonols
O
OH
O
Flavanones
O
Flavanols
O
OH
Flavanolols
O
OH
O
Isoflavones
O
O-Me = Methoxy
23
Table 2. Interactions of flavonoids with P-gp

Flavonoid
Cell Line Used
Substrates
Flavones
Apigenin
MCF-7 breast cancer cells
Daunomycin
Activity
81

MBEC4 endothelial cells
Daunomycin
Vincristine
Activity
Activity
81
Luteolin
Daunomycin
Activity
81
Diosmin
Daunomycin
Activity
81
KB-C2 carinoma cells

Daunorubicin
Daunomycin
Activity
Activity
50

Rat hepatocytes
Daunomycin
Rhodamine123/Doxorubicin
Adriamycin
Expression
or Activity
(Substrate-dependent)
Activity
81
Rhodamine-123
Daunorubicin
Daunomycin
Vincristine
Rhodamine-123/
Doxorubicin
Adriamycin
Activity
Activity
Activity
Biphasic effect
or Activity
Activity
50
50
Chrysin
Flavonols
Fisetin
Galangin
HCT-15 colon cells

Kaempferol

Rat hepatocytes
HCT-15 colon cells
Effect on P-gp
Ref
47
81
48
45
81
47
48
45

MCF-7 and MDA435/LCC6 breast
cancer cells
Daunorubicin
Daunomycin
Activity
Activity
Daunomycin
Activity

Daunorubicin
Daunomycin
Activity
Activity
50
Rhodamine-123
Daunorubicin
Daunomycin
Adriamycin
Rhodamine-123
Vincristine
Hoechst 33342
Adriamycin
Activity
Activity
Activity
Activity
50
Biphasic effect
Activity
or Activity
Activity
47
Activity
Activity
Activity
Activity
50

Rhodamine-123
Daunorubicin
Daunomycin
Vincristine

Vincristine
Daunomycin
Activity
Activity
81
Hesperidin
Vincristine
Activity
47
Naringenin

Daunorubicin
Daunomycin
Activity
Activity
50
Morin
Myricetin
Quercetin

CHRC5
Rat hepatocytes
HCT-15 colon cells
Rutin
Flavanones
Hesperitin
24
82
81
81
81,83
46
49
48
45
81
47
47
83
Naringin
Flavanols
Catechin
Vincristine
Daunomycin
Vincristine
Activity
Activity
Activity
Activity
81

CHRC5 cells
Rhodamine-123
Activity
84
47
81
47
CG
CH C5 cells
Rhodamine-123
Activity
84
Epicatechin
Rhodamine-123
Daunorubicin
Rhodamine-123
Activity
Activity
Activity
85
Rhodamine-123
Daunorubicin
Rhodamine-123
Activity
Activity
Activity
85
Rhodamine-123
Daunorubicin
Rhodamine-123
Daunomycin
Activity
Activity
Activity
Activity
85
Doxorubicin
Rhodamine-123
Daunorubicin
Rhodamine-123
86
Daunomycin
Activity
Digoxin
Vinblastine
Daunomycin
Activity
Activity
Activity
Daunomycin
Activity
Digoxin
Vinblastine
Daunomycin
Activity
Activity
Activity
Daunomycin
Rhodamine-123
Daunorubicin
Activity
Activity
CHRC5 cells
ECG

CHRC5 cells
EGC

CHRC5 cells
EGCG
KB-A1 carcinoma cells

CHRC5 cells
Flavanolols
Silibinin/
silymarin

cancer cells
Caco-2 cells
Isoflavones
Biochanin A

cancer cells
Caco-2 cells
Genistein

BC19/3 breast cancer cells
25
Activity
Activity
Activity
Activity
84
84
84
81
85
84
82,83
87
81
82,83
87
81
81
88
Table 3. Interactions of flavonoids with MRPs

Flavonoid
Flavones
Apigenin
Chrysin
Luteolin
Diosmetin
Diosmin
Flavonols
Fisetin
Galangin
Kaempferol
Cell Line Used
Substrates
GLC4/ADR lung cancer cells (MRP1)

GLC4/ADR lung cancer cells /membrane
vesicles (MRP1)
Vesicles from Hela-MRP1 cells
Panc-1 cells (MRP1)
Daunorubicin
Daunorubicin
Human erythrocytes
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
MCF7-pMTG5 cells
MDCK-MRP1
MDCK-MRP2
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
Panc-1 cells (MRP1)
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
MCF7-pMTG5 cells
MDCK-MRP1
MDCK-MRP2
Vesicles from GLC4/ADR cells
GLC4/ADR lung cancer cells/ membrane
vesicles (MRP1)
Panc-1 cells (MRP1)

MCF7-pMTG5 cells
MDCK-MRP1
MDCK-MRP2
Glutathione
LTC4/E217G
DNM
VBL
BCPCF
Calcein
Calcein
DNM/VBL
Calcein
Calcein
DNM
VBL
DNP-SG
Calcein
Calcein
Calcein
Calcein
DNM/VBL
DNM/VBL
Calcein
Calcein
DNM
VBL
DNP-SG
Calcein
Calcein
Daunorubicin
LTC4/E217G
Glutathione
Morin
Panc-1 cells (MRP1)
DNM/VBL
DNP-SG
Calcein
Calcein
DNM/VBL
Myricetin
MCF7-pMTG5 cells
Human erythrocytes
MDCK-MRP1
MDCK-MRP2
DNP-SG
BCPCF
Calcein
Calcein
LTC4
26
Effect on MRPs
Ref
Accumulation
Accumulation
ATPase Activity
GSH transport
Transport
Accumulation
Accumulation
Efflux via MRP1
Efflux
Efflux
Accumulation
Efflux
Efflux
Accumulation
Accumulation
Efflux via MRP1
Efflux
Efflux
Efflux
Efflux
Accumulation
89
Accumulation
Efflux
Efflux
Accumulation
Accumulation
Efflux via MRP1
Efflux
Efflux
ATPase Activity
Accumulation
ATPase Activity
Transport
GSH transport
ATPase Activity
ADP trapping
Accumulation
Efflux via MRP1
Efflux
Efflux
Accumulation
GSH level
Efflux via MRP1
Efflux via MRP1
Efflux
Efflux
Transport
29
90
59,91
59
29
62
61
29
61
29
60
61
61
29
61
29
60
61
92
90
59
29
60
61
29
60
62
61
59
MCF7-pMTG5 cells
MDCK-MRP1/2
MDCK-MRP1/2
E217G
Glutathione
DNM
VBL
DNP-SG
Vincristine
Calcein
Daunorubicin
Glutathione
LTC4/E217G

Panc-1 cells (MRP1)
DNM/VBL
Panc-1 cells (MRP1)
Quercetin
MCF7-pMTG5 cells
Vesicles from human erythrocytes
HEK293-MRP1
HEK293-MRP5
MDCK-MRP1
MDCK-MRP2
Rutin
Flavanones
Hesperitin
Naringenin
Panc-1 cells (MRP1)
DNM
VBL
Panc-1 cells (MRP1)

DNM/VBL
DNP-SG
cGMP
Calcein
BCECF
Glutathione
LTC4/E217G
HEK293-MRP1
HEK293-MRP5
Panc-1 cells (MRP1)
Naringin
Flavanols
Catechin
EGC
Flavanolols
Silibinin/
silymarin
DNP-SG
DNP-SG
cGMP
Calcein
BCECF
Calcein
Calcein
HEK293-MRP1
HEK293-MRP5
Panc-1 cells (MRP1)
DNM/VBL
DNP-SG
cGMP
Calcein
BCECF
LTC4
DNM/VBL
MCF7-pMTG5 cells
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
DNP-SG
Calcein
Calcein
DNM
VBL
Panc-1 cells (MRP1)
DNM/VBL
Vesicles from BHK-21-MRP1 cells

Human erythrocytes
LTC4
BCPCF
DNP-SG
cGMP
Calcein
BCECF
HEK293-MRP1
HEK293-MRP5
27
Transport
GSH transport
Accumulation
Accumulation
Efflux via MRP1
Efflux
Efflux
Accumulation
GSH transport
Transport
ATPase Activity
ADP trapping
MRP1 mRNA level
Accumulation
GSH level
Efflux via MRP1
Transport via MRP1
Transport via MRP4
Accumulation
Accumulation
Efflux
Efflux
Accumulation
Accumulation
Accumulation
Transport via MRP1
Transport via MRP4
Accumulation
Accumulation
GSH transport
Transport
ATPase Activity
ADP trapping
Accumulation
Transport via MRP1
Transport via MRP4
Accumulation
Accumulation
Transport
Accumulation
29
60
93
61
89
59,91
59
94
29
60
95
61
29
29
95
59,91
59
29
95
59
29
Efflux via MRP1

Efflux
Efflux
Accumulation
Accumulation
60
Accumulation
GSH level
Transport
Efflux via MRP1
Transport via MRP1
Transport via MRP4
Accumulation
Accumulation
29
61
29
58
62
95
Isoflavones
Biochanin A
Genistein
Genistin

Panc-1 cells (MRP1)
Daunorubicin
DNM/VBL
Daunorubicin
K562/TPA leukemia cells (MRP1)

vesicles (MRP1)
Panc-1 cells (MRP1)
Adriamycin
LTC4
Glutathione
DNM/VBL
Human erythrocytes
BCPCF

vesicles (MRP1)
28
Daunorubicin
Daunorubicin
LTC4
Accumulation
Accumulation
GSH level
Accumulation
Transport
Accumulation
ATPase Activity
Accumulation
ATPase Activity
Transport
GSH transport
Accumulation
GSH level
Efflux via MRP1
89
ATPase Activity
Accumulation
ATPase Activity
Transport
92
29
89
96
97
92
90
59
91
29
62
90
59
Table 4. Interactions of flavonoids with BCRP

Flavonoid
Cell Line Used
Substrates
MCF-7/MX100
K562/BCRP
Mitoxantrone
SN38
Accumulation
Reverse resistance
30
MCF-7/MX100
K562/BCRP
Mitoxantrone
SN38
Accumulation
Reverse resistance
30
MCF-7/MX100
K562/BCRP
Mitoxantrone
SN38
Accumulation
Reverse resistance
30
Diosmetin
K562/BCRP
SN38
Reverse resistance
74
Diosmin
K562/BCRP
SN38
No effects
74
MCF-7/MX100
K562/BCRP
Mitoxantrone
SN38
Accumulation
No effects
30
Galangin
K562/BCRP
SN38
Reverse resistance
74
Kaempferol
MCF-7/MX100
K562/BCRP
Mitoxantrone
Mitoxantrone/SN38
Accumulation
Reverse resistance
30
Morin
MCF-7/MX100
Mitoxantrone
Accumulation
30
Myricetin
MCF-7/MX100
K562/BCRP
Mitoxantrone
SN38
Accumulation
No effects
30
MCF-7/MX100
MCF-7/MR and K562/BCRP
Accumulation
Accumulation
30
K562/BCRP
Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Moderate reversal
74
K562/BCRP
SN38
No effects
74
MCF-7/MX100
Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Accumulation
Accumulation
30
Reverse resistance
Accumulation
Reverse resistance
Accumulation
Accumulation
30
MCF-7/MX100
Mitoxantrone
Mitoxantrone/SN38
Topotecan
Mitoxantrone
K562/BCRP
SN38
No effects
74
EGC
MCF-7/MX100
Mitoxantrone
Accumulation
30
EGCG
MCF-7/MX100
Mitoxantrone
Accumulation
30
MCF-7/MX100
Accumulation
Accumulation
30
K562/BCRP
Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Moderate reversal
74
Isoflavones
Biochanin A
MCF-7/MX100
Mitoxantrone
Accumulation
30
Genistein
MCF-7/MX100
Mitoxantrone
Accumulation
30
Flavones
Apigenin
Chrysin
Luteolin
Flavonols
Fisetin
Quercetin
Rutin
Flavanones
Hesperitin
K562/BCRP
Naringenin
Naringin
Flavanols
Catechin
Flavanolols
Silibinin/
silymarin
MCF-7/MX100
K562/BCRP
29
Effect on BCRP
Ref
74
74
74
74
74
74
28
28
74
74
30
28
K562/BCRP
Daidzein
MCF-7/MX100
K562/BCRP
30
Mitoxantrone/SN38
Topotecan
Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Reversal resistance
Accumulation
Accumulation
Accumulation
Moderate reversal
74
30
28
74
MCT1
MCT1
Figure 1. Expression of drug transporters in different organs. OCT, Organic cation

transporter; OAT, organic anion transporter; NTCP, sodium-taurocholate cotransporting
polypeptide; MDR, multidrug resistance protein; BSEP, bile salt export pump; OATP,
organic anion transporting polypeptide; MRP, multidrug resistanceassociated protein;
BCRP, breast cancer resistance protein; PEPT, peptide transporter; MCT,
monocarboxylate transporter; ASBT, apical sodium-dependent bile acid transporter
(adapted from Figure 1 in ref 36 and ref 35,80).
31
CHAPTER 2
Flavonoids as a Novel Class of Human Organic Anion

Transporting Polypeptide OATP1B1 (OATP-C) Modulators
This chapter has been published in Drug Metab Dispos, 33(11): 1666-72, 2005.
32
ABSTRACT
Flavonoids are a class of polyphenolic compounds widely present in the diet and herbal
products. The interactions of flavonoids with certain efflux transporters (e.g., Pglycoprotein), multidrug resistance-associated protein 1 (MRP1), and breast cancer
resistance protein (BCRP)) have been reported; however, their interactions with uptake
transporters are largely unknown. Organic anion transporting polypeptide OATP1B1 is a
liver-specific uptake transporter important in hepatic drug disposition. Our objective was
to evaluate the effects of 20 naturally occurring flavonoids, and some of their
corresponding glycosides, on the uptake of [3H]dehydroepiandrosterone sulfate (DHEAS)
in OATP1B1-expressing and OATP1B1-negative HeLa cells. Many of the tested
flavonoids (including biochanin A, genistein, and epigallocatechin-3-gallate)
significantly inhibited [3H]DHEAS uptake in a concentration-dependent manner in
OATP1B1-expressing cells, with biochanin A being one of the most potent inhibitors
with an IC50 of 11.3 3.22 M. The flavonoids had negligible or small effects in
OATP1B1-negative cells. Four of the eight pairs of tested flavonoids and their
glycosides, namely, genistein/genistin, diosmetin/diosmin,
epigallocatechin/epigallocatechin-3-gallate, and quercetin/rutin, exhibited distinct effects
on [3H]DHEAS uptake. For example, genistin did not inhibit DHEAS uptake while
genistein did and rutin stimulated uptake while quercetin had no effect. [3H]biochanin A
uptake was similar in OATP1B1-expressing and OATP1B1-negative cells, suggesting
that it is not a substrate for OATP1B1. A kinetic study revealed that biochanin A
inhibited [3H]DHEAS uptake in a noncompetitive manner with a Ki of 10.2 1.89 M.
Taken together, these results indicate that flavonoids are a novel class of OATP1B1
33
modulators, suggesting the potential for diet-drug interactions.
34
INTRODUCTION
Organic anion transporting polypeptides OATPs are important membrane transporters
expressed in various organs critical in drug disposition, such as liver, intestine, bloodbrain barrier, kidney, placenta and other organs (Tamai et al., 2000; Hagenbuch and
Meier, 2004). This transporter family mediates sodium-independent uptake of a broad
spectrum of amphipathic organic anions as well as steroid conjugates, organic cations,
and xenobiotics (Hagenbuch and Meier, 2004; Mikkaichi et al., 2004). A number of
OATPs share some overlap in substrate specificity with certain efflux transporters such as
P-glycoprotein (Cvetkovic et al., 1999), multidrug resistance-associated protein 2
(MRP2) (Su et al., 2004; Spears et al., 2005), and breast cancer resistance protein
(BCRP) (Nozawa et al., 2005). To date, thirty-six OATPs have been identified in human,
rat, and mouse, and eleven of them were cloned from human tissues (Hagenbuch and
Meier, 2004). Among all the human OATPs, OATP1B1 (previously known as LST-1,
OATP-C, OATP-2, and SLC21A6) that has been identified by a number of groups (Abe et
al., 1999; Hsiang et al., 1999; Konig et al., 2000; Tamai et al., 2000) represents one of the
best characterized human OATPs. Molecular characterization revealed that OATP1B1
consists of 691 amino acids with an apparent molecular mass of 84 kDa, which is reduced
to 54 kDa after deglycosylation (Konig et al., 2000). OATP1B1 is specifically expressed
on the basolateral (sinusoidal) membrane of human hepatocytes and transports a broad
range of compounds such as bile acids, conjugated steroids, thyroid hormones, peptides,
and drugs including rifampicin and pravastatin (Abe et al., 1999; Konig et al., 2000;
Hagenbuch and Meier, 2004). Due to its liver-specific expression and its capacity for
transporting a large number of structurally different compounds, it has been suggested
35
that OATP1B1 plays an important role in hepatic drug uptake and elimination (Kim,
2003). Thus, modulators of OATP1B1 may alter the pharmacokinetics of OATP1B1
substrate drugs, causing potential drug-drug interactions, which is exemplified by the
interactions between cerivastatin and cyclosporine A (Shitara et al., 2003) and between
cerivastatin and gemfibrozil (Shitara et al., 2004).
Flavonoids are a class of polyphenolic compounds widely present in fruits, vegetables

and plant-derived beverages, and in many herbal products marketed as over-the-counter
dietary supplements. The average daily intake of total flavonoids from the U.S. diet was
estimated to be 1 g (Kuhnau, 1976), although this may be an overestimation and actual
intake may be lower (Harborne and Williams, 2000). Flavonoids have long been
associated with a variety of biochemical and pharmacological properties including
antioxidant, antiviral, anticarcinogenic and anti-inflammatory activities, with no or low
toxicity (Middleton et al., 2000; Havsteen, 2002). These health-promoting activities
indicate that flavonoids may play a protective role in the prevention of cancer, coronary
heart disease as well as other age-related degenerative diseases (Hertog et al., 1993;
Havsteen, 2002; Kohno et al., 2002). Recently, numerous studies have indicated that
flavonoids could interact with several efflux transporters such as P-glycoprotein, MRP1,
and BCRP, suggesting potential drug-flavonoid interactions (Conseil et al., 1998; Leslie
et al., 2001; Zhang and Morris, 2003b; Zhang et al., 2004d). However, the interactions
between flavonoids and uptake tranporters, especially OATPs, have not been well
characterized and remain largely unknown.
36
Recent in vitro and in vivo fruit juice and fexofenadine interaction studies have indicated
that fruit juices and constituents preferentially inhibit OATPs rather than P-glycoprotein
(Dresser et al., 2002; Kamath et al., 2005). Fruit juices at 5% of normal strength potently
inhibit human OATP1A2 (OATP-A) and at least 2 rat oatp transporters. Several
flavonoids (quercetin, naringenin and hesperitin) and their glycosides, at a concentration
of 50 M, significantly inhibit fexofenadine uptake mediated by rat Oatp1a5 (Oatp3)
(Dresser et al., 2002), suggesting that flavonoids could interact with some OATP
isoforms. However, to our knowledge, the interaction between flavonoids and human
OATP1B1 has not been reported. Based on the studies with fruit juices, we hypothesized
that some naturally occurring flavonoids may also interact with OATP1B1. In the
present study, we examined the effects of 20 flavonoids on the uptake of
dehydroepiandrosterone sulfate (DHEAS), a well-known OATP1B1 substrate, in both
OATP1B1-expressing and OATP1B1-negative cells, and investigated the potential
mechanism of interaction between flavonoids and OATP1B1.
37
MATERIALS AND METHODS

Materials
[3H]DHEAS (60 Ci/mmol, > 97% purity) was purchased from Perkin Elmer Life
Sciences (Boston, MA). [3H]biochanin A (14 Ci/mmol, > 97% purity) was purchased
from Moravek Biochemicals (Brea, CA). Flavonoids and their glycosides were
purchased from Sigma-Aldrich (St. Louis, MO) and Indofine (Hillsborough, NJ).
Silymarin is comprised of silibinin (major component), silydianin, and silychristin, and
the molar concentration was calculated based on the molecular weight of silibinin. All
other reagents were of either HPLC or analytical grade, and unless otherwise stated, were
purchased from Sigma-Aldrich (St. Louis, MO). Dulbeccos Modified Eagle Medium
(DMEM), fetal bovine serum, penicillin, and streptomycin were purchased from
Invitrogen (Carlsbad, CA).
Cell Culture
HeLa cells stably transfected with human OATP1B1 were cultured under similar
conditions as described previously. Under these conditions, the expression of OATP1B1
in these HeLa cells has been shown to be induced significantly and can be readily
detected by Western blot analysis (Wang et al., 2003). Briefly, HeLa cells were cultured
in 75-cm2 flasks with DMEM medium supplemented with 10% fetal bovine serum at
37oC in a humidified 5% CO2 /95 % air atmosphere. The culture medium also contained
100 units/ml penicillin and 100 g/ml streptomycin. For uptake and inhibition
experiments, the cells were seeded at a density of 5 105 cells per 35-mm (diameter)
petri-dish. For induction of OATP1B1, cells were cultured in medium containing 100
38
M ZnSO4. After a 24-hour incubation, an additional 50 M ZnSO4 was added to this

medium for the final 24 hours before use.
Uptake Studies
Cells were washed three times with 2 ml of uptake buffer (135 mM NaCl, 1.2 mM
MgCl2, 0.81 mM MgSO4, 27.8 mM glucose, 2.5 mM CaCl2, and 25 mM HEPES, pH
7.2). They were then preincubated with 1 ml of uptake buffer at 37oC for 10 min. At the
end of preincubation period, the uptake of [3H]DHEAS (0.5 M) was performed by
incubating cells with 1 ml uptake buffer containing [3H]DHEAS together with 50 M
flavonoids or the vehicle (0.3 % dimethyl sulfoxide at 37oC for 5 min. Rifampicin (100
M) was used as a positive control. The uptake reaction was stopped by aspiration,
followed by washing with 3 ml ice-cold buffer (137 mM NaCl, and 14 mM Tris base, pH
7.2) three times. The same procedures were used for the [3H]biochanin A (1 M) uptake
experiments. Cells were then solubilized using a solution of 1 ml 0.3N NaOH and 1%
sodium dodecyl sulfate (SDS). Aliquots were used to determine the radioactivity by
liquid scintillation counting (1900 CA, Tri-Carb liquid scintillation analyzer;
PerkinElmer Life Sciences). Results were normalized by protein content determined by
bicinchoninic acid protein assay. The uptake of [3H]DHEAS was expressed as
percentage of the control (uptake in OATP1B1-negative cells in the presence of 0.3%
dimethyl sulfoxide).
39
Concentration-Dependent Inhibition Studies

Concentration-dependent studies were performed following the same procedures as
described above except that the effects of flavonoids were tested at varying
concentrations (0.3 50 M). Concentration-dependent effects were determined for
three flavonoids (biochanin A, genistein, and epigallocatechin-3-gallate (EGCG)).
Specific uptake was obtained by subtracting [3H]DHEAS uptake into OATP1B1-negative
cells from the uptake into OATP1B1-expressing cells. The specific uptake of
[3H]DHEAS was expressed as percentage of the control (in the presence of 0.3%
DMSO). The IC50 value, the concentration of flavonoid required to inhibit 50% of
specific [3H]DHEAS uptake, was obtained by fitting data with the following equation
using WinNonlin Professional 2.1 (Pharsight, Mountain View, CA):
F = 100 (1
I max C
IC50 + C
),
where C is the concentration of flavonoid, F is the percentage of the specific uptake of

[3H]DHEAS, Imax is the maximum percentage of inhibition, and is the Hill coefficient.
For each flavonoid, the IC50 value (expressed as mean S.D.) was determined from three
separate experiments and each experiment had triplicate measurements.
Inhibition of DHEAS Uptake by Biochanin A

To determine the inhibition kinetics, [3H]DHEAS (0.5, 1, and 5 M) uptake was
determined at 5 min both in the absence and presence of varying concentrations of
biochanin A. The specific uptake was obtained by subtracting [3H]DHEAS uptake into
OATP1B1-negative cells from the uptake into OATP1B1-expressing cells.
40
Kinetic
parameters were obtained using nonlinear regression fitting (WinNonlin) according to the
following equations:
v=
Vmax /(1 + I / K i ) C
(for noncompetitive kinetics)
Km + C
v=
Vmax C
(for competitive kinetics)
K m /(1 + I / K i ) + C
where v is the specific uptake velocity of the substrate (pmol/mg protein/min), Vmax is
the maximum specific uptake rate (pmol/mg protein/min), C is the substrate
concentration (M), Km is the Michaelis-Menton constant (M), I is the inhibitor
concentration, and Ki is the biochanin A inhibition constant. The kinetic model selection
was based on visual examination and the goodness of fit (CV % and Akaikes
Information Criterion). Kinetic parameters (expressed as mean S.D.) were determined
from three separate triplicate experiments.
Statistical Analysis
Data were analyzed for statistically significant differences using ANOVA test followed
by a Dunnetts post hoc test or by a Students t test. P values < 0.05 were considered
statistically significant.
41
RESULTS
Effects of Flavonoids on [3H]DHEAS Uptake. To determine whether flavonoids
modulate OATP1B1 activity, uptake studies were performed with DHEAS (a known
OATP1B1 substrate) in OATP1B1-expressing (induced by Zn2+) and OATP1B1-negative
(non-induced) HeLa cells in the presence or absence of flavonoids (50 M) (Fig.1). The
expression of OATP1B1 under the same culture condition has been characterized
previously (Wang et al., 2003). The time-course study of [3H]DHEAS uptake indicated
that the uptake of DHEAS was linear within a 5 min time period (data not shown).
Therefore, we chose 5 min as an appropriate time for [3H]DHEAS uptake studies. As
shown in Table 1, in the absence of flavonoids, [3H]DHEAS uptake in OATP1B1expressing cells was significantly higher than that in OATP1B1-negative control cells
(452 105 % versus 100 7.93 % of the control, p < 0.001). Rifampicin (100 M) (a
known OATP1B1 inhibitor) significantly decreased [3H]DHEAS uptake in OATP1B1expressing cells (p < 0.01, % decrease in mean value: -56.8 % ) with no significant
effects on [3H]DHEAS uptake in OATP1B1-negative cells ( p > 0.05, % decrease in
mean value: -2.88 %). All the tested flavonoids except for benzoflavone, chrysin,
galangin, and kaempferol, produced a significant decrease in [3H]DHEAS uptake in
OATP-expressing cells with no or small effects on [3H]DHEAS uptake in OATP1B1negative cells. The flavonoids biochanin A, fisetin, silibinin, and silymarin produced the
greatest effects, resulting in significant decreases in [3H]DHEAS uptake (p < 0.01, %
decrease in mean values: -68.6 %, -56.5 %, -66.5 %, and 68.9 %, respectively)
comparable to or even greater than that caused by rifampicin (-56.8 %) in OATP1B1expressing cells. In OATP1B1-negative cells, the flavonoids apigenin, chrysin, fisetin,
42
galangin, luteolin, and silibinin, at a concentration of 50 M, produced statistically

significant increases in [3H]DHEAS uptake (p < 0.01 or p < 0.05, % increase in mean
values: +92.8 %, +17.5 %, +37.9 %, +17.8 %, +17.8 %, and +13.9 %, respectively).
Effects of Flavonoids and Their Glycosides on [3H]DHEAS Uptake. Many flavonoids

exist in both aglycone and glycone forms. Different biochemical and pharmacological
activities between these two forms have been reported for a number of flavonoids (Kwon
et al., 2004; Lin et al., 2005). To compare the effects of flavonoids and their glycosides
on [3H]DHEAS uptake, eight pairs of flavonoids and corresponding glycosides were
characterized in both OATP1B1-expressing and OATP1B1-negative cells (Fig. 2). As
shown in Table 2, in OATP1B1-expressing cells, both daidzein and its glycoside daidzin
had no effects on [3H]DHEAS uptake (p > 0.05, % change in mean values: +4.34 % and
2.94 %, respectively). Three pairs of flavonoids and their corresponding glycosides
(hesperitin/hesperidin, naringenin/naringin, and phloretin/phloridzin) all significantly
decreased [3H]DHEAS uptake (p< 0.01). On the other hand, genistein and diosmetin
significantly inhibited [3H]DHEAS uptake (p < 0.01 or p < 0.05, % decrease in mean
values: -61.6 % and 20.6 %, respectively) with no significant effects found for their
glycosides genistin and diosmin (p > 0.05, % change in mean values: +4.88 % and 1.36
%, respectively). In contrast, epigallocatechin (EGC) and quercetin had no significant
effects (p > 0.05, % change in mean values: -6.70 % and 11.8 %, respectively) on
[3H]DHEAS uptake while their glycosides epigallocatechin-3-gallate (EGCG) and rutin
significantly decreased and increased [3H]DHEAS uptake, respectively (p < 0.01, %
change in mean values: -51.0 % and +81.1 %, respectively). In OATP1B1-negative
43
control cells, most flavonoids and their glycosides had negligible effects on [3H]DHEAS
uptake. However, diosmetin, hesperitin, and naringenin, at a concentration of 50 M,
produced small but statistically significant increases in [3H]DHEAS uptake (p < 0.01, %
increase in mean values: +59.1 %, +27.5 %, and +26.0 %, respectively). EGC, EGCG,
and genistin resulted in small but statistically significant decreases in [3H]DHEAS uptake
(p < 0.01 or p < 0.05, % decrease in mean values: -16.1 %, -17.6 %, and 12.8 %,
respectively).
Concentration-Dependent Effects of Flavonoids on [3H]DHEAS Uptake. The

concentration-dependent effects of flavonoids on [3H]DHEAS uptake were investigated
for three flavonoids, biochanin A, genistein, and EGCG, which demonstrated high
OATP1B1 inhibition activity when tested at a concentration of 50 M. As shown in Fig.
3, the inhibition of [3H]DHEAS uptake by biochanin A, genistein, and EGCG were
flavonoid concentration-dependent. At 10 M, all the tested flavonoids had significant
effects on [3H]DHEAS uptake (p < 0.001). In addition, 3 M biochanin A significantly
decreased [3H]DHEAS uptake (Fig.3, 85.8 4.14 % of the control, p < 0.001). The IC50
values for biochanin A, genistein, and EGCG for inhibiting [3H]DHEAS uptake were
11.3 3.22, 14.9 3.76, 14.1 1.44 M, respectively.
Uptake of [3H]biochanin A by OATP1B1. To further characterize the OATP1B1modulating activities of the flavonoids, biochanin A was used as a model flavonoid and
the uptake of [3H]biochanin A was studied in both OATP1B1-expressing and OATP1B1negative cells. As shown in Fig. 4, [3H]biochanin A uptake was similar in OATP1B1-
44
expressing and OATP1B1-negative HeLa cells in the absence of 100 M rifampicin (1.27
0.23 and 1.20 0.22 nmol/mg protein, respectively, p > 0.05), as well as in the
presence of 100 M rifampicin (1.30 0.23 and 1.23 0.21 nmol/mg protein,
respectively, p > 0.05).
Inhibition of OATP1B1-mediated Uptake of [3H]DHEAS by Biochanin A. To gain

insight into the mechanism underlying the inhibition of OATP1B1-mediated uptake of
DHEAS by biochanin A, [3H]DHEAS uptake kinetics in the absence and presence of
biochanin A was determined by Dixon plot analysis as shown in Fig. 5. Based on visual
examination and computer fitting results (data not shown), the kinetic study revealed that
biochanin A inhibited OATP1B1-mediated [3H]DHEAS uptake in a noncompetitive
manner with a Ki value of 10.2 1.89 M, and a Vmax value of 154 7.61 pmol/mg
protein.
45
DISCUSSION
OATPs are key membrane transporters that mediate the sodium-independent uptake of a
wide range of endogenous and exogenous compounds, including drugs in clinical use
(Hagenbuch and Meier, 2004). The importance of this transporter family in drug
disposition has been increasingly recognized. The co-expression of OATPs with some
efflux transporters in multiple tissues and their partially overlapping substrate specificity
suggest that OATPs may play an important role together with efflux transporters in
overall drug absorption and disposition (Kim, 2003). OATP1B1, a well studied human
OATP, is one of the major sodium-independent bile salt transporters localized on the
basolateral membrane of human hepatocytes (Trauner and Boyer, 2003). OATP1B1
transports a large number of structurally divergent compounds and has been implicated as
an important determinant in hepatic drug uptake and elimination (Kim, 2003). Thus,
analogous to the cases of efflux transporters, modulation of OATP1B1 may alter the
pharmacokinetics of OATP1B1 substrate drugs, causing potential drug-drug interactions.
Flavonoids are a class of polyphenolic compounds widely present in diet and herbal
products with exceptional safety records (Havsteen, 2002). In addition to their many
anticancer properties, flavonoids have been found to interact with several efflux
transporters (Conseil et al., 1998; Leslie et al., 2001; Zhang et al., 2004d). Recent studies
have indicated that several flavonoids rich in fruit juices significantly inhibit
fexofenadine uptake mediated by rat Oatp1a5 (Oatp3) at a concentration of 50 M
(Dresser et al., 2002). Therefore, it is likely that some flavonoids may also be modulators
of OATP1B1. The elucidation of the effects of flavonoids on OATP1B1-mediated
46
transport may help to better predict potential flavonoid-drug pharmacokinetic

interactions, which is an important issue considering the wide-spread consumption of
large amounts of flavonoids in food or herbal products.
In the present study, we examined the effects of 20 flavonoids, representing all the
chemical subclasses of flavonoids, on OATP1B1-mediated DHEAS transport. DHEAS,
one of the major steroids in the systemic circulation, is a known substrate for several
OATPs (Kim, 2003) as well as MRPs (Zelcer et al., 2003). For OATP1B1, a 3.2 to 7.3
fold ratio of DHEAS uptake in OATP1B1-expressing cells over control cells was
observed in different experimental systems (Konig et al., 2000; Kullak-Ublick et al.,
2001). The high uptake ratio of DHEAS indicates that it is a suitable substrate for the
study of OATP1B1-mediated transport. In this study, we used a OATP1B1-expressing
HeLa cell line, in which the expression of OATP1B1 can be substantially induced by
Zn2+ (Wang et al., 2003). Although very low levels of some endogenous transporters,
such as P-glycoprotein and MRP1/2 (Sakaeda et al., 2002), are present in parent HeLa
cells and may possibly contribute to the DHEAS transport, we observed a 3 to 4 fold net
difference in DHEAS uptake between OATP1B1-expressing and OATP1B1-negative
cells in the absence of flavonoids. This net difference in DHEAS uptake is most likely
due to OATP1B1 since substantial OATP1B1 expression is induced in the presence of
Zn2+and may represent the major difference between OATP1B1-expressing and
OATP1B1-negative cells. In OATP1B1-expressing cells, the flavonoids biochanin A,
fisetin, silibinin, and silymarin (50 M) produced significant decreases in [3H]DHEAS
uptake, which were comparable to or even greater than that caused by 100 M
47
rifampicin, indicating that biochanin A, fisetin, silibinin, and silymarin strongly inhibited
the OATP1B1-mediated uptake of [3H]DHEAS. The flavonoids apigenin, luteolin,
morin, and myricetin produced smaller, but statistically significant decreases in
[3H]DHEAS uptake in OATP1B1-expressing cells with no or small effects in OATP1B1negative cells, suggesting that these flavonoids are also OATP1B1 inhibitors. Some
flavonoids (apigenin, chrysin, fisetin, galangin, luteolin, and silibinin), at a concentration
of 50 M, produced statistically significant increases in [3H]DHEAS uptake in
OATP1B1-negative cells. The exact reason(s) for this is currently unknown but might be
possibly due to alterations in cell membrane permeability by these flavonoids at high
concentrations and/or the interaction of these flavonoids with an endogenous
transporter(s) expressed in HeLa cells that mediates [3H]DHEAS transport. A very low
level of endogenous MRP1, which was detected by real time polymerase chain reaction
(RT-PCR) in HeLa cells (Sakaeda et al., 2002), may be responsible for the small changes
in [3H]DHEAS uptake observed since some flavonoids may also interact with MRP1
(Nguyen et al., 2003) and DHEAS seems to be transported by MRP1 as well (Zelcer et
al., 2003).
To compare the effects of flavonoids and their glycosides on [3H]DHEAS uptake, eight
pairs of flavonoids and their corresponding glycosides were characterized in both
OATP1B1-expressing and OATP1B1-negative cells. In OATP1B1-expressing cells,
three pairs of flavonoids and glycosides (hesperitin/hesperidin, naringenin/naringin, and
phloretin/phloridzin) all significantly decreased [3H]DHEAS uptake (p< 0.01) when
compared to the uptake value in the absence of flavonoids. Fruit juices and their major
48
flavonoid components hesperitin, naringenin, and their glycosides have been shown to
significantly inhibit fexofenadine uptake mediated by rat Oatp1a5 (Oatp3) (Dresser et al.,
2002). The potent inhibitory effects of these flavonoids on OATP1B1-mediated uptake
of [3H]DHEAS indicate that fruit juices may also represent potent inhibitors of
OATP1B1, suggesting the potential for hepatic drug interactions. Interestingly, both
daidzein and its glycoside daidzin had no effect on [3H]DHEAS uptake. Daidzein is an
isoflavone with a similar chemical structure to that of biochanin A and genistein, except
that no hydroxyl group is present at the 5-position of the A-ring in the daidzein structure.
Considering the potent inhibitory effects of biochanin A and genistein on [3H]DHEAS
uptake, it is reasonable to speculate that attachment of a hydroxyl group at 5-position
may markedly attenuate or totally abolish OATP1B1-inhibitory activity of the flavonoids.
On the other hand, genistein and diosmetin significantly inhibited [3H]DHEAS uptake
with no significant effects found for their glycosides genistin and diosmin. In contrast,
EGC and quercetin had no significant effects on [3H]DHEAS uptake while their
glycosides EGCG and rutin significantly decreased and increased [3H]DHEAS uptake,
respectively. These results indicate that attachment of a sugar moiety may differently
modulate OATP1B1 activity, suggesting glycosidation of flavonoid aglycones should be
considered as an important modulator of the biological activities of flavonoids.
Interestingly, rutin is the only flavonoid identified in this study that can stimulate
OATP1B1 activity. Although the reason for such increase is not clear at present,
increased substrate uptake has been reported for some other OATP isoforms, such as rat
Oatp1a4 (Oatp2) (Sugiyama et al., 2002) and human OATP2B1 (OATP-B) (Tamai et al.,
2001). Future studies will be essential to clarify the underlying mechanisms.
49
It should be noted that, in OATP1B1-negative control cells, most flavonoids and their
glycosides had negligible effects on [3H]DHEAS uptake with the exception of diosmetin,
hesperitin, and naringenin, which, at a concentration of 50 M, produced small but
statistically significant increases in [3H]DHEAS uptake. Interestingly, EGC, EGCG, and
genistin resulted in small but statistically significant decreases in [3H]DHEAS uptake.
The exact reason(s) for these changes is currently unknown, but may possibly be due to
inhibition of a low endogenous OATP1B1 expression in OATP1B1-negative HeLa cells,
resulting in lower uptake, although OATP1B1 expression in these cells was not
detectable by Western blot analysis (Wang et al., 2003). Additionally, inhibition of an
efflux transporter such as MRP1 may result in higher uptake values.
To investigate the concentration-dependent effects of flavonoids on OATP1B1-mediated

transport, the effects of three flavonoids with strong OATP1B1-inhibitory activities when
tested at a concentration of 50 M, namely, biochanin A, genistein, and EGCG, were
evaluated. These three flavonoids demonstrated concentration-dependent inhibition of
OATP1B1. Biochanin A seems to be one of the most potent OATP1B1 inhibitors with
an IC50 value of 11.3 3.22 M. Significant inhibition of OATP1B1 by biochanin A
could be produced at concentrations as low as 3 M. Our laboratory, as well as others,
previously investigated flavonoids as inhibitors of P-glycoprotein, MRP-, and BCRPmediated drug resistance in cancer cell lines and demonstrated that some flavonoids, such
as biochanin A and genistein, can also inhibit P-glycoprotein, MRP1, and BCRP (Castro
and Altenberg, 1997; Nguyen et al., 2003; Zhang and Morris, 2003b; Zhang et al.,
50
2004d), indicating that these flavonoids may potentially affect in vivo drug disposition
via inhibiting different transporter systems. Some other flavonoids, however,
preferentially inhibit one major transporter over the others. For example, chrysin has
been shown as a potent BCRP inhibitor (Zhang et al., 2004d) while it has no OATP1B1
inhibitory activity, as shown in the present study. In contrast, EGCG is not a BCRP
inhibitor (Zhang et al., 2004d) while it can effectively inhibit OATP1B1 activity. Futures
studies regarding the structure activity relationships for the flavonoids-mediated
inhibition of OATP1B1 together with the existing information on some major efflux
transporters may help us better understand and design inhibitors with preferred potency
and specificity.
To understand the mechanism of OATP1B1 inhibition by flavonoids, biochanin A was

selected as a model flavonoid and the interaction between biochanin A and OATP1B1
was further characterized. The uptake studies indicated that [3H]biochanin A uptake was
similar in OATP1B1-expressing and OATP1B1-negative HeLa cells in the absence of
100 M rifampicin, as well as in the presence of 100 M rifampicin, suggesting that
biochanin A is not likely a substrate of OATP1B1. This is expected since biochanin A
itself is a neutral compound and most substrates of OATP1B1 identified so far are
organic anions (Hagenbuch and Meier, 2004). The kinetics study revealed that biochanin
A inhibited OATP1B1-mediated [3H]DHEAS uptake in a noncompetitive manner with a
Ki value of 10.2 1.89 M, and a Vmax value of 154 7.61 pmol/mg protein. This is in
general agreement with some other OATP1B1 inhibitors, such as cyclosporin A and
51
indinavir (Tirona et al., 2003), which have been shown to inhibit OATP1B1-mediated
estradiol glucuronide uptake in a noncompetitive manner as well (Campbell et al., 2004).
In conclusion, many naturally occurring flavonoids can modulate OATP1B1-mediated

uptake of [3H]DHEAS, indicating they are a novel class of OATP1B1 modulators. From
studies with biochanin A, investigating the possible mechanism(s) of this interaction, we
have found that biochanin A is not a substrate for OATP1B1 and it represents a
noncompetitive inhibitor. From the drug interaction point of view, considering the high
consumption of flavonoids in the diet and in herbal products, hepatic uptake interactions
of these flavonoids with drugs that are OATP1B1 substrates, may result following
coadministration.
52
REFERENCES
Abe T, Kakyo M, Tokui T, Nakagomi R, Nishio T, Nakai D, Nomura H, Unno M, Suzuki
M, Naitoh T, Matsuno S and Yawo H (1999) Identification of a novel gene family
encoding human liver-specific organic anion transporter LST-1. J Biol Chem
274:17159-17163.
Campbell SD, de Morais SM and Xu JJ (2004) Inhibition of human organic anion
transporting polypeptide OATP 1B1 as a mechanism of drug-induced
hyperbilirubinemia. Chem Biol Interact 150:179-187.
Castro AF and Altenberg GA (1997) Inhibition of drug transport by genistein in
multidrug-resistant cells expressing P-glycoprotein. Biochem Pharmacol 53:8993.
Conseil G, Baubichon-Cortay H, Dayan G, Jault JM, Barron D and Di Pietro A (1998)
Flavonoids: a class of modulators with bifunctional interactions at vicinal ATPand steroid-binding sites on mouse P-glycoprotein. Proc Natl Acad Sci U S A
95:9831-9836.
Cvetkovic M, Leake B, Fromm MF, Wilkinson GR and Kim RB (1999) OATP and Pglycoprotein transporters mediate the cellular uptake and excretion of
fexofenadine. Drug Metab Dispos 27:866-871.
Dresser GK, Bailey DG, Leake BF, Schwarz UI, Dawson PA, Freeman DJ and Kim RB
(2002) Fruit juices inhibit organic anion transporting polypeptide-mediated drug
uptake to decrease the oral availability of fexofenadine. Clin Pharmacol Ther
71:11-20.
Hagenbuch B and Meier PJ (2004) Organic anion transporting polypeptides of the OATP/
nomenclature and molecular/functional properties. Pflugers Arch 447:653-665.
Harborne JB and Williams CA (2000) Advances in flavonoid research since 1992.
Phytochemistry 55:481-504.
Havsteen BH (2002) The biochemistry and medical significance of the flavonoids.
Pharmacol Ther 96:67-202.
Hertog MG, Feskens EJ, Hollman PC, Katan MB and Kromhout D (1993) Dietary
antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly
Study. Lancet 342:1007-1011.
Hsiang B, Zhu Y, Wang Z, Wu Y, Sasseville V, Yang WP and Kirchgessner TG (1999)
A novel human hepatic organic anion transporting polypeptide (OATP2).
Identification of a liver-specific human organic anion transporting polypeptide
and identification of rat and human hydroxymethylglutaryl-CoA reductase
inhibitor transporters. J Biol Chem 274:37161-37168.
Kamath AV, Yao M, Zhang Y and Chong S (2005) Effect of fruit juices on the oral
bioavailability of fexofenadine in rats. J Pharm Sci 94:233-239.
Kim RB (2003) Organic anion-transporting polypeptide (OATP) transporter family and
drug disposition. Eur J Clin Invest 33 Suppl 2:1-5.
Kohno H, Tanaka T, Kawabata K, Hirose Y, Sugie S, Tsuda H and Mori H (2002)
53
azoxymethane-induced colon carcinogenesis in male F344 rats. Int J Cancer

101:461-468.
Konig J, Cui Y, Nies AT and Keppler D (2000) A novel human organic anion
transporting polypeptide localized to the basolateral hepatocyte membrane. Am J
Physiol Gastrointest Liver Physiol 278:G156-164.
Kuhnau J (1976) The flavonoids. A class of semi-essential food components: their role in
human nutrition. World Rev Nutr Diet 24:117-191.
Kullak-Ublick GA, Ismair MG, Stieger B, Landmann L, Huber R, Pizzagalli F, Fattinger
K, Meier PJ and Hagenbuch B (2001) Organic anion-transporting polypeptide B
(OATP-B) and its functional comparison with three other OATPs of human liver.
Gastroenterology 120:525-533.
Kwon YS, Kim SS, Sohn SJ, Kong PJ, Cheong IY, Kim CM and Chun W (2004)
Modulation of suppressive activity of lipopolysaccharide-induced nitric oxide
production by glycosidation of flavonoids. Arch Pharm Res 27:751-756.
Leslie EM, Mao Q, Oleschuk CJ, Deeley RG and Cole SP (2001) Modulation of
multidrug resistance protein 1 (MRP1/ABCC1) transport and atpase activities by
interaction with dietary flavonoids. Mol Pharmacol 59:1171-1180.
Lin HY, Shen SC and Chen YC (2005) Anti-inflammatory effect of heme oxygenase 1:
glycosylation and nitric oxide inhibition in macrophages. J Cell Physiol 202:579590.
Middleton E, Jr., Kandaswami C and Theoharides TC (2000) The effects of plant
flavonoids on mammalian cells: implications for inflammation, heart disease, and
cancer. Pharmacol Rev 52:673-751.
Mikkaichi T, Suzuki T, Tanemoto M, Ito S and Abe T (2004) The organic anion
transporter (OATP) family. Drug Metab Pharmacokinet 19:171-179.
Nguyen H, Zhang S and Morris ME (2003) Effect of flavonoids on MRP1-mediated
transport in Panc-1 cells. J Pharm Sci 92:250-257.
Nozawa T, Minami H, Sugiura S, Tsuji A and Tamai I (2005) Role of organic anion
transporter OATP1B1 (OATP-C) in hepatic uptake of irinotecan and its active
metabolite, 7-ethyl-10-hydroxycamptothecin: in vitro evidence and effect of
single nucleotide polymorphisms. Drug Metab Dispos 33:434-439.
Sakaeda T, Nakamura T, Hirai M, Kimura T, Wada A, Yagami T, Kobayashi H, Nagata
S, Okamura N, Yoshikawa T, Shirakawa T, Gotoh A, Matsuo M and Okumura K
(2002) MDR1 up-regulated by apoptotic stimuli suppresses apoptotic signaling.
Pharm Res 19:1323-1329.
Shitara Y, Hirano M, Sato H and Sugiyama Y (2004) Gemfibrozil and its glucuronide
inhibit the organic anion transporting polypeptide 2
(OATP2/OATP1B1:SLC21A6)-mediated hepatic uptake and CYP2C8-mediated
metabolism of cerivastatin: analysis of the mechanism of the clinically relevant
drug-drug interaction between cerivastatin and gemfibrozil. J Pharmacol Exp
Ther 311:228-236.
Shitara Y, Itoh T, Sato H, Li AP and Sugiyama Y (2003) Inhibition of transportermediated hepatic uptake as a mechanism for drug-drug interaction between
cerivastatin and cyclosporin A. J Pharmacol Exp Ther 304:610-616.
Spears KJ, Ross J, Stenhouse A, Ward CJ, Goh LB, Wolf CR, Morgan P, Ayrton A and
Friedberg TH (2005) Directional trans-epithelial transport of organic anions in
54
porcine LLC-PK1 cells that co-express human OATP1B1 (OATP-C) and MRP2.
Su Y, Zhang X and Sinko PJ (2004) Human organic anion-transporting polypeptide
OATP-A (SLC21A3) acts in concert with P-glycoprotein and multidrug resistance
protein 2 in the vectorial transport of Saquinavir in Hep G2 cells. Mol Pharm
1:49-56.
Sugiyama D, Kusuhara H, Shitara Y, Abe T and Sugiyama Y (2002) Effect of 17 betaestradiol-D-17 beta-glucuronide on the rat organic anion transporting polypeptide
2-mediated transport differs depending on substrates. Drug Metab Dispos 30:220223.
Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M and Tsuji A (2000) Molecular
identification and characterization of novel members of the human organic anion
transporter (OATP) family. Biochem Biophys Res Commun 273:251-260.
Tamai I, Nozawa T, Koshida M, Nezu J, Sai Y and Tsuji A (2001) Functional
characterization of human organic anion transporting polypeptide B (OATP-B) in
comparison with liver-specific OATP-C. Pharm Res 18:1262-1269.
Tirona RG, Leake BF, Wolkoff AW and Kim RB (2003) Human organic anion
transporting polypeptide-C (SLC21A6) is a major determinant of rifampinmediated pregnane X receptor activation. J Pharmacol Exp Ther 304:223-228.
Trauner M and Boyer JL (2003) Bile salt transporters: molecular characterization,
function, and regulation. Physiol Rev 83:633-671.
Wang P, Kim RB, Chowdhury JR and Wolkoff AW (2003) The human organic anion
transport protein SLC21A6 is not sufficient for bilirubin transport. J Biol Chem
278:20695-20699.
Zelcer N, Reid G, Wielinga P, Kuil A, van der Heijden I, Schuetz JD and Borst P (2003)
Steroid and bile acid conjugates are substrates of human multidrug-resistance
protein (MRP) 4 (ATP-binding cassette C4). Biochem J 371:361-367.
Zhang S and Morris ME (2003) Effects of the flavonoids biochanin A, morin, phloretin,
and silymarin on P-glycoprotein-mediated transport. J Pharmacol Exp Ther
304:1258-1267.
Zhang S, Yang X and Morris ME (2004) Flavonoids are inhibitors of breast cancer
resistance protein (ABCG2)-mediated transport. Mol Pharmacol 65:1208-1216.
55
TABLE 1.
Effects of flavonoids on the uptake of [3H]DHEAS in both OATP1B1-expressing and
OATP1B1-negative cells
OATP1B1-expressing cells
mean S.D. (% change in mean)
Control
452 105
100 7.93
Rifampicin
195 78.5**
(-56.8 %)
97.1 10.9
(-2.88 %)
Apigenin
362 49.8*
(-19.8 %)
193 42.3**
(+92.8 %)
Biochanin A
142 35.0**
(-68.6 %)
90.1 19.6
(-9.95 %)
Benzoflavone
445 29.3
(-1.53 %)
112 4.48
(+11.9 %)
Chrysin
477 77.0
(+5.53 %)
117 10.6**
(+17.5 %)
Fisetin
197 22.8**
(-56.5 %)
138 13.0**
(+37.9 %)
Galangin
391 19.6
(-13.5 %)
118 9.49*
(+17.8 %)
Kaempferol
378 68.8
(-16.5 %)
85.9 3.75
(-14.1 %)
Luteolin
217 44.6**
(-52.0 %)
118 6.41**
(+17.8 %)
Morin
252 20.0**
(-44.2 %)
87.7 11.8
(-12.3 %)
Myricetin
305 24.0**
(-32.5 %)
94.0 10.5
(-6.03 %)
Silibinin
152 15.2**
(-66.5 %)
114 5.70*
(+13.9 %)
Silymarin
140 11.6**
(-68.9 %)
96.0 12.8
(-3.96 %)
The uptake of [3H]DHEAS (0.5 M) in both OATP1B1-expressing and OATP1B1negative cells in the absence or presence of flavonoids (50 M) was performed as
described under Materials and Methods. Rifampicin (100 M) was used as a positive
control. The uptake of [3H]DHEAS was expressed as the percentage of control (uptake
in OATP1B1-negative cells in the presence of 0.3% DMSO). The data were expressed as
56
mean S.D., n 6. *, P < 0.05, **, P < 0.01 compared with the control of either
OATP1B1-expressing or OATP1B1-negative cells. The values in parenthesis represent
the percentage of decrease or increase in means relative to the control value for either
OATP1B1-expressing or OATP1B1-negative cells.
57
TABLE 2.
Effects of flavonoids and their glycosides on the uptake of [3H]DHEAS in both
OATP1B1-expressing and OATP1B1-negative cells
OATP1B1-expressing cells
Control
379 49.3
100 7.03
Rifampin
141 16.1**
(-62.9 %)
106 8.16
(+5.75 %)
Daidzein
395 109
(+4.34 %)
99.3 6.27
(-0.67 %)
Daidzin
368 38.1
(-2.94 %)
97.7 8.48
(-2.34 %)
Diosmetin
301 26.7*
(-20.6 %)
159 7.31**
(+59.1 %)
Diosmin
374 88.4
(-1.36 %)
106 7.99
(+5.57 %)
EGC
354 15.0
(-6.70 %)
83.9 7.35**
(-16.1 %)
EGCG
186 24.6**
(-51.0 %)
82.4 6.39**
(-17.6 %)
Genistein
146 8.05**
(-61.6 %)
99.5 5.23
(-0.50 %)
Genistin
398 13.9
(+4.88 %)
87.2 4.62*
(-12.8 %)
Hesperitin
292 22.5**
(-22.9 %)
127 6.67**
(+27.5 %)
Hesperidin
288 16.2**
(-24.0 %)
94.3 5.53
(-5.67 %)
Naringenin
178 17.9**
(-53.1 %)
126 9.23**
(+26.0 %)
Naringin
167 6.94**
(-55.9 %)
90.7 6.69
(-9.23 %)
Phloretin
230 14.9**
(-39.3 %)
90.7 9.09
(-9.27 %)
Phloridzin
203 17.0**
(-46.6 %)
103 18.7
(+3.23 %)
Quercetin
334 28.4
(-11.8 %)
96.2 5.77
(-3.85 %)
Rutin
686 52.7**
(+81.1 %)
105 12.5
(+5.24 %)
The uptake of [3H]DHEAS (0.5 M) in both OATP1B1-expressing and OATP1B1negative cells in the absence or presence of flavonoids (50 M) was performed as
described under Materials and Methods. Rifampicin (100 M) was used as a positive
58
control. The uptake of [3H]DHEAS was expressed as the percentage of control (uptake
in OATP1B1-negative cells in the presence of 0.3% DMSO). The data were expressed as
mean S.D., n 6. *, P < 0.05, **, P < 0.01 compared with the control of either
OATP1B1-expressing or OATP1B1-negative cells. The values in parenthesis represent
the percentage of decrease or increase in means relative to the control value for either
OATP1B1-expressing or OATP1B1-negative cells. EGC, epigallocatechin; EGCG,
epigallocatechin-3-gallate.
59
FIGURE LEGEND
Figure 1. Effects of flavonoids on the uptake of [3H]DHEAS in both OATP1B1expressing (solid bar) and OATP1B1-negative HeLa cells (shadowed bar). The uptake of
[3H]DHEAS (0.5 M) in both OATP1B1-expressing and OATP1B1-negative cells in the
absence or presence of flavonoids (50 M) was performed as described under Materials
and Methods. Rifampicin (100 M) was used as a positive control. The uptake of
[3H]DHEAS is expressed as the percentage of control (uptake in OATP1B1-negative
cells in the presence of 0.3% DMSO). The data are expressed as mean S.D., n 6. *,
P < 0.05, **, P < 0.01 compared with the control of either OATP1B1-expressing or
OATP1B1-negative cells. In the absence of flavonoids, the quantitative level of
[3H]DHEAS uptake in OATP1B1-expressing and OATP1B1-negative HeLa cells was
15.0 9.10 and 3.08 1.22 pmol/mg protein, respectively (mean S.D., P < 0.001).
Figure 2. Effects of flavonoids and their glycosides on the uptake of [3H]DHEAS in

both OATP1B1-expressing (solid bar) and OATP1B1-negative HeLa cells (shadowed
bar). The uptake of [3H]DHEAS (0.5 M) in both OATP1B1-expressing and
OATP1B1-negative cells in the absence or presence of flavonoids (50 M) was
performed as described under Materials and Methods. The two flavonoids with a
connecting line represent a flavonoid and its corresponding glycoside. Rifampicin (100
M) was used as a positive control. The uptake of [3H]DHEAS is expressed as the

percentage of control (uptake in OATP1B1-negative cells in the presence of 0.3%
DMSO). The data are expressed as mean S.D., n 6. EGC, epigallocatechin; EGCG,
60
epigallocatechin-3-gallate. *, P < 0.05, **, P < 0.01 compared with the control of either
OATP1B1-expressing or OATP1B1-negative cells.
Figure 3. Concentration-dependent effects of flavonoids on [3H]DHEAS uptake in HeLa

cells. Concentration-dependent effects were determined for three flavonoids (biochanin
A (A), genistein (B), and EGCG (C)) in the presence of varying concentrations (0.3 50
M) of flavonoids or vehicle (0.3% DMSO). Specific uptake was obtained by

subtracting DHEAS uptake into OATP1B1-negative HeLa cells from the uptake into
OATP1B1-expressing HeLa cells. The specific uptake of [3H]DHEAS is expressed as
percentage of the control (in the presence of 0.3% DMSO). Each data point represents
the mean S.D., determined from three separate triplicate experiments.
Figure 4. Uptake of [3H]biochanin A in both OATP1B1-expressing (solid bar) and

OATP1B1-negative HeLa cells (opened bar). The uptake of [3H]biochanin A (1 M) in
both OATP1B1-expressing and OATP1B1-negative cells in the absence or presence of
rifampicin (100 M) was performed as described under Materials and Methods. The
uptake of [3H]biochanin A is given as mean S.D., n = 6.
Figure 5. Dixon plot analysis of inhibition of OATP1B1-mediated uptake of

[3H]DHEAS by biochanin A in HeLa cells. The uptake of [3H]DHEAS at the fixed
concentrations of 0.5 M (), 1 M (), and 5 M (S), was measured both in the
absence and presence of the indicated inhibitor concentration. Data represent the
reciprocal of the uptake rate difference between OATP1B1-expressing cells and OATP-
61
negative cells. Each data point represents the mean S.D., determined from three
separate triplicate experiments.
62
63
64
65
66
67
CHAPTER 3
Monocarboxylate Transporter 1 (MCT1)-Mediated

Pharmacokinetic Interaction for the Flavonoid Luteolin:
Model Fitting and Simulation
This chapter has been submitted for publication in Pharm Res.

We acknowledge the assistance of Dr. Qi Wang in the animal studies.
68
ABSTRACT
Monocarboxylate transporter 1 (MCT1) has been previously reported as an important
determinant of the renal reabsorption of the drug of abuse, -hydroxybutyrate (GHB).
Luteolin is a potent MCT1 inhibitor, inhibiting the uptake of GHB with an IC50 of 0.41
M in MCT1-transfected MDA-MB231 cells. The objectives of this study were to

characterize the effects of luteolin on GHB pharmacokinetics and pharmacodynamics in
rats, and to investigate the mechanism of the interaction using model-fitting methods.
GHB (400 and 1000 mg/kg) and luteolin (0, 4 and 10 mg/kg) were administered to rats
via i.v. bolus doses. The plasma or urine concentrations of luteolin and GHB were
determined by HPLC and LC/MS/MS, respectively. The pharmacodynamic parameter
sleep time after GHB administration was recorded. A pharmacokinetic model containing
capacity-limited renal reabsorption and metabolic clearance was constructed to
characterize the in vivo interaction. Luteolin significantly decreased the plasma
concentration and AUC, and increased the total and renal clearances of GHB in a dosedependent manner. Moreover, luteolin significantly shortened the duration of GHB
(1000 mg/kg)-induced sleep in rats (161 16, 131 14 and 121 5 min for control,
luteolin 4 and 10 mg/kg groups, respectively, p<0.01). An uncompetitive inhibition
model, with an inhibition constant of 1.1 M, best described the in vivo pharmacokinetic
interaction. The results of this study indicated that luteolin significantly altered the
pharmacokinetics of GHB by uncompetitively inhibiting its MCT1-mediated transport.
The interaction between luteolin and GHB may offer a potential clinical detoxification
strategy to treat GHB overdoses.
69
INTRODUCTION
Monocarboxylate transporters (MCTs), which belong to the solute carrier 16 (SLC 16)
gene family, play an essential role in cellular metabolism (Halestrap and Meredith, 2004).
Of the now 14 identified MCT members, MCT 1-4 have been shown to be protoncoupled monocarboxylate transporters, important in the transport of endogenous
monocarboxylates, such as pyruvate, lactate and ketone bodies (Halestrap and Price,
1999; Halestrap and Meredith, 2004), as well as clinically relevant drugs, such as
foscarnet (Tamai et al., 1999), nateglinide (Okamura et al., 2002) and lovastatin
(Nagasawa et al., 2002). Among these four MCTs, MCT1 is expressed nearly
ubiquitously throughout the body, including erythrocytes, muscle, heart, kidney,
intestine, liver and brain (Halestrap and Price, 1999). Transport of lactate by MCT1
across the plasma membrane to maintain cytosolic pH is physiologically important to
mammalian cells under hypoxia conditions (Halestrap and Meredith, 2004).
The typical inhibitors of MCT1 include 4, 4-diisothiocyanostilbene-2, 2-disulphonate

(DIDS) and bulky monocarboxylates such as -cyano-4-hydroxycinnamate (CHC)
(Halestrap and Meredith, 2004). Recently, we reported that flavonoids, a class of
polyphenolic compounds present in the diet and herbal products, are potent MCT1
inhibitors as well. Among all the flavonoids tested, luteolin (3,4,5,7tetrahydroxyflavone), a naturally occurring flavonoid abundant in vegetables (Manach et
al., 2004) with reported anti-oxidant (Horvathova et al., 2004) and anti-inflammatory
activities (Kotanidou et al., 2002), represents one of the most potent inhibitors, with an
70
IC50 of 0.41 M in inhibiting the uptake of GHB in MCT1-transfected MDA-MB231

cells (Wang and Morris, 2007a).
Recently, due to the popular use of dietary supplements and herbal medicines, flavonoiddrug interactions have increasingly been observed in preclinical and clinical studies
(Dresser et al., 2002; Choi et al., 2004). These interactions, in some cases, can cause
serious or life-threatening adverse effects, especially when flavonoids are used together
with narrow therapeutic index drugs (Wang et al., 2004). On the other hand, flavonoids
have been shown to be able to improve the bioavailability of the coadministered drugs,
resulting in beneficial flavonoid-drug interactions (Choi et al., 2004; Wang and Morris,
2007b). The mechanisms underlying these flavonoid-drug interactions are presumably
due to the inhibition of drug metabolizing enzymes and efflux drug transporters, such as
P-glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) (Choi et al.,
2004; Wang et al., 2004; Wang and Morris, 2007b). However, far less information is
available on flavonoid-drug interactions involving uptake transporters, including the
monocarboxylate transporters.
The physiological substrates of MCT1 include pyruvate, lactate and ketone bodies
(Halestrap and Meredith, 2004). In recent studies, we demonstrated that MCT1 also
represents an important transporter for -hydroxybutyrate (GHB) (Wang et al., 2006), a
drug used to treat the sleep disorder narcolepsy (Mamelak et al., 1986). GHB is also a
drug of abuse (Drasbek et al., 2006), and GHB abuse and subsequent overdoses may lead
to serious adverse effects, such as coma, seizure, respiratory arrest and even death
71
(Mason and Kerns, 2002). Current treatment of GHB overdoses mainly consists of
supportive care and no specific detoxification strategies have been developed for clinical
use (Mason and Kerns, 2002).
The pharmacokinetics of GHB are nonlinear in rats and humans, due to its capacitylimited metabolism (Lettieri and Fung, 1976; Lettieri and Fung, 1979; Morris et al.,
2005), capacity-limited oral absorption (Arena and Fung, 1980; Palatini et al., 1993), and
capacity-limited renal clearance (Morris et al., 2005). At higher doses when the
metabolic clearance is saturated, the renal clearance of GHB following intravenous
administration becomes more pronounced and significantly contributes to the overall
elimination of GHB (Morris et al., 2005). An important, if not the only mechanism
underlying this capacity-limited renal clearance of GHB, is the extensive renal
reabsorption mediated by monocarboxylate transporters in the kidney proximal tubules
(Morris et al., 2005; Wang et al., 2006). Given the potent inhibitory effects of luteolin on
MCT1 and significant contribution of MCT1 in GHB renal reabsorption, it is very likely
that a MCT1-mediated luteolin and GHB interaction might occur in vivo. In a previous
preliminary study, we have demonstrated that luteolin (10 mg/kg, i.v. bolus) significantly
decreased the plasma concentration of GHB, increased its renal clearance and shortened
the duration of GHB-induced sleep in rats (Wang and Morris, 2007a).
The objectives of this study were 1) to characterize the pharmacokinetic interaction

between luteolin and GHB; 2) to construct a pharmacokinetic model for the interaction
between luteolin and GHB, and use this model to discern the in vivo inhibition
72
mechanism; and 3) to use our model to predict the pharmacokinetic consequences of

MCT1-mediated drug-drug interactions.
73

Chemicals and Reagents
Luteolin, apigenin, -hydroxybutyrate, hydroxypropyl--cyclodextrin were purchased
from Sigma-Aldrich (St. Louis, MO). The internal standard GHB-D6 was purchased
from Cerilliant (Round Rock, TX). Saline was purchased from Henry-Schein (Melville,
NY). All other reagents or solvents used were either analytical or high-performance
liquid chromatography (HPLC) grade.
Animals and Surgery

Male Sprague-Dawley (SD) rats (body weight ~ 300 g) were purchased from Harlan
(Indianapolis, IN). Animals were housed in a temperature and humidity-controlled
environment with a 12-h light/dark cycle and received a standard diet with free access to
tap water. Animals were acclimatized to this environment for at least one week before
experiments. All protocols of animal studies were reviewed and approved by the
University at Buffalo Institutional Animal Care and Use Committee. The rats had
cannulas implanted in the right jugular vein and bladder under anesthesia (ketamine 90
mg/kg and xylazine 10 mg/kg, i.m. injection) as previously described (Morris et al.,
2005), and were kept in individual cages for recovery from surgery for three days.
Pharmacokinetic Studies
Pharmacokinetic studies were performed as previously reported (Wang and Morris,
2007a). The rats were kept in metabolic cages for blood and urine collection throughout
the study. GHB was dissolved in sterile water as a 200 mg/ml solution and was given to
74
rats via an i.v. bolus injection for specified doses. Luteolin was dissolved in DMSO as a
50 mg/ml stock solution and was further diluted with hydroxypropyl--cyclodextrin
(25%) to such concentrations that specified doses were delivered as 2 l/g body weight
drug solution. The luteolin solution was injected intravenously right after the GHB
injection. For the control group, GHB (400 or 1000 mg/kg) and the luteolin control
vehicle were given to rats. For the luteolin treatment group, GHB (400 or 1000 mg/kg)
and luteolin (4 or 10 mg/kg) were administered to rats. Blood samples (200 l ) were
collected from the jugular vein cannula at 0 (predose), 2, 5, 10, 30, 60, 120, 150, 180,
210, 240, and 300 min after GHB administration. The plasma was separated from the
whole blood by centrifugation at 2,000 g for 5 min at 4oC. The urine samples were
collected over 6 hr and the volume was measured. All plasma and urine samples were
stored at -80oC until analysis. The hypnotic effects of GHB following various treatments
were determined by the sleep time, which was measured as the difference in the time
between the loss of righting reflex (LRR) and return of righting reflex (RRR).
HPLC and LC/MS/MS Assay

The concentration of luteolin in plasma samples was determined by a previously
published method with minor modifications (Li et al., 2005; Chen et al., 2007). Briefly,
100 l of 6.0% perchloric acid was added to 100 l plasma sample to precipitate protein.
10 l of apigenin solution (100 g /ml) was added as the internal standard. Luteolin was
extracted by adding 1.5 ml of ethyl acetate followed by vigorous vortexing. The mixture
was centrifuged at 2,000 g for 10 min. The supernatant was transferred to a new tube and
dried under a stream of nitrogen. The residue was reconstituted in 100 l of mobile phase
75
followed by centrifugation at 22,000 g for 10 min and 60 l of supernatant was injected

into HPLC for analysis.
The HPLC analysis was performed on a system consisting of a Waters 1525 pump, a
Waters BreezeTM workstation, 717 plus autosampler, a 2847 UV detector, and an Alltech
Alltima C18 column (125 4.6 mm, 5-m particle size). The composition of the mobile
phase was methanol/0.2% phosphoric acid solution (60:40, v/v). The mobile phase was
delivered isocratically with a flow rate of 1.0 ml/min. UV absorbance was measured at
350 nm. Luteolin appeared on the chromatograph at approximately 7 min with no
interfering peaks. The standard curve was linear over the concentration range of 0.01 to
20 g/ml with a regression coefficient greater than 0.99. The lower limit of quantitation
of luteolin was 10 ng/ml.
The concentration of GHB in plasma and urine samples was determined by a validated
liquid chromatography-tandem mass spectrometry (LC/MS/MS) assay as previous
described with minor modifications (Fung et al., 2004; Wang and Morris, 2007a).
Briefly, to generate a calibration curve, a GHB-D6 stock solution (6 mM, 5 l) and GHB
stock solutions with varying concentrations were added into blank plasma or urine (50 l;
appropriately diluted with water) in order to prepare standards of GHB (60, 240, 600,
1200, 2400, 4800, and 7200 M as final concentrations). To each plasma or urine
sample, the internal standard GHB-D6 was added in the same concentration and volume.
The protein present in plasma and urine samples was precipitated with methanol (1
volume of methanol added to 1 volume sample). After vigorous vortexing, the mixtures
76
were centrifuged at 22,000 g for 20 min, the supernatant was collected for LC/MS/MS
assay.
The LC/MS/MS assay of GHB was performed on a PE SCIEX API 3000 triple-quadruple
tandem mass spectrometer system equipped with a turbo ion spray (Applied Biosystems,
Foster City, CA), a Series 200 PE autosampler (Perkin-Elmer, Shelton, CT), and a Series
200 PE micro pump (Perkin-Elmer, Shelton, CT). The sample separation by liquid
chromatography was conducted using a reversed-phase Aqua C18 5 m 125 column
(150 4.6 mm, Phenomenex, Torrance, CA) connected with a C18 5 m guard column
cartridge system (Phenomenex, Torrance, CA). The mobile phase consisting of 5 mM
formic acid/methanol (33:67, v/v) was delivered isocratically. The flow rate was 0.75
ml/min and the injection volume was 10 l. The actual flow into the mass-spectrum was
achieved by using a splitter, which accounted for one third of total flow. The turbo ion
spray was operated in the positive mode with interface temperature set at 400oC. The
declustering potential and collision energy for fragmentation was set at 30 and 13 eV,
respectively. Multiple reaction monitoring was applied to detect GHB-D6 and GHB by
measuring the ion pair transitions from m/z 111 (parent ion) to m/z 93 (product ion) and
from m/z 105 (parent ion) to m/z 87 (product ion), respectively. The retention time of
GHB-D6 and GHB was 2.6 min with no interfering peaks found in plasma and urine
samples. The Analyst software 1.4.1 (Applied Biosystems, Foster City, CA) was used for
data quantitation and instrument control.
Pharmacokinetic Analysis
77
The total clearance of GHB (CL) was determined from Dose/AUC, where AUC is the
area under the plasma concentration-time curve. Renal clearance (CLR) was determined
by Ae/AUC, where Ae is the amount of GHB excreted into the urine. The fraction of the
dose eliminated by renal excretion (fe) was determined by Ae/Dose. The metabolic
clearance (CLm) was calculated using the equation CLm = CL - CLR, assuming that total
clearance of GHB is equal to renal clearance plus metabolic clearance.
To better understand the in vivo interaction between luteolin and GHB, pharmacokinetic
modeling was conducted to characterize both luteolin and GHB kinetics. First, luteolin
data were fitted with different pharmacokinetic models, e.g. one-compartment, twocompartment and three-compartment models with linear or nonlinear elimination. The
final pharmacokinetic model for luteolin (equation 1 and 2) was selected based on the
goodness-of-fit criteria including coefficient variation of the estimates (CV %), R2,
Akaikes Information Criterion (AIC) and Schwarz Criterion (SC) by using ADAPTII
software (BMSR, University of South California, Los Angeles CA).
(1)
(2)
dX C _ Lut
dt
dX T _ Lut
dt
= k10 X C _ Lut k12 X C _ Lut + k 21 X T _ Lut
= k12 X C _ Lut k 21 X T _ Lut
( IC = Dose)
( IC = 0)
where XC_Lut and XT-Lut represents the luteolin amount in the central and peripheral
compartment, respectively. k10 represents the elimination rate constant of the central
78
compartment. k12 and k21 represent the first-order rate constants of distribution between
the two compartments. The initial condition for each equation is shown in parenthesis.
The pharmacokinetics of GHB has previously been characterized by a one-compartment

nonlinear model (Lettieri and Fung, 1979). The nonlinearity in GHB kinetics, following
intravenous administration, is related to the capacity-limited metabolism (Lettieri and
Fung, 1976; Lettieri and Fung, 1979; Morris et al., 2005) and capacity-limited renal
reabsorption (Morris et al., 2005). Therefore, a one-compartment model was used to
describe the GHB pharmacokinetics with the incorporation of capacity-limited
metabolism, glomerular filtration, and capacity-limited reabsorption, as shown in
equation 3 and fig 4. In this model, several assumptions were made: 1) no or minimum
effects of luteolin on GHB metabolism; 2) no or minimum renal secretion of GHB; and
3) only parent luteolin inhibits MCT1. To our knowledge, there is no report in the
literature of luteolin affecting the metabolism of GHB. The negligible renal secretion of
GHB was assumed based on a previous study, in which a lack of renal secretion was
reported for the ketone bodies of -hydroxybutyrate or the congener of GHB with the
hydroxyl group at carbon 3 (Ferrier et al., 1992). We also assumed only parent luteolin
could inhibit MCT1 since flavonoid glycosides were reported previously with no or
minimum MCT1 inhibitory activity (Wang and Morris, 2007a).
The equation 3 describes the pharmacokinetics of GHB in the absence of luteolin, while
equation 4 describes the pharmacokinetics of GHB in the presence of luteolin as a
competitive inhibitor on GHB renal clearance. Equation 5 and 6 describe the
79
pharmacokinetics of GHB in the presence of luteolin as a noncompetitive and

uncompetitive inhibitor on GHB renal clearance, respectively. Simultaneous fitting was
conducted based on the integrated luteolin model and GHB model to evaluate the in vivo
inhibition mechanism. The final pharmacokinetic model for luteolin and GHB was
selected based on the goodness-of-fit criteria (CV % of the estimates, R-square, AIC and
SC) by using ADAPTII software.
(3)
dX GHB
Vmax X GHB
=
dt
k m VGHB + X GHB
(
(4)
( IC = Dose)
dX GHB
Vmax X GHB
=
dt
k m VGHB + X GHB
(
(5)
Vmax_ ren
GFR
) X GHB
VGHB k ren VGHB + X GHB
GFR
VGHB
Vmax_ ren
) X GHB
I
k ren VGHB (1 + ) + X GHB
Ki
( IC = Dose)
dX GHB
Vmax X GHB
=
dt
k m VGHB + X GHB
I
)
Ki
) X GHB
+ X GHB
Vmax_ ren /(1 +
(6)
GFR
VGHB k ren VGHB
( IC = Dose)
dX GHB
Vmax X GHB
=
dt
k m VGHB + X GHB
(
GFR
VGHB
Vmax_ ren /(1 +
I
)
Ki
I
k ren VGHB /(1 + ) + X GHB
Ki
80
) X GHB
( IC = Dose)
where XGHB represents the GHB amount in the central compartment. VGHB represents the
volume of distribution of GHB. Vmax represents the maximal metabolic rate of GHB and
km represents the concentration of GHB at 50% of Vmax. GFR represents the glomerular
filtration rate in the kidney. Vmax_ren represents the maximal rate of GHB renal
reabsorption and kren represents the concentration of GHB at 50% of the maximal
reabsorption rate. I represents luteolin in the central compartment (XC_Lut) that inhibits
GHB reabsorption and Ki represents the inhibition constant.
To determine the effects of varying doses of luteolin on GHB detoxification at different

dose levels of GHB, model simulations were conducted using the parameters obtained
from the above nonlinear regression analysis with the incorporation of a 10% variance
using ADAPT II software. Three doses of GHB (200, 400 and 1000 mg/kg by i.v. bolus)
and five doses of luteolin (0, 2, 4, 10, and 20 mg/kg by i.v. bolus) were simulated based
on the proposed pharmacokinetic model.
Data were analyzed for statistically significant differences using ANOVA (Prism 3.0
software, GraphPad, San Diego, CA) followed by a Dunnetts post hoc test or by the
Students t test. p values < 0.05 were considered statistically significant.
81
RESULTS
Pharmacokinetics of luteolin
To characterize the interaction between luteolin and GHB, it is essential to determine the
pharmacokinetics of luteolin. Therefore, we measured the plasma concentrations of
luteolin over time following two i.v. doses. As shown in figure 1, after intravenous
administration, luteolin plasma concentrations could be described by a biexponential
equation, exhibiting a rapid decrease at the early time points followed by a slower
decrease beyond 90 min, indicating the involvement of multiple compartment
pharmacokinetics. Following doses of 4 mg/kg and 10 mg/kg, the AUC of luteolin was
189 9.20 and 541 173 gxml-1xmin (mean SD), respectively. The clearance of
luteolin was similar at these two doses (21.2 1.06 and 19.7 5.50 mlxmin-1xkg-1 for the
4 and 10 mg/kg doses, respectively).
Effects of luteolin on GHB pharmacokinetics and pharmacodynamics
To further investigate the in vivo MCT1-mediated interaction, GHB (400 or 1000 mg/kg)
and luteolin (0, 4 or 10 mg/kg) were administered to rats intravenously. As shown in
figure 2, GHB plasma concentration versus time profile exhibited nonlinear kinetics,
which is consistent with previous reports (Lettieri and Fung, 1979; Wang and Morris,
2007a). When GHB was given at the dose of 1000 mg/kg, luteolin effectively decreased
the plasma concentrations of GHB. Compared with the GHB alone (control) group, the
AUC of GHB was significantly decreased from 170 39.8 mgxml-1xmin in the control rats
to 117 28.6 mgxml-1xmin (p < 0.05) and 113 20.8 mgxml-1xmin (p < 0.05) for luteolin 4
mg/kg and 10 mg/kg group, respectively (table 1). In contrast, the total clearance of
82
GHB was significantly increased from 6.19 1.59 mlxmin-1xkg-1 in the control group to
8.92 2.02 mlxmin-1xkg-1 (p < 0.05) in luteolin 4 mg/kg group and 9.05 1.43 mlxmin1
-1
xkg
(p < 0.05) in luteolin 10 mg/kg group. Similarly, the renal clearance of GHB was
significantly increased in luteolin 10 mg/kg group when compared with the control group
(p < 0.05, 1.64 0.62 and 3.84 1.21 mlxmin-1xkg-1 for the control and luteolin 10 mg/kg
group, respectively). However, the metabolic clearance of GHB was not significantly
altered among the control and luteolin treatment groups. In addition to the effects of
luteolin on GHB pharmacokinetics, luteolin significantly shortened the duration of GHBinduced sleep in rats. Compared with the GHB only group, the total sleep time was
decreased from 161 16 min in the control rats to 131 14 min (p < 0.01) in the luteolin
4 mg/kg group and 121 5 min (p < 0.01) in the luteolin 10 mg/kg group. At the lower
dose of GHB (400 mg/kg), the AUC of GHB in control rats was 56.6 6.31 mgxml-1xmin,
which is similar to literature data (Van Sassenbroeck et al., 2003). At this GHB dose
level, similar effects of luteolin (10 mg/kg) on GHB pharmacokinetics and
pharmacodynamics were observed (table 1). However, the decrease in GHB AUC and
increase in GHB total and renal clearances were less pronounced when compared with
the changes at higher dose of GHB. At the GHB dose of 400 mg/kg, the time point at the
loss of righting reflex (onset of sleep) in rats was later (10 4 min) than that observed in
the GHB 1000 mg/kg group (~ 2 min). Interestingly, luteolin (10 mg/kg) significantly
delayed the onset of GHB (400 mg/kg)-induced sleep to 23 8 min (p < 0.05, compared
to 10 4 min), with no evident effects on the onset of GHB (1000 mg/kg)-induced sleep
(~ 2 min, similar to that in GHB 1000 mg/kg only group). Consistent with the results
from the GHB 1000 mg/kg study, the total sleep time induced by 400 mg/kg GHB, which
83
was calculated as the difference between the time points at the return and loss of righting
reflex in rats, was significantly decreased in the 10 mg/kg luteolin treated rats (p < 0.05,
60 2 min and 41 12 min for control and luteolin 10 mg/kg group, respectively).
When the AUC of GHB was plotted against GHB-induced total sleep time, a good
correlation between these two parameters was observed with a R2 value of 0.915 (figure
3), indicating the total sleep time in rats was closely related to the total exposure of GHB
in the plasma. At the return of righting reflex time point, there was no significant
difference in GHB plasma concentrations among all control and treatment groups (~ 0.3
to 0.4 mg/ml, estimated from the data in figure 2).
Model fitting for GHB pharmacokinetics in the presence of luteolin
To further understand the in vivo luteolin-GHB interaction, model-fitting technique was

applied to discern the inhibition mechanism. A number of pharmacokinetic models (e.g.,
a one-, two- or three-compartment model with linear or nonlinear elimination) were first
constructed to describe luteolin kinetics. Based on the fitting criteria (e.g., CV % of the
estimates, R2 and AIC), a two-compartment model with linear elimination best
characterized luteolin pharmacokinetics (data not shown). The final fitted
pharmacokinetic parameters for luteolin, as shown in table 2, were 0.124 min-1, 0.120
min-1, 0.0224 min-1 and 204 ml/kg for k10, k12, k21 and VC_Lut, respectively. Using the
selected luteolin model as a base model, an integrated pharmacokinetic model (figure 4)
was proposed to describe the interaction between luteolin and GHB by incorporating a
one-compartment GHB model with capacity-limited metabolism, glomerular filtration,
and capacity-limited reabsorption. The parameters of GHB capacity-limited metabolism
84
and glomerular filtration in rats have been previously determined (Morris et al., 2005)
and were fixed in the model fitting (table 3). In the inhibition model, luteolin was
assumed not to inhibit GHB metabolism but to inhibit GHB renal reabsorption via
different mechanisms, e.g., competitive, noncompetitive or uncompetitive inhibition
(equation 4, 5 and 6). Simultaneous fitting was conducted to compare the goodness-of-fit
to these models. Based on the fitting criteria (e.g., CV % of the estimates, R2, AIC and
SC), an uncompetitive inhibition model gave the best fitting results (table 3). The model
fitted data captured well the experimental data (figure 5 and 6). The fitted
pharmacokinetic parameters of GHB in the final model were 49.2 mgxmin-1xkg-1, 5.54
mg/ml, 578 ml/kg, and 63.3 g/kg for Vmax_ren, kren, VGHB, and Ki, respectively.
Model simulation for luteolin and GHB pharmacokinetic interaction
To determine the effects of varying doses of luteolin on GHB detoxification at different

dose levels of GHB, model simulations were conducted using the parameters obtained
from the above nonlinear regression analysis with the incorporation of a 10% variance
using ADAPT II software. GHB was given by simulated i.v. bolus at three hypothesized
doses (200, 400 and 1000 mg/kg) and luteolin was given by i.v. bolus at five
hypothesized doses (0, 2, 4, 10, and 20 mg/kg). As shown in figure 7 and table 4, the
simulation results indicated that luteolin significantly decreased the AUC of GHB and
increased the clearance of GHB. The changes were more evident at a higher dose of
GHB (1000 mg/kg) than at a lower dose of GHB (200 mg/kg). For example, at the dose
of 10 mg/kg, luteolin decreased the AUC of GHB by approximately 20%, 30% and 40%
for GHB doses of 200, 400 and 1000 mg/kg, respectively.
85
DISCUSSION
Flavonoid-drug interactions with favorable or adverse effects have increasingly been

observed in recent years, due to the widespread use of flavonoid-enriched dietary
supplements and herbal medicines for disease prevention and treatment (Havsteen, 2002;
Kruijtzer et al., 2002; Sparreboom et al., 2004). The mechanisms underlying most of
these interactions are effects (inhibition/induction) on drug metabolizing enzymes and/or
efflux drug transporters, such as P-glycoprotein and BCRP (Choi et al., 2004; Wang et
al., 2004; Wang and Morris, 2007b). Limited information is available concerning
interactions between flavonoids and uptake transporters. In a previous study, we found
that flavonoids represent a class of potent inhibitors of monocarboxylate transporter-1
(MCT1) (Wang and Morris, 2007a), an uptake transporter important in the transport of
endogenous monocarboxylates as well as clinically important drugs (Nagasawa et al.,
2002; Halestrap and Meredith, 2004). Therefore, the objectives of this study were to
further characterize the flavonoid-drug interactions mediated by MCT1 using GHB as a
model substrate, and to discern the in vivo inhibition mechanism using pharmacokinetic
model fitting methods.
The flavonoid luteolin is a potent MCT1 inhibitor, with an IC50 of 0.41 M in inhibiting
the uptake of GHB in rat MCT1-transfected MDA-MB231 cells (Wang and Morris,
2007a). In the present study, the plasma concentrations of luteolin in rats after an
intravenous dose of 10 mg/kg are well above 0.41 M (0.12 g/ml). At a dose of 4
mg/kg, the plasma concentrations of luteolin are significantly higher than 1 M (~ 0.3
g/ml) for the first 60 min, and remain above 0.36 M (~ 0.1 g/ml) at later time points
86
(figure 1), indicating in vivo MCT1 inhibition by luteolin is likely to occur under our
experimental conditions.
In this study, GHB was selected as a model MCT1 substrate. Previous studies have
demonstrated that in vivo GHB elimination following intravenous administration is due to
capacity-limited metabolism and capacity-limited renal clearance (Lettieri and Fung,
1979; Morris et al., 2005). In the kidney, GHB is extensively reabsorbed from urine into
blood (Morris et al., 2005) and this process is mediated by a family of monocarboxylate
transporters. In studies using rat kidney membrane vesicles and human kidney HK-2
cells, MCT1 and MCT2 have been shown as the important transporters responsible for
GHB renal reabsorption (Wang et al., 2006; Wang et al., 2007). However, the
contribution of MCT2 in the overall GHB renal transport in HK-2 cells is minor when
compared to that of MCT1 (Wang et al., 2007). Moreover, a single transport process for
GHB was observed in rat kidney membrane vesicles based on Eadie-Hofstee plot, and
similar transport kinetic parameters of GHB were obtained from both rat kidney
membrane vesicles and rat MCT1-transfected cells (Wang et al., 2006). Therefore,
MCT1 appears to represent a major, if not the only transporter governing GHB renal
reabsorption.
Given the potent MCT1 inhibition activity of luteolin and the significant contribution of
MCT1 in GHB renal reabsorption, it is likely that the interaction of luteolin with MCT1
might have significant effects on GHB pharmacokinetics. This hypothesis was tested in a
previous report (Wang and Morris, 2007a) and is confirmed and further characterized in
87
this study. As shown in figure 2 and table 1, luteolin significantly decreased GHB (1000
mg/kg) plasma concentration and AUC. Luteolin at the dose of 10 mg/kg significantly
increased the total and renal clearances of GHB (1000 mg/kg) by about 1.5-fold and 2fold, respectively. The contribution of renal clearance of GHB to its total clearance was
increased from 27 % in control group to 42 % in the luteolin 10 mg/kg treatment group.
However, luteolin had no significant effects on the metabolic clearance of GHB. All
these results clearly indicated that luteolin altered GHB pharmacokinetics via increasing
GHB renal clearance, which is consistent with the inhibition of MCT1-mediated renal
reabsorption. To our knowledge, there is no report of luteolin affecting the metabolism
of GHB, which is in agreement with the lack of changes observed in GHB metabolic
clearance when determined with or without luteolin treatment. It is recognized, though,
that GHB metabolism following a 1000 mg/kg dose is saturated (table 1) (Morris et al.,
2005) and, as a result, no changes in metabolic clearance might be readily detected upon
the treatment of luteolin. At a lower GHB dose level (400 mg/kg), luteolin treatment
increased the total and renal clearances of GHB, although the changes were not
statistically significant (table 1). The less pronounced effect at a lower GHB dose is
probably due to the nonlinearity in GHB metabolic clearance. At lower doses, GHB
metabolism is not saturated, and most GHB ( ~ 90%) is eliminated via metabolic
transformation (table 1). Consistent with previous studies, the contribution of GHB renal
clearance is less significant at lower GHB doses (Morris et al., 2005), and therefore, the
effects of luteolin on GHB clearance are smaller in magnitude.
88
Consistent with the effects on GHB pharmacokinetics, luteolin also significantly

decreased the GHB-induced total sleep time in rats. The AUC of GHB correlated well
with GHB-induced sleep time (figure 3, R2 = 0.915), indicating the hypnotic effects of
GHB are directly related to the plasma concentration of GHB. Interestingly, luteolin (10
mg/kg) significantly delayed the loss of righting reflex induced by 400 mg/kg GHB (10
4 versus 23 8 min for the control and treatment group, respectively), with no effects on
the loss of righting reflex induced by 1000 mg/kg GHB (~ 2 min for both control and
treatment group). This is likely due to the higher concentrations of luteolin at early time
points that can affect renal reabsorption and possibly inhibit MCT1-mediated brain
uptake (Bhattacharya and Boje, 2004). This inhibition could be more readily achieved at
a relatively lower GHB dose. At the return of righting reflex time point, there was no
significant difference in GHB plasma concentrations among all control and treatment
groups (~ 0.3 to 0.4 mg/ml, estimated from the data in figure 2), indicating this
concentration might represent the wake-up threshold concentration in rats.
To better understand the interaction between luteolin and GHB, an integrated

pharmacokinetic model was constructed to describe the in vivo data. First of all, luteolin
pharmacokinetics was determined based on the fitting results from different models (data
not shown). A two-compartment linear model provides the best fit, which is consistent
with literature reports of other flavonoid analogues, such as quercetin (Ferry et al., 1996)
and puerarin (Li et al., 2006). In addition, the dose-proportional AUC and constant
clearance at different luteolin doses in this study further supported the linear kinetics of
luteolin. As for the pharmacokinetics of GHB, a one-compartment model was selected
89
with the incorporation of capacity-limited metabolism, glomerular filtration, and

capacity-limited reabsorption, according to the previous reports (Lettieri and Fung, 1979;
Morris et al., 2005). In this model, we assumed MCT1 represented the major player
determining GHB renal reabsorption, based on the results in HK-2 cells and the observed
single transport process of GHB in rat kidney membrane vesicles (Wang et al., 2006;
Wang et al., 2007).
The final fitting results from the integrated model showed that the uncompetitive
inhibition model gave the best fitting. This is consistent with several other studies, in
which luteolin has been reported as an uncompetitive inhibitor of N-acetyltransferase (Li
et al., 2001) and tyrosinase (Xie et al., 2003). However, this result differs from our
previous in vitro study showing that luteolin competitively inhibited GHB uptake into
MDA-MB231 cells with a Ki value approximately of 10 M (Wang and Morris, 2007a).
In our in vitro study, only one luteolin concentration (50 M) was used to discern the
inhibition kinetics (Wang and Morris, 2007a). In addition, the protein binding of luteolin
might be different for the in vitro and in vivo studies. Moreover, the concentrations of
luteolin at the renal reabsorption site are likely different from the plasma concentrations,
which might cause the difference in the estimation of Ki values. Further studies are
needed to elucidate the discrepancy. As shown in figure 5 and 6, the fitted results
captured well the experimental data with small CV % values (table 3), indicating the
proposed model reasonably described the in vivo interaction. The value of VGHB
determined in this study was very close to a previous reported value (Lettieri and Fung,
1979). We observed a 10-fold difference in the estimated Ki values among three
90
different models. This is probably due to the model fittings from the equations with
different inhibition mechanisms. The Ki value determined from uncompetitive model is
63.3 g/kg, which is an apparent value. If we normalize it with luteolin volume of
distribution (204 ml/kg), the true Ki value will be around 1.1 M, which is close to the in
vitro IC50 value (0.41 M). If we consider the contribution of protein binding of luteolin
in plasma, the true Ki value will be even smaller. In a previous study, the free fraction
(fu) of quercetin, an analogue of luteolin, in plasma has been reported around 1 %
(Boulton et al., 1998). Therefore, if we assume the similar protein binding of quercetin
and luteolin, the true Ki value determined in vivo will be close to 0.01 M (Kixfu/VC_LUT),
indicating luteolin is a more potent MCT1 inhibitor in vivo comparing to its in vitro
inhibition. In a previous study, the protein binding of GHB was reported to be negligible
(Morris et al., 2005). Therefore, the GHB concentrations determined in the study (total
concentration) also represented the unbound concentrations of GHB in the plasma.
In the model simulations using the parameters obtained from model fitting, luteolin was
shown to significantly decrease the AUC of GHB and increase the clearance of GHB.
The changes were more evident at higher doses of GHB (1000 mg/kg) than at lower
doses of GHB (200 mg/kg). This is possibly due to the more significant contribution of
GHB renal clearance to the total clearance, when metabolic clearance is saturated.
In summary, the results of this study characterized the effects of the flavonoid luteolin on
GHB pharmacokinetics and pharmacodynamics mediated by MCT1. The
pharmacokinetic interaction was well described by an uncompetitive inhibition model
91
consisting of capacity-limited metabolic clearance and capacity-limited renal clearance.

Inhibiting MCT1 by the flavonoid luteolin might represent a potential mechanism for
GHB detoxification following overdoses of GHB.
92
REFERENCES:
Arena C and Fung HL (1980) Absorption of sodium gamma-hydroxybutyrate and its

prodrug gamma-butyrolactone: relationship between in vitro transport and in vivo
absorption. J Pharm Sci 69:356-358.
Bhattacharya I and Boje KM (2004) GHB (gamma-hydroxybutyrate) carrier-mediated
transport across the blood-brain barrier. J Pharmacol Exp Ther 311:92-98.
Boulton DW, Walle UK and Walle T (1998) Extensive binding of the bioflavonoid
quercetin to human plasma proteins. J Pharm Pharmacol 50:243-249.
Chen T, Li LP, Lu XY, Jiang HD and Zeng S (2007) Absorption and excretion of luteolin
and apigenin in rats after oral administration of Chrysanthemum morifolium
extract. J Agric Food Chem 55:273-277.
Choi JS, Choi HK and Shin SC (2004) Enhanced bioavailability of paclitaxel after oral
coadministration with flavone in rats. Int J Pharm 275:165-170.
Drasbek KR, Christensen J and Jensen K (2006) Gamma-hydroxybutyrate--a drug of
abuse. Acta Neurol Scand 114:145-156.
71:11-20.
Ferrier B, Martin M, Janbon B and Baverel G (1992) Transport of beta-hydroxybutyrate
and acetoacetate along rat nephrons: a micropuncture study. Am J Physiol
262:F762-769.
Ferry DR, Smith A, Malkhandi J, Fyfe DW, deTakats PG, Anderson D, Baker J and Kerr
DJ (1996) Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and
evidence for in vivo tyrosine kinase inhibition. Clin Cancer Res 2:659-668.
Fung HL, Haas E, Raybon J, Xu J and Fung SM (2004) Liquid chromatographic-mass
spectrometric determination of endogenous gamma-hydroxybutyrate
concentrations in rat brain regions and plasma. J Chromatogr B Analyt Technol
Biomed Life Sci 807:287-291.
Halestrap AP and Meredith D (2004) The SLC16 gene family-from monocarboxylate
transporters (MCTs) to aromatic amino acid transporters and beyond. Pflugers
Arch 447:619-628.
Halestrap AP and Price NT (1999) The proton-linked monocarboxylate transporter
(MCT) family: structure, function and regulation. Biochem J 343 Pt 2:281-299.
Horvathova K, Novotny L, Tothova D and Vachalkova A (2004) Determination of free
radical scavenging activity of quercetin, rutin, luteolin and apigenin in H2O2treated human ML cells K562. Neoplasma 51:395-399.
Kotanidou A, Xagorari A, Bagli E, Kitsanta P, Fotsis T, Papapetropoulos A and Roussos
C (2002) Luteolin reduces lipopolysaccharide-induced lethal toxicity and
expression of proinflammatory molecules in mice. Am J Respir Crit Care Med
165:818-823.
Kruijtzer CM, Beijnen JH, Rosing H, ten Bokkel Huinink WW, Schot M, Jewell RC,
Paul EM and Schellens JH (2002) Increased oral bioavailability of topotecan in
93
combination with the breast cancer resistance protein and P-glycoprotein inhibitor
GF120918. J Clin Oncol 20:2943-2950.
Lettieri J and Fung HL (1976) Absorption and first-pass metabolism of 14C-gammahydroxybutyric acid. Res Commun Chem Pathol Pharmacol 13:425-437.
Lettieri JT and Fung HL (1979) Dose-dependent pharmacokinetics and hypnotic effects
of sodium gamma-hydroxybutyrate in the rat. J Pharmacol Exp Ther 208:7-11.
Li L, Jiang H, Wu H and Zeng S (2005) Simultaneous determination of luteolin and
apigenin in dog plasma by RP-HPLC. J Pharm Biomed Anal 37:615-620.
Li Y, Pan WS, Chen SL, Xu HX, Yang DJ and Chan AS (2006) Pharmacokinetic, tissue
distribution, and excretion of puerarin and puerarin-phospholipid complex in rats.
Drug Dev Ind Pharm 32:413-422.
Li YC, Hung CF, Yeh FT, Lin JP and Chung JG (2001) Luteolin-inhibited arylamine Nacetyltransferase activity and DNA-2-aminofluorene adduct in human and mouse
leukemia cells. Food Chem Toxicol 39:641-647.
Mamelak M, Scharf MB and Woods M (1986) Treatment of narcolepsy with gammahydroxybutyrate. A review of clinical and sleep laboratory findings. Sleep 9:285289.
Manach C, Scalbert A, Morand C, Remesy C and Jimenez L (2004) Polyphenols: food
sources and bioavailability. Am J Clin Nutr 79:727-747.
Mason PE and Kerns WP, 2nd (2002) Gamma hydroxybutyric acid (GHB) intoxication.
Acad Emerg Med 9:730-739.
Morris ME, Hu K and Wang Q (2005) Renal clearance of gamma-hydroxybutyric acid in
rats: increasing renal elimination as a detoxification strategy. J Pharmacol Exp
Ther 313:1194-1202.
Nagasawa K, Nagai K, Sumitani Y, Moriya Y, Muraki Y, Takara K, Ohnishi N,
Yokoyama T and Fujimoto S (2002) Monocarboxylate transporter mediates
uptake of lovastatin acid in rat cultured mesangial cells. J Pharm Sci 91:26052613.
Okamura A, Emoto A, Koyabu N, Ohtani H and Sawada Y (2002) Transport and uptake
of nateglinide in Caco-2 cells and its inhibitory effect on human monocarboxylate
transporter MCT1. Br J Pharmacol 137:391-399.
Palatini P, Tedeschi L, Frison G, Padrini R, Zordan R, Orlando R, Gallimberti L, Gessa
GL and Ferrara SD (1993) Dose-dependent absorption and elimination of gammahydroxybutyric acid in healthy volunteers. Eur J Clin Pharmacol 45:353-356.
Sparreboom A, Cox MC, Acharya MR and Figg WD (2004) Herbal remedies in the
United States: potential adverse interactions with anticancer agents. J Clin Oncol
22:2489-2503.
Tamai I, Sai Y, Ono A, Kido Y, Yabuuchi H, Takanaga H, Satoh E, Ogihara T, Amano
O, Izeki S and Tsuji A (1999) Immunohistochemical and functional
characterization of pH-dependent intestinal absorption of weak organic acids by
the monocarboxylic acid transporter MCT1. J Pharm Pharmacol 51:1113-1121.
Van Sassenbroeck DK, De Paepe P, Belpaire FM and Buylaert WA (2003)
Characterization of the pharmacokinetic and pharmacodynamic interaction
between gamma-hydroxybutyrate and ethanol in the rat. Toxicol Sci 73:270-278.
94
Wang Q, Darling IM and Morris ME (2006) Transport of gamma-hydroxybutyrate in rat

kidney membrane vesicles: Role of monocarboxylate transporters. J Pharmacol
Exp Ther 318:751-761.
Wang Q, Lu Y and Morris ME (2007) Monocarboxylate Transporter (MCT) Mediates the
Transport of gamma-Hydroxybutyrate in Human Kidney HK-2 cells. Pharm Res
24:1067-1078.
Wang Q and Morris ME (2007a) Flavonoids modulate monocarboxylate transporter-1mediated transport of gamma-hydroxybutyrate in vitro and in vivo. Drug Metab
Dispos 35:201-208.
Wang X and Morris ME (2007b) Effects of the flavonoid chrysin on nitrofurantoin
pharmacokinetics in rats: potential involvement of ABCG2. Drug Metab Dispos
35:268-274.
Wang YH, Chao PD, Hsiu SL, Wen KC and Hou YC (2004) Lethal quercetin-digoxin
interaction in pigs. Life Sci 74:1191-1197.
Xie LP, Chen QX, Huang H, Wang HZ and Zhang RQ (2003) Inhibitory effects of some
flavonoids on the activity of mushroom tyrosinase. Biochemistry (Mosc) 68:487491.
95
Table 1. Effects of luteolin on the pharmacokinetics and pharmacodynamics of GHB in SD rats.

GHB (1000 mg/kg, i.v.)
GHB (400 mg/kg, i.v.)
Control
Luteolin (4 mg/kg)
Luteolin (10 mg/kg)
Control
Luteolin (10 mg/kg)
(n = 6)
(n = 5)
(n = 4)
(n = 4)
(n = 3)
AUC (mgxml-1xmin)
170 39.8
117 28.6 *
113 20.8 *
56.6 6.31
46.6 6.58
Cl (mlxmin-1xkg-1)
6.19 1.59
8.92 2.02 *
9.05 1.43 *
7.13 0.73
8.70 1.33
Urinary recovery %
27.1 9.04
29.9 19.6
42.1 10.3
8.77 4.64
10.9 3.39
ClR (mlxmin-1xkg-1)
1.64 0.62
2.46 1.31
3.84 1.21 *
0.63 0.36
0.93 0.22
Clm (mlxmin-1xkg-1)
4.55 1.36
6.46 2.85
5.22 1.19
6.50 0.70
7.77 1.37
Sleep Time (min)
161 16
131 14 **
121 5 **
60 2
39 11 **
Parameters
The plasma concentrations of GHB in the absence or presence of luteolin were determined as described under Materials
and Methods. The pharmacokinetic parameters were obtained by noncompartmental analysis using WinNonlin. Data were
presented as mean SD. The number of animals for each group was specified in parentheses after the corresponding
group names (*: p < 0.05, **: p < 0.01 compared with the corresponding control group).
96
Table 2. Fitted pharmacokinetic parameters of luteolin following intravenous

administration in SD rats.
Luteolin (2 doses)
Parameters
Fitted values
CV %
k10 (min-1)
0.124
16.4
k12 (min-1)
0.120
17.3
k21 (min-1)
0.0224
12.2
204
20.0
VC_Lut (ml/kg)
Model fitting of luteolin pharmacokinetics at the doses of 4 mg/kg and 10 mg/kg was
conducted as described under Materials and Methods. Several models were constructed
to characterize luteolin pharmacokinetics. The final pharmacokinetic model for luteolin
(equation 1 and 2) was selected based on the goodness-of-fit criteria (coefficient variation
of the estimates, R2, Akaikes Information Criterion and Schwartz Criterion).
97
Table 3. Fitted pharmacokinetic parameters from the integrated GHB and luteolin model.
Competitive
Noncompetitive
Uncompetitive
Fitted
values
CV %
Fitted
values
CV %
Fitted
values
CV %
Vmax (mgxmin-1xkg-1)
2.27
Fixed
2.27
Fixed
2.27
Fixed
km (mg/ml)
0.063
Fixed
0.063
Fixed
0.063
Fixed
VGHB (ml/kg)
567
4.20
563
4.12
578
3.63
GFR (mlxmin-1xkg-1)
10
Fixed
10
Fixed
10
Fixed
Vmax_ren (mgxmin-1xkg-1)
24.8
36.2
21.0
28.3
49.2
38.5
kren (mg/ml)
2.56
44.7
2.05
36.0
5.54
43.5
Ki (g/kg)
572
20.7
686
17.5
63.3
35.2
AIC
-117
-122
-141
SC
-104
-109
-127
Simultaneous fitting of luteolin and GHB pharmacokinetics was conducted as described

under Materials and Methods. Competitive, noncompetitive and uncompetitive models
were constructed to characterize luteolin inhibition. The final pharmacokinetic model for
luteolin and GHB interaction was selected based on the goodness-of-fit criteria
(coefficient variation of the estimates, R2, Akaikes Information Criterion and Schwartz
Criterion).
98
Table 4. Model simulation of luteolin-GHB interaction following i.v. bolus of GHB and luteolin in SD rats.
GHB (200 mg/kg, i.v. bolus)
Luteolin
(i.v. bolus)
AUC
-1
(mgxml xmin)
Cl
-1

AUC
-1
(mlxmin xkg )
-1
(mgxml xmin)
Cl
-1
AUC
-1
(mlxmin xkg )
-1
(mgxml xmin)
Cl
(mlxmin-1xkg-1)
0 mg/kg
17.8 3.65
11.6 2.16
52.0 5.98
7.78 0.85
184 9.46
5.45 0.28
2 mg/kg
16.0 3.32
12.9 2.41
44.0 5.22 **
9.20 1.03 *
146 8.21 **
6.88 0.38 **
4 mg/kg
15.2 3.14
13.6 2.52
40.8 4.84 **
9.91 1.10 **
132 7.49 **
7.62 0.42 **
10 mg/kg
14.0 2.82
14.7 2.67
36.2 4.21 **
11.2 1.22 **
113 6.26 **
8.91 0.48 **
20 mg/kg
13.1 2.54 *
15.7 2.77 **
32.9 3.67 **
12.3 1.29 **
100 5.32 **
10.0 0.51 **
Model simulation of luteolin-GHB interaction was conducted using the parameters obtained from the model fitting with
the incorporation of a 10% variance using ADAPT II software. Three doses of GHB (200, 400 and 1000 mg/kg) and five
doses of luteolin (0, 2, 4, 10 and 20 mg/kg) were used in the simulation. Number of simulation = 8. Data were presented
as mean SD. *: p < 0.05, **: p < 0.01 compared with the corresponding luteolin 0 mg/kg group.
99
FIGURE LEGEND:
Figure 1. Pharmacokinetics of luteolin in rats following intravenous administration.

Plasma concentrations of luteolin at the doses of 4 mg/kg (z) and 10 mg/kg () were
plotted as mean SD, n = 3 for both groups.
Figure 2. Effects of luteolin on GHB pharmacokinetics in rats following intravenous

coadministration. Plasma concentrations of GHB (1000 mg/kg) in the absence (z) or
presence of luteolin at the doses of 4 mg/kg (T) and 10 mg/kg () were plotted as mean
SD. Data points shown as () and (S) represent the plasma concentrations of GHB
(400 mg/kg) in the absence or presence of luteolin (10 mg/kg), respectively. n = 3-6 for
each group.
Figure 3. Pharmacokinetic/pharmacodynamic correlation of GHB. AUC of GHB (xaxis) was plotted against GHB-induced sleep time (y-axis). Data were presented as mean
SD, n = 3-6 for each data point. Some of the data in the figure were obtained from a
previous study (Wang and Morris, 2007a). Some of the data in the figure were obtained
from L-lactate study (unpublished data). The doses of GHB are 400 and 1000 mg/kg.
The solid line represents the linear regression of the data. The dashed lines represent the
95% confidence intervals.
Figure 4. The proposed model for luteolin and GHB pharmacokinetics. CC_Lut and CT_Lut
represent luteolin concentration in the central and peripheral compartment, respectively.
VC_Lut and VT_Lut represent the volume of distribution of luteolin in the central and
100
peripheral compartment, respectively. CGHB and VGHB represent GHB plasma

concentration and volume of distribution, respectively. k10 represents luteolin elimination
rate constant of the central compartment. k12 and k21 represent luteolin first-order
distribution rate constants between the two compartments. Vmax represents the maximal
metabolic rate of GHB and km represents the concentration of GHB at 50% of the
maximal metabolic rate. GFR represents the glomerular filtration rate in the kidney.
Vmax_ren represented the maximal renal reabsorption rate of GHB and kren represents the
concentration of GHB at 50% of the maximal reabsorption rate. I represents luteolin in
the central compartment that inhibits GHB reabsorption and Ki represents the inhibition
constant at 50% of inhibition.
Figure 5. Model fitting of luteolin pharmacokinetics at the doses of 4 mg/kg (z) and 10
mg/kg (). Experimentally determined data were plotted as mean SD (n = 3). The
solid and dashed lines represent the fitted lines for the doses of 4 mg/kg and 10 mg/kg,
respectively. The model fitting results were obtained using equation 1 and 2.
Figure 6. Model fitting of GHB pharmacokinetics in the absence and presence of

luteolin. Experimentally determined data were plotted as mean SD (n = 3-6). Data
shown as (z), (T), and () represent the plasma concentrations of GHB (1000 mg/kg)
in the absence or presence of 4 mg/kg and 10 mg/kg luteolin, respectively. Data points
shown as () and (S) represent the plasma concentrations of GHB (400 mg/kg) in the
absence or presence of luteolin (10 mg/kg), respectively. The lines represent the fitted
lines obtained from the simultaneous fitting using equation 1-6.
101
Figure 7. Model simulation of luteolin-GHB interaction following i.v. bolus of GHB (A,
B and C for 1000, 400 and 200 mg/kg, respectively) in the presence of various doses of
luteolin (z: 0 mg/kg; {: 2 mg/kg; T: 4 mg/kg; V: 10 mg/kg; : 20 mg/kg). Model
simulations were conducted using the parameters obtained from the model fitting with the
incorporation of a 10% variance using ADAPT II software.
102
Figure 1.
103
Figure 2.
104
Figure 3.
105
Figure 4.
106
Figure 5.
107
Figure 6.
108
Figure 7.
109
CHAPTER 4
Effects of the Flavonoid Chrysin on Nitrofurantoin

Pharmacokinetics in Rats: Potential Involvement of ABCG2
This chapter has been published in Drug Metab Dispos, 35(2): 268-74, 2007.
110
ABSTRACT
Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette efflux

transporter, important in drug disposition and in the development of multidrug resistance
in cancer. Flavonoids, a large class of natural compounds widely present in the diet and
herbal products, have been demonstrated in vitro as BCRP inhibitors. The flavonoid
chrysin is a potent inhibitor of BCRP, inhibiting the efflux of mitoxantrone with an IC50
of 0.39 M in BCRP-overexpressing human MCF-7 breast cancer cells. The purpose of
this study was to investigate the potential pharmacokinetic interactions between chrysin
and nitrofurantoin (a specific BCRP substrate) in rats. In MDCK cells expressing human
BCRP or murine Bcrp1, the polarized transport of nitrofurantoin was effectively inhibited
by chrysin at concentrations of 20 and 100 M. Compared with the vehicle-treated
group, oral coadministration of chrysin (200 mg/kg) significantly increased the AUC and
Cmax of nitrofurantoin (10 mg/kg) by 1.76 (p<0.01) and 1.72-fold (p<0.05), respectively.
When nitrofurantoin (2 mg/kg) was given intravenously, administration of chrysin (50
mg/kg, i.p.) significantly increased the AUC of nitrofurantoin (123 34.0 versus 91.5
18.0 g/mlmin in controls, p<0.05). Moreover, the cumulative hepatobiliary excretion
of nitrofurantoin (1.5 mg/kg, i.v.) was significantly decreased by approximately 75% at
the end of 120 min following the coadministration of chrysin (50 mg/kg, i.p.). Taken
together, these results indicate that the flavonoid chrysin significantly inhibits
nitrofurantoin transport mediated by human BCRP and murine Bcrp1. Bcrp1 inhibition
by chrysin is likely one potential mechanism for the observed chrysin-nitrofurantoin
pharmacokinetic interactions in rats.
111
INTRODUCTION
Flavonoids, a large class of polyphenolic compounds present in the diet and many herbal
products, have long been associated with a variety of biochemical and pharmacological
activities important in cancer prevention and health promotion (Middleton et al., 2000;
Havsteen, 2002). The flavonoid chrysin (5,7-dihydroxyflavone) is present at high levels
in honey and propolis (Siess et al., 1996), and in many plant extracts (Williams et al.,
1997). In addition to its reported anti-carcinogenic (Cardenas et al., 2006), anti-viral
(Critchfield et al., 1996), anti-oxidant (Lapidot et al., 2002), and anti-inflammatory (Cho
et al., 2004) activities, chrysin has been shown as a potent inhibitor of aromatase (Jeong
et al., 1999), an enzyme responsible for the conversion of androstenedione and
testosterone into estrone and estradiol, respectively (Meinhardt and Mullis, 2002). As
such, chrysin acts as a testosterone-boosting agent, and is currently available as dietary
supplements used for increasing lean body mass. The products on the market typically
contain 500 mg chrysin per capsule (iHerb Inc., Monrovia, CA; VitaDigest, Walnut, CA)
and the suggested dosage can be as high as 6 capsules per day. Upon consumption of
these dietary supplements, high concentrations of chrysin in the intestine would be
expected, raising the potential for flavonoid-drug interactions.
Recently, due to an increased public interest in alternative medicine and disease

prevention, the use of dietary supplements and herbal preparations containing high doses
of flavonoids for health maintenance has become very popular (Sparreboom et al., 2004).
As a result, flavonoid-drug interactions have been increasingly evidenced in animal and
clinical studies (Dresser et al., 2002; Choi et al., 2004b). In some cases, these flavonoid-
112
drug interactions can be serious or even life-threatening. This is true especially when
flavonoids or dietary supplements are used concomitantly with narrow therapeutic index
drugs, such as digoxin (Wang et al., 2004b). On the other hand, coadministration of drug
candidates with flavonoids, which are inhibitors of known drug transporters and
metabolizing enzymes, may represent a strategy to improve the bioavailability of the
coadministered drug. For example, flavonoid flavone has been shown to increase the
bioavailability of paclitaxel (a dual substrate of P-glycoprotein and cytochrome P450
(CYP) 3A) in rats in a concentration-dependent manner (Choi et al., 2004b).
The mechanisms underlying most of the reported flavonoid-drug interactions have been
ascribed to the inhibition of P-glycoprotein and/or drug metabolizing enzymes. Very
little information is currently available on flavonoid-drug interactions mediated by other
major efflux drug transporters. Breast cancer resistance protein (BCRP) is a recently
identified ATP-binding cassette transporter, important in conferring cellular resistance to
many chemotherapeutic agents, such as anthracyclines, camptothecin derivatives,
mitoxantrone, and nucleoside analogs (Mao and Unadkat, 2005). In addition, BCRP has
been shown to play a critical role in the disposition of dietary carcinogens (van
Herwaarden et al., 2003), and several clinically important drugs, such as imatinib
mesylate (Breedveld et al., 2005), topotecan, and cimetidine (Jonker et al., 2005). In
recent pre-clinical and clinical studies, inhibition of BCRP by GF120918 has been shown
to significantly increase the bioavailability and decrease the biliary excretion of topotecan
(a BCRP substrate) (Jonker et al., 2000; Kruijtzer et al., 2002). Therefore, inhibition of
BCRP might represent an additional mechanism underlying drug-drug interactions.
113
Flavonoids represent a class of BCRP inhibitors, with chrysin being one of the most
potent ones in inhibiting the efflux of mitoxantrone with an IC50 of 0.39 0.13 M
(Zhang et al., 2004d; Zhang et al., 2004c). Therefore, we hypothesized that inhibition of
BCRP by the flavonoid chrysin might alter the pharmacokinetics of drugs that are BCRP
substrates. In a recent study, nitrofurantoin, an antibiotic used in the treatment of urinary
tract infections, was shown to be specifically transported by human BCRP and murine
Bcrp1, but not by P-glycoprotein and multidrug resistance-associated protein 2 (MRP2)
(Merino et al., 2005). Moreover, chrysin was shown to exhibit very weak, if any,
inhibitory effects on MRP1 and MRP2 (van Zanden et al., 2005b). Additionally, studies
in MRP1-expressing H69-AR cells have found no increased accumulation of
nitrofurantoin in the presence of cyclophosphamide, further suggesting that
nitrofurantoin may not be a substrate for MRP1 (Wang and Morris, unpublished results).
Given the transporter specificity of nitrofurantoin and inhibition spectrum of chrysin,
BCRP inhibition by chrysin is likely an important, if not the only, mechanism underlying
the interaction between chrysin and nitrofurantoin.
The objective of this study was to examine the effects of chrysin on human BCRP- and
murine Bcrp1-mediated transport of nitrofurantoin using polarized cells lines, and to
investigate the potential pharmacokinetic interaction between chrysin and nitrofurantoin
in rats.
114
METERIALS AND METHODS

Animals
Female Sprague-Dawley (SD) rats (body weight ~ 220 g) were purchased from Harlan
(Indianapolis, IN). Animals were housed in a temperature-controlled environment with a
12-h light/dark cycle and received a standard diet with free access to tap water. Animals
were acclimatized to this environment for at least one week. All animal studies were
reviewed and approved by the University at Buffalo Institutional Animal Care and Use
Committee.
Materials
Chrysin, glycofurol, polyethylene glycol 400, nitrofurantoin, and furazolidone were

purchased from Sigma-Aldrich (St. Louis, MO). Dulbeccos Modified Eagle Medium
(DMEM), Hanks buffered salt solution (HBSS), fetal bovine serum, penicillin, and
streptomycin were purchased from Invitrogen (Carlsbad, CA). Fumitremorgin C (FTC)
was a kind gift from Dr. Susan E. Bates (National Cancer Institute, Bethesda, MD). All
other reagents or solvents used were either analytical or high-performance liquid
chromatography (HPLC) grade.
Cell Culture
The polarized Madin-Darby canine kidney cell line (MDCK-II) was used in the transport
studies. MDCK-mock (MDCK-II cells transfected with empty vector), MDCK-Bcrp1
(MDCK-II cells transfected with murine Bcrp1), and MDCK-BCRP (MDCK-II cells
transfected with human BCRP) cells were kind gifts from Dr. Alfred Schinkel
115
(Netherlands Cancer Institute, Amsterdam, The Netherlands). The expression of human

BCRP and murine Bcrp1 in transfected MDCK cells has been characterized previously
(Jonker et al., 2000; Pavek et al., 2005), and was confirmed in our lab (data not shown).
The MDCK cells were cultured in 75-cm2 flasks with DMEM medium supplemented
with 10% fetal bovine serum at 37oC in a humidified 5% CO2 /95 % air atmosphere. The
culture medium also contained 100 units/ml penicillin and 100 g/ml streptomycin. The
cells were trypsinized every 3 to 4 days for subculturing.
Nitrofurantoin Bidirectional Transport Studies
Transport studies using MDCK cell monolayers were performed as described previously
(Zhang et al., 2005a) with minor modifications. Briefly, MDCK-bcrp1, MDCK-BCRP
and MDCK-mock cells were seeded on Transwell polycarbonate inserts (six-well, 0.4-m
pore size; Transwell 3412, Corning Glassworks, Corning, NY) at a density of
approximately 106 cells/well. Cells were grown for 5 ~ 7 days, and medium was replaced
every day. On the day of the transport study, cell monolayers were first washed with
HBSS (pH 7.2) twice, and incubated at 37C for a total of 30 min. Transport buffer
(HBSS, pH 7.2) containing specified concentrations of chrysin or 10 M FTC or 0.1%
dimethyl sulfoxide (control) was then loaded into both apical (1.5 ml) and basolateral (2.6
ml) chambers. After a 15-min of incubation, nitrofurantoin (10 M) was added to either
the apical or basolateral chamber (donor chamber). The aliquots (100 l) were taken
from the opposite chamber (receiver chamber) at 30, 60, and 90 min after the addition of
nitrofurantoin and replaced with the same volume of fresh transport buffer. The samples
were stored at -80C until HPLC analysis. The integrity of the monolayer was measured
116
by monitoring the flux of [3H]mannitol, a paracellular marker, across cell monolayers.

The apparent permeability coefficients (Papp) of mannitol in both apical to basolateral
(Papp, AP-BL) and basolateral to apical (Papp, BL-AP) directions were measured in triplicate,
and these values were all lower than 1.0 x 106 cm/s. The apparent permeability
coefficient was calculated as follows:
Papp =
Q
1
t A C 0
where Q/t is the rate of nitrofurantoin appearing in the receiver chamber, which was
obtained from the slope of the regression line on the transport-time profile of
nitrofurantoin across MDCK cell monolayers, C0 is the initial concentration of
nitrofurantoin loaded in the donor chamber, and A is the cell monolayer surface area
(4.71 cm2).
Pharmacokinetic studies were performed in a manner similar to that previously described

(Merino et al., 2005; Zhang et al., 2005a) with some modifications. Briefly, jugular vein
cannulation was performed in rats following an intramuscular injection of 90 mg/kg
ketamine and 10 mg/kg xylazine (Henry Schein, Melville, NY) at least 3 days before
pharmacokinetic studies. For oral drug interaction studies, rats were kept fasting
overnight with free access to tap water. A specified dose of the test compounds (chrysin
dissolved in glycofurol) or the vehicle glycofurol (as control) was administered to rats by
gavage. Chrysin was dissolved in glycofurol at such a concentration that the specified
dose was delivered as 10 l/g body weight drug solution. Five minutes later, the animals
were administered 10 mg/kg nitrofurantoin orally (3 mg/ml dissolved in 50% (v/v)
117
ethanol and 50% (v/v) polyethylene glycol 400). The blood samples (150 l) were taken
from the jugular vein cannula at 0 (predose), 5, 15, 30, 60, 120, 240, 360, 480, and 720
min after nitrofurantoin administration. For intravenous administration of nitrofurantoin,
a dose of 50 mg/kg chrysin (dissolved in dimethyl sulfoxide at a concentration of 20
mg/ml) or vehicle (dimethyl sulfoxide) was first given to rats via intraperitoneal
injection. Five minutes later, a dose of 2 mg/kg nitrofurantoin (1 mg/ml dissolved in
10% (v/v) ethanol, 40% (v/v) polyethylene glycol 400, and 50% phosphate-buffered
saline) was given to the rats via jugular vein cannula. The blood samples (150 l) were
taken at 0 (predose), 2, 7, 15, 30, 60, and 120 min after nitrofurantoin administration.
Urine samples were collected over the time periods of 0 to 2, 2 to 4, and 4 to 8 h after
nitrofurantoin administration. All samples were stored at 80C until the time of HPLC
analysis. Four to seven rats were used for each group.
Bile Duct Cannulation Experiment
Bile duct cannulation was carried out as described previously (Jackson et al., 2000) under
anesthesia (a combination of 90 mg/kg ketamine and 10 mg/kg xylazine ). A dose of 50
mg/kg chrysin (dissolved in dimethyl sulfoxide at a concentration of 20 mg/ml) or
vehicle (dimethyl sulfoxide) was first given to rats via intraperitoneal injection. Five
minutes later, a dose of 1.5 mg/kg nitrofurantoin (1 mg/ml dissolved in 10% (v/v)
ethanol, 40% (v/v) polyethylene glycol 400, and 50% phosphate-buffered saline) was
administered via tail vein injection. Bile was collected in 15-min fractions for 120 min
after the administration of nitrofurantoin. Bile samples were stored at 80C until HPLC
analysis. Four to five animals were used for each group.
118
HPLC Analysis
The concentration of nitrofurantoin in rat plasma, urine, bile, and buffer samples from
transport studies was analyzed by a previously published method (Merino et al., 2005)
with minor modifications. Briefly, an aliquot of 50 l sample was spiked with 5 l of
12.5 g/ml furazolidone solution (internal standard), and then 50 l of cold methanol (
20oC) was added. The mixture was vortexed vigorously for 60s and incubated at 20oC
for 15 min. The organic and water phases were separated by centrifugation at 16,000g
for 10 min at 4oC, and 30 l of the supernatant was injected into HPLC system. The
HPLC analysis was performed using an Alltech Alltima C18 column (125 4.6 mm, 5m particle size). The system consisted of a Waters 1525 pump, 717 plus autosampler, a
2847 UV detector, and a Waters BreezeTM workstation. The composition of the mobile
phase was 25 mM potassium phosphate buffer (pH3)/acetonirile (75:25). The mobile
phase was delivered isocratically with a flow rate of 1.0 ml/min. UV absorbance was
measured at 366 nm. Nitrofurantoin appeared on the chromatograph at approximately 8
min with no interference peaks. The standard curve was linear over the concentration
range of 0.01 to 20 g/ml with a regression coefficient greater than 0.99. The lower limit
of quantitation of nitrofurantoin was 10 ng/ml.
The pharmacokinetic parameters of nitrofurantoin were obtained by noncompartmental

analysis using WinNonlin version 2.1 (Pharsight, Mountain View, CA). The area under
the plasma concentration-time curve (AUC) was calculated using trapezoidal method.
119
The terminal half-life (t1/2) was calculated as ln2/k, and k was determined from the slope
of the terminal regression line. The systemic clearance (CL) and oral clearance (CL/F)
were calculated as the intravenous and oral dose divided by AUC, respectively. The
bioavailability (F) was determined by (AUCp.o./Dosep.o.)/ (AUCi.v./Dosei.v.).
Data were analyzed for statistically significant differences using ANOVA followed by a
Bonferroni or Dunnetts post hoc test or by the two-sided unpaired Students t test. p
values < 0.05 were considered statistically significant.
120
RESULTS
Effects of Chrysin on In Vitro Transport of Nitrofurantoin. Flavonoid chrysin has
been shown to inhibit BCRP-mediated efflux of mitoxantrone (Zhang et al., 2004d);

however, its effects on BCRP-mediated efflux of nitrofurantoin have not been evaluated.
Therefore, we first looked at the effects of chrysin on nitrofurantoin transport in both
MDCK parental cells (MDCK-mock) and MDCK cells expressing either murine Bcrp1
(MDCK-Bcrp1) or human BCRP (MDCK-BCRP). As shown in Fig. 1, the polarized
transport of nitrofurantoin (10 M) was observed in both MDCK-Bcrp1 and MDCKBCRP cells in the absence of inhibitors, with Papp, BL-AP values significantly higher than
the corresponding Papp, AP-BL values (9.69 0.41 versus 0.70 0.23 10-6 cm/s, p < 0.001
and 11.7 0.80 versus 2.91 0.21 10-6 cm/s, p < 0.001 for MDCK-Bcrp1 and MDCKBCRP cells, respectively). When the specific Bcrp1/BCRP inhibitor FTC (10 M) was
used, the Bcrp1/BCRP-mediated transport was completely inhibited, resulting in vectorial
transport patterns similar to that of the MDCK parental cells. In the presence of chrysin
(20 and 100 M), apically directed transport of nitrofurantoin was significantly decreased
and basolaterally directed transport was significantly increased in both MDCK-Bcrp1 and
MDCK-BCRP cells, resulting in efflux ratios (Papp, BL-AP / Papp, AP-BL) close to unity. For
example, in MDCK-Bcrp1 cells, the efflux ratios were decreased from 13.9 (without
inhibitors) to 1.75 (with 20 M chrysin) and 1.29 (with 100 M chrysin); and in MDCKBCRP cells, the efflux ratios were decreased from 4.02 (without inhibitors) to 1.08 (with
20 M chrysin) and 0.77 (with 100 M chrysin). These results suggest that chrysin
indeed inhibits both murine Bcrp1 and human BCRP-mediated transport of nitrofurantoin
in MDCK cells. In MDCK parental cells, nitrofurantoin was consistently transported
121
somewhat more efficiently in the basolateral direction than in the apical direction,
indicating low endogenous basolaterally directed transport.
Effects of Chrysin on Nitrofurantoin Pharmacokinetics in SD Rats. The expression
of Bcrp1 has been previously reported at high levels in the intestinal, renal and hepatic
tissues in female Sprague-Dawley rats (Tanaka et al., 2005). To assess whether the in
vitro chrysin-nitrofurantoin interactions via Bcrp1/BCRP also occurred in vivo, the
pharmacokinetics of nitrofurantoin, following its oral and intravenous administration,
were determined in SD female rats with and without the coadministration of chrysin. As
shown in Fig. 2, oral coadministration of chrysin (200 mg/kg) significantly increased the
AUC and Cmax of nitrofurantoin (10 mg/kg) by 1.76-fold (605 163 in the chrysintreated group versus 344 101 g/mlmin in the vehicle-treated (control) group, p<0.01)
and 1.72-fold (1.73 0.52 in the chrysin-treated group versus 1.01 0.47 g/ml in the
vehicle-treated group, p<0.05), respectively. In contrast, the apparent oral clearance of
nitrofurantoin (CL/F) was significantly decreased from 31.0 8.65 ml/min/kg in the
control group to 17.9 6.58 ml/min/kg in the 200 mg/kg chrysin-treated group (p<0.05).
The terminal t1/2, however, was not significantly changed following the coadministration
of chrysin. At a lower oral dose of 50 mg/kg, chrysin had no significant effects on
nitrofurantoin pharmacokinetics (Table 1). When nitrofurantoin (2 mg/kg) was given
intravenously, administration of chrysin (50 mg/kg) intraperitoneally significantly
increased the AUC of nitrofurantoin (123 34.0 versus 91.5 18.0 g/mlmin in
controls, p<0.05) (Fig. 3). Taking the AUC of intravenously administered nitrofurantoin
in vehicle-treated rats as 100 %, the bioavailability of nitrofurantoin administered orally
122
was significantly increased from 60.2 15.3 % in the control group to 114 17.9 % in
the 200 mg/kg chrysin-treated group (p<0.01). These results indicate that chrysin indeed
interacts with nitrofurantoin in vivo, altering both the bioavailability and elimination of
nitrofurantoin.
Effects of Chrysin on the Hepatobiliary and Urinary Excretion of Nitrofurantoin in

SD Rats. To investigate the effects of chrysin on nitrofurantoin hepatobiliary excretion,
1.5 mg/kg nitrofurantoin was administered to bile duct cannulated rats via a tail vein
injection 5 min after the intraperitoneal injection of vehicle or 50 mg/kg chrysin. Biliary
excretion of nitrofurantoin was measured in 15-min bile collections, obtained over 120
min. As shown in Fig. 4, the hepatobiliary excretion rate of nitrofurantoin was
substantially decreased in chrysin-treated rats for the first 60 min. After that, the
excretion rate in both groups gradually became similar. The cumulative hepatobiliary
excretion of nitrofurantoin was significantly decreased from 20.1 4.54 g (or 5.16
0.96 % of dose) in control rats to 5.39 2.24 g (or 1.21 0.50 % of dose) in chrysintreated rats (p<0.001) at the end of 120 min, indicating that approximately 75% of the
biliary excretion of nitrofurantoin over 2 h was abolished upon the administration of
chrysin.
To assess the effects of chrysin on the urinary excretion of nitrofurantoin, a 2 mg/kg dose
of nitrofurantoin was administered to SD rats via intravenous injection 5 min after the
intraperitoneal administration of control vehicle or 50 mg/kg chrysin. Levels of
nitrofurantoin in urine samples were measured by HPLC. Most of the nitrofurantoin in
123
urine was excreted during the first 0 to 8 h. In contrast to the biliary excretion of
nitrofurantoin, chrysin had no significant effects on the renal excretion of nitrofurantoin.
The percentage of the dose excreted in urine at the end of 8h was 39.0 10.2 % in
control rats and 35.8 8.79 % in chrysin-treated rats (p>0.05).
124
DISCUSSION
The recent resurgence of public interest in flavonoids has resulted in a dramatic increase
in the consumption of flavonoid-containing products for health maintenance and disease
prevention (Havsteen, 2002; Sparreboom et al., 2004). Increasing evidence from recent
animal and human studies has indicated that flavonoids may interact with many clinically
important drugs, causing favorable or adverse pharmacokinetic interactions (Kruijtzer et
al., 2002; Choi et al., 2004b; Wang et al., 2004b). However, the mechanisms underlying
most of these reported interactions are largely related to the modulation of drug
metabolizing enzymes and/or P-glycoprotein (Harris et al., 2003; Sparreboom et al.,
2004). To date, far less information is available on flavonoid-drug interactions mediated
by other major efflux drug transporters. Therefore, the objective of this study was to
determine the potential in vitro and in vivo flavonoid-drug interactions mediated by
BCRP a recently identified efflux transporter important in conferring multidrug
resistance in cancer therapy and in determining in vivo disposition of drugs and
xenotoxins (Mao and Unadkat, 2005; van Herwaarden and Schinkel, 2006).
The flavonoid selected for this study is chrysin, which was shown to be a potent human
BCRP inhibitor (Zhang et al., 2004c). The BCRP/Bcrp1 substrate, nitrofurantoin used in
this investigation, was previously reported as a specific substrate of BCRP/Bcrp1
(Merino et al., 2005). To determine whether chrysin interacts with nitrofurantoin in vivo,
we first investigated the effects of chrysin on the transport of nitrofurantoin across
MDCK-BCRP and MDCK-Bcrp1 cell monolayers. As shown in Fig. 1, chrysin, at
concentrations of 20 and 100 M, significantly decreased apically directed transport and
125
increased basolaterally directed transport of nitrofurantoin in both MDCK-Bcrp1 and

MDCK-BCRP cells, resulting in efflux ratios close to unity. These results suggested that
chrysin indeed effectively inhibited both Bcrp1 and BCRP-mediated transport of
nitrofurantoin in MDCK cells. Consistent with a previous report (Merino et al., 2005), in
MDCK parental cells, nitrofurantoin was consistently transported somewhat more
efficiently in the basolateral direction than in the apical direction, indicating the presence
of low endogenous basolaterally directed transporter(s). Further study is needed to
identify this unknown endogenous transporter(s). Similar to the results reported
previously (Merino et al., 2005), the efflux ratio of nitrofurantoin in MDCK-Bcrp1 cells
was higher than that in MDCK-BCRP cell (13.9 versus 4.02). As suggested in other
studies, this is possibly due to an effectively lower expression level or reduced transport
efficiency of human BCRP in the cell line used (Merino et al., 2006). Compared to the
inhibitory effects of chrysin on murine Bcrp1, chrysin appeared to be a more efficient
inhibitor of human BCRP. At the concentrations of 20 and 100 M, chrysin almost
completely inhibited the activity of human BCRP, resulting in vectorial transport patterns
similar to that of the MDCK parental cells.
To further investigate the interactions of chrysin with nitrofurantoin in vivo,

pharmacokinetic studies of nitrofurantoin after oral and intravenous administration were
performed in rats with and without the coadministration of chrysin. Following the oral
administration of nitrofurantoin, chrysin (200 mg/kg) significantly increased the AUC
and Cmax, and decreased the apparent oral clearance (CL/F) of nitrofurantoin. Compared
to a 4-fold increase in nitrofurantoin AUC after oral administration in Bcrp1 knockout
126
mice (Merino et al., 2005), a 1.76-fold increase in AUC observed in this study suggested
that the in vivo Bcrp1 inhibition by chrysin (200 mg/kg) might not have been complete, if
we assume the similar contribution of Bcrp1 to the bioavailability and clearance of
nitrofurantoin in both rats and mice. However, previous pharmacokinetic studies in rats
and mice using topotecan as a probe substrate have shown that the contribution of Bcrp1
in mice is relatively larger than that in rats (> 1.5-fold) (Jonker et al., 2000; Zhang et al.,
2005a). Therefore, we cannot exclude the possibility that a 1.76-fold increase in
nitrofurantoin AUC in rats might still represent the nearly complete Bcrp1 inhibition by
chrysin, possibly attributed to the enhanced intestinal absorption and/or decreased
systemic elimination of nitrofurantoin. Considering the potentially high intestinal
concentrations of chrysin after oral administration and low bioavailability of chrysin
(Walle et al., 2001), the altered oral pharmacokinetics of nitrofurantoin is more likely due
to the local interactions of chrysin with nitrofurantoin in the intestine. At a lower oral
dose of 50 mg/kg, chrysin had no significant effects on nitrofurantoin pharmacokinetics.
Given the fact that chrysin potently inhibited nitrofurantoin transport in vitro, the lack of
in vivo effects of chrysin at a lower dose might be possibly due to its extensive metabolic
degradation in the intestine (Galijatovic et al., 1999). To determine whether chrysin also
affects the systemic elimination of nitrofurantoin, we administered chrysin via
intraperitoneal injection to ensure some of the dose of chrysin would reach the systemic
circulation. Nitrofurantoin was quickly eliminated in rats after intravenous
administration with t1/2 values of approximately 25 min, consistent with the results in a
previous report (Buzard et al., 1961). It should be noted that the t1/2 values of
nitrofurantoin following intravenous and oral administration were different (~ 25 min and
127
160 min, respectively), indicating the likely flip-flop oral kinetics of nitrofurantoin.
Administration of chrysin (50 mg/kg) intraperitoneally significantly increased the AUC
of nitrofurantoin, indicating chrysin might also inhibit nitrofurantoin elimination. Taking
the AUC of intravenously administered nitrofurantoin in control rats as 100 %, the
bioavailability of nitrofurantoin administered orally was significantly increased from 60.2
15.3 % in control rats to 114 17.9 % in 200 mg/kg chrysin-treated rats (p<0.01). The
apparent nitrofurantoin bioavailability value of greater than 100% may indicate that the
increase in nitrofurantoin AUC resulted not only from the increased intestinal absorption
but also from the decreased systemic elimination, consistent with the mdr1a/1b (-/-)
mouse study and our previous topotecan study (Jonker et al., 2000; Zhang et al., 2005a).
A previous study has shown that Bcrp1 plays a predominant role (>98 %) in
nitrofurantoin hepatobiliary excretion (Merino et al., 2005). In our study, consistent with
this result, the administration of chrysin remarkably inhibited nitrofurantoin hepatobiliary
excretion by 75 % (Fig. 4). This inhibition is likely due to the inhibition of Bcrp1mediated excretion of nitrofurantoin by chrysin, since nitrofurantoin is not transported by
other major efflux transporters present on the hepatic canalicular membrane, such as Pglycoprotein and MRP1/2. It has been reported that nitrofurantoin undergoes
enterohepatic cycling (Conklin et al., 1973). Therefore, inhibiting Bcrp1 by chrysin
might also possibly interfere with nitrofurantoin enterohepatic circulation.
It has been reported that Bcrp1 is present at high levels in the kidney in rats, suggesting
that Bcrp1 might play an important role in the renal excretion of substrate drugs (Tanaka
128
et al., 2005). Therefore, administration of chrysin with nitrofurantoin would be expected

to affect the urinary excretion of nitrofurantoin. In the control rats of this study, the
urinary excretion of unchanged nitrofurantoin after intravenous administration was 39.0
10.2 % of dose, which is close to the results reported in the literature (Paul et al., 1960;
Veronese et al., 1974). However, different from the results we have seen in the
hepatobiliary excretion study, chrysin had no significant effects on nitrofurantoin urinary
excretion (35.8 8.79 % of dose in chrysin-treated rats versus 39.0 10.2 % in control
rats, p>0.05). The exact mechanisms for this still remain to be elucidated. Interestingly,
similar results were reported in other studies, in which Bcrp1 was shown to play a
predominant role in determining the plasma concentration and hepatobiliary, but not
urinary, excretion of topotecan and nitrofurantoin (Jonker et al., 2000; Merino et al.,
2005). A possible explanation to this might be due to the relatively large variability in
urinary excretion data or possibly due to the involvement of other potential transport
mechanisms in the urinary excretion of nitrofurantoin (Moller and Sheikh, 1982).
It was previously shown that nitrofurantoin is likely a CYP 1A substrate and chrysin is a
CYP1A inhibitor with a Ki value in the low micromolar range (Jonen et al., 1980; Moon
et al., 1998). Therefore, inhibition of CYP1A enzymes by chrysin might also be
responsible for the increased plasma concentrations of nitrofurantoin. However, a
previous report has demonstrated that CYP1A-mediated metabolism of nitrofurantoin
does not represent a major metabolic pathway in CYP1A un-induced rats (Jonen et al.,
1980). Moreover, our LC/MS analysis of the samples from pharmacokinetic studies
indicated that the levels of 4-hydroxy-nitrofurantoin (the CYP1A metabolite of
129
nitrofurantoin) were much lower than those of nitrofurantoin (< 3% of parent drug)(data
not shown). Therefore, the contribution of CYP1A-mediated metabolism of
nitrofurantoin in our rat studies seems minor and inhibition of CYP1A by chrysin might
not have a significant role in nitrofurantoin pharmacokinetics.
In conclusion, we have demonstrated in this study that the flavonoid chrysin significantly
inhibited nitrofurantoin transport mediated by human BCRP and murine Bcrp1 in vitro.
More importantly, chrysin and nitrofurantoin pharmacokinetic interactions were observed
in vivo, likely due to the Bcrp1 inhibition by chrysin. These findings provide proof-of concept for in vivo inhibition of Bcrp1 by flavonoids, and lend insight into the potential
clinical interactions following concomitant use of these agents.
130
REFERENCES:
Breedveld P, Pluim D, Cipriani G, Wielinga P, van Tellingen O, Schinkel AH and

Schellens JH (2005) The effect of Bcrp1 (Abcg2) on the in vivo pharmacokinetics
and brain penetration of imatinib mesylate (Gleevec): implications for the use of
breast cancer resistance protein and P-glycoprotein inhibitors to enable the brain
penetration of imatinib in patients. Cancer Res 65:2577-2582.
Buzard JA, Conklin JD, O'Keefe E and Paul MF (1961) Studies on the absorption,
distribution and elimination of nitrofurantoin in the rat. J Pharmacol Exp Ther
131:38-43.
Cardenas M, Marder M, Blank VC and Roguin LP (2006) Antitumor activity of some
natural flavonoids and synthetic derivatives on various human and murine cancer
cell lines. Bioorg Med Chem.
Cho H, Yun CW, Park WK, Kong JY, Kim KS, Park Y, Lee S and Kim BK (2004)
Modulation of the activity of pro-inflammatory enzymes, COX-2 and iNOS, by
chrysin derivatives. Pharmacol Res 49:37-43.
Choi JS, Choi HK and Shin SC (2004) Enhanced bioavailability of paclitaxel after oral
Conklin JD, Sobers RJ and Wagner DL (1973) Further studies on nitrofurantoin excretion
in dog hepatic bile. Br J Pharmacol 48:273-277.
Critchfield JW, Butera ST and Folks TM (1996) Inhibition of HIV activation in latently
infected cells by flavonoid compounds. AIDS Res Hum Retroviruses 12:39-46.
71:11-20.
Galijatovic A, Otake Y, Walle UK and Walle T (1999) Extensive metabolism of the
flavonoid chrysin by human Caco-2 and Hep G2 cells. Xenobiotica 29:12411256.
Harris RZ, Jang GR and Tsunoda S (2003) Dietary effects on drug metabolism and
transport. Clin Pharmacokinet 42:1071-1088.
131
Jackson GD, Dai Y and Sewell WA (2000) Bile mediates intestinal pathology in
endotoxemia in rats. Infect Immun 68:4714-4719.
Jeong HJ, Shin YG, Kim IH and Pezzuto JM (1999) Inhibition of aromatase activity by
flavonoids. Arch Pharm Res 22:309-312.
Jonen HG, Oesch F and Platt KL (1980) 4-Hydroxylation of nitrofurantoin in the rat. A
3-methylcholanthrene-inducible pathway of a relatively nontoxic compound.
Drug Metab Dispos 8:446-451.
Jonker JW, Merino G, Musters S, van Herwaarden AE, Bolscher E, Wagenaar E,
Mesman E, Dale TC and Schinkel AH (2005) The breast cancer resistance protein
BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins into milk. Nat
Med 11:127-129.
Jonker JW, Smit JW, Brinkhuis RF, Maliepaard M, Beijnen JH, Schellens JH and
Schinkel AH (2000) Role of breast cancer resistance protein in the bioavailability
and fetal penetration of topotecan. J Natl Cancer Inst 92:1651-1656.
GF120918. J Clin Oncol 20:2943-2950.
Lapidot T, Walker MD and Kanner J (2002) Antioxidant and prooxidant effects of
phenolics on pancreatic beta-cells in vitro. J Agric Food Chem 50:7220-7225.
Mao Q and Unadkat JD (2005) Role of the breast cancer resistance protein (ABCG2) in
drug transport. Aaps J 7:E118-133.
Meinhardt U and Mullis PE (2002) The essential role of the aromatase/p450arom. Semin
Reprod Med 20:277-284.
Merino G, Alvarez AI, Pulido MM, Molina AJ, Schinkel AH and Prieto JG (2006) Breast
Cancer Resistance Protein (BCRP/ABCG2) transports fluoroquinolone antibiotics
and affects their oral availability, pharmacokinetics and milk secretion. Drug
Metab Dispos.
Merino G, Jonker JW, Wagenaar E, van Herwaarden AE and Schinkel AH (2005) The
breast cancer resistance protein (BCRP/ABCG2) affects pharmacokinetics,
hepatobiliary excretion, and milk secretion of the antibiotic nitrofurantoin. Mol
Pharmacol 67:1758-1764.
132
Moller JV and Sheikh MI (1982) Renal organic anion transport system: pharmacological,
physiological, and biochemical aspects. Pharmacol Rev 34:315-358.
Moon JY, Lee DW and Park KH (1998) Inhibition of 7-ethoxycoumarin O-deethylase
activity in rat liver microsomes by naturally occurring flavonoids: structureactivity relationships. Xenobiotica 28:117-126.
Paul MF, Paul HE, Bender RC, Kopko F, Harrington CM, Ells VR and Buzard JA (1960)
Studies on the distribution and excretion of certain nitrofurans. Antibiot
Chemother 10:287-302.
Pavek P, Merino G, Wagenaar E, Bolscher E, Novotna M, Jonker JW and Schinkel AH
(2005) Human breast cancer resistance protein: interactions with steroid drugs,
hormones, the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5b)pyridine, and transport of cimetidine. J Pharmacol Exp Ther 312:144-152.
Siess MH, Le Bon AM, Canivenc-Lavier MC, Amiot MJ, Sabatier S, Aubert SY and
Suschetet M (1996) Flavonoids of honey and propolis: characterization and
effects on hepatic drug-metabolizing enzymes and benzo[a]pyrene-DNA binding
in rats. J Agric Food Chem 44:2297-2301.
22:2489-2503.
Tanaka Y, Slitt AL, Leazer TM, Maher JM and Klaassen CD (2005) Tissue distribution
and hormonal regulation of the breast cancer resistance protein (Bcrp/Abcg2) in
rats and mice. Biochem Biophys Res Commun 326:181-187.
van Herwaarden AE, Jonker JW, Wagenaar E, Brinkhuis RF, Schellens JH, Beijnen JH
and Schinkel AH (2003) The breast cancer resistance protein (Bcrp1/Abcg2)
restricts exposure to the dietary carcinogen 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine. Cancer Res 63:6447-6452.
van Herwaarden AE and Schinkel AH (2006) The function of breast cancer resistance
protein in epithelial barriers, stem cells and milk secretion of drugs and
xenotoxins. Trends Pharmacol Sci 27:10-16.
van Zanden JJ, Wortelboer HM, Bijlsma S, Punt A, Usta M, Bladeren PJ, Rietjens IM
and Cnubben NH (2005) Quantitative structure activity relationship studies on the
flavonoid mediated inhibition of multidrug resistance proteins 1 and 2. Biochem
Pharmacol 69:699-708.
Veronese M, Salvaterra M, Barzaghi D and Setnikar I (1974) Urinary excretion in the rat
of nifurpipone (NP) and of nitrofurantoin (NTF) administered by different routes.
Arzneimittelforschung 24:39-43.
133
Walle T, Otake Y, Brubaker JA, Walle UK and Halushka PV (2001) Disposition and
metabolism of the flavonoid chrysin in normal volunteers. Br J Clin Pharmacol
51:143-146.
Williams CA, Harborne JB, Newman M, Greenham J and Eagles J (1997) Chrysin and
other leaf exudate flavonoids in the genus Pelargonium. Phytochemistry 46:13491353.
Zhang S, Wang X, Sagawa K and Morris ME (2005) Flavonoids chrysin and
benzoflavone, potent breast cancer resistance protein inhibitors, have no
significant effect on topotecan pharmacokinetics in rats or mdr1a/1b (-/-) mice.
Zhang S, Yang X and Morris ME (2004a) Combined effects of multiple flavonoids on
breast cancer resistance protein (ABCG2)-mediated transport. Pharm Res
21:1263-1273.
Zhang S, Yang X and Morris ME (2004b) Flavonoids are inhibitors of breast cancer
134
FIGURE LEGEND
Figure 1. Transepithelial transport of 10 M nitrofurantoin in MDCK-mock (parent)
(A), MDCK-Bcrp1 (B to E), and MDCK-BCRP (F to I) monolayers. The studies were

started with the addition of nitrofurantoin to one donor compartment (basolateral or
apical). At 30, 60, and 90 min, the amount of nitrofurantoin appeared in the opposite
compartment was measured by HPLC. BCRP inhibitor FTC (C and G) was used as the
positive control. Chrysin (D and E, H and I) was studied as the compound of interest.
The data are expressed as mean S.D., n = 3 or 6. z, transport from the basolateral to
apical compartment; {, transport from the apical to basolateral compartment.
Figure 2. Plasma concentration versus time profile of nitrofurantoin after oral
coadministration of nitrofurantoin (10 mg/kg) with control vehicle ({) or chrysin (50
mg/kg, z) or chrysin (200 mg/kg, S) in SD female rats. Plasma levels of nitrofurantoin
were determined by HPLC. The data are expressed as mean S.D.; n = 6 for control
group, n = 4 for 50 mg/kg chrysin group, and n = 5 for 200 mg/kg chrysin group (**,
p<0.01; ***, p<0.001 compared with the control group).
Figure 3. Plasma concentration versus time profile of nitrofurantoin after intravenous
injection of nitrofurantoin (2 mg/kg), 5 min following the intraperitoneal administration

of control vehicle ({) or chrysin (50 mg/kg, z) in SD female rats. Plasma levels of
nitrofurantoin were determined by HPLC. The data are expressed as mean S.D.; n = 7
for both control and 50 mg/kg chrysin group.
135
Figure 4. Cumulative biliary excretion of nitrofurantoin after tail vein injection of
nitrofurantoin (1.5 mg/kg), 5 min following the intraperitoneal administration of control

vehicle ({) or chrysin (50 mg/kg, z) in SD female rats. Levels of nitrofurantoin were
determined in bile fractions by HPLC. The data are expressed as mean S.D.; n = 4 for
control group, n = 5 for 50 mg/kg chrysin group. At all time points p<0.001 except at 15
min p<0.01.
136
TABLE 1.
Pharmacokinetic parameters of nitrofurantoin in SD female rats after oral and intravenous administration of nitrofurantoin with or
without the coadministration of chrysin.
Nitrofurantoin (10 mg/kg, p.o.)
Parameters
AUC0-720 (g/mlmin)
Control
(n = 6)
306 83.3
Chrysin (50 mg/kg, p.o.)

(n = 4)
308 60.1
Nitrofurantoin (2 mg/kg, i.v.)
Chrysin (200 mg/kg, p.o.)

(n = 5)
Control
(n = 7)
Chrysin (50 mg/kg, i.p.)

(n = 7)
527 139 **
90.3 17.8
122 32.9 *
91.5 18.0
123 34.0 *
17.9 6.58 *
22.7 4.95
17.5 5.35
23.6 5.34
21.3 2.28
AUC0-120 (g/mlmin)
AUC0- (g/mlmin)
344 101
366 55.9
605 163 **
Cmax (g/ml)
1.01 0.47
1.01 0.33
1.73 0.52 *
CL/F or CL (ml/min/kg)
31.0 8.65
t1/2 (min)
166 67.4
145 13.1
164 73.1
Bioavailability (%)
60.2 15.3
61.8 8.75
114 17.9 **
27.8 4.13
The plasma concentration-time profile of nitrofurantoin in SD rats after oral and intravenous administration with control vehicle or the
specified doses of chrysin was determined as described under Materials and Methods. The pharmacokinetic parameters were obtained
by noncompartmetal analysis using WinNonlin. Data are expressed as mean S.D. The number of animals for each treatment group
is specified in parentheses after the corresponding group names (* p<0.05; ** p<0.01 compared with the corresponding control
group).
137
138
139
140
141
142
CHAPTER 5
Pharmacokinetics and Bioavailability of 7,8-Benzoflavone

in Rats
This chapter has been submitted for publication in J Pharm Sci.
143
ABSTRACT
The flavonoid 7,8-benzoflavone was recently identified as one of the most potent
inhibitors of breast cancer resistance protein (BCRP); however, little is known of the in
vivo disposition of 7,8-benzoflavone. The objective of this study was to investigate the
pharmacokinetics and bioavailability of 7,8-benzoflavone in rats. Three intravenous (5,
10, and 25 mg/kg) and three oral (12.5, 25, and 50 mg/kg) doses were administered to
female Sprague-Dawley rats. Plasma samples were analyzed by high-performance liquid
chromatography. Pharmacokinetic analysis was conducted by WinNonlin and ADAPT
II. The dose-normalized plasma concentration vs. time curves did not superimpose with
each other, indicating the nonlinear pharmacokinetics of 7,8-benzoflavone. 7,8benzoflavone exhibited a large volume of distribution (Vss ~ 1.5 L/kg) and rapid oral
absorption (tmax < 30 min). The bioavailability of 7,8-benzoflavone was low (0.61 % ~
13.2 %) and dose-dependent. A pharmacokinetic model with linear absorption, nonlinear
intestinal metabolism and nonlinear elimination best described the pharmacokinetic
profiles of 7,8-benzoflavone. The mechanism underlying the nonlinear pharmacokinetics
of 7,8-benzoflavone is likely due to its capacity-limited metabolism and BCRP-mediated
elimination. Using a 50 mg/kg oral dose of 7,8-benzoflavone, we could significantly
increase the AUC for the BCRP substrate nitrofurantoin, demonstrating the potential for
BCRP-mediated drug interactions.
144
INTRODUCTION
7,8-benzoflavone (-naphthoflavone), a flavonoid present in the root of Russian

Knapweed 1, has attracted considerable attention due to its potent cytochrome P450
(CYP) modulating activities, such as inhibiting CYP1A1, CYP1A2 and CYP3A6 and
activating CYP3A4
2-4
. In addition, 7,8-benzoflavone has been reported as a potent
aromatase inhibitor 5, an enzyme responsible for the conversion of androstenedione and

testosterone into estrone and estradiol, respectively 6. Consistent with its activity as an
aromatase enzyme inhibitor, a 7,8-benzoflavone moiety isolated from the plant Passiflora
incarnata has exhibited virility-enhancing activity in 2-year-old male rats 7.
Recently, we reported that flavonoids represent a class of inhibitors of breast cancer

resistance protein (BCRP), an efflux transporter important in determining drug
disposition 8,9. Among all the flavonoids tested, 7,8-benzoflavone and chrysin (Figure 1)
are two of the most potent flavonoids for inhibiting the efflux of mitoxantrone (IC50
values of 0.07 0.02 and 0.39 0.13 M, respectively 10). In an in vivo study, the
flavonoid chrysin, at the oral dose of 200 mg/kg, significantly increased the
bioavailability and decreased the apparent oral clearance of nitrofurantoin, a BCRP
substrate, in rats. However, at the lower chrysin dose of 50 mg/kg, no significant
interaction between BCRP and chrysin was observed 11. One explanation for this lack of
interaction might be the poor bioavailability of chrysin is due to the extensive metabolism
of chrysin in the intestine as a result of enzymatic conjugation reactions occurring at the
hydroxyl groups 12. The chemical structure of 7,8-benzoflavone differs from that of
chrysin in that it lacks hydroxyl groups susceptible for conjugation reactions. Therefore,
145
it is possible that the bioavailability of 7,8-benzoflavone may be greater than that

reported for other flavonoids such as biochanin A (1.2 % at 50 mg/kg) 13 or chrysin (< 1
%) 14. Therefore, 7,8-benzoflavone might have significant effects on BCRP in vivo at
relevant physiological or pharmacological dose, resulting in BCRP-mediated flavonoiddrug interactions.
The objective of this study was to investigate the pharmacokinetics and bioavailability of
7,8-benzoflavone in rats, to develop a pharmacokinetic model to characterize the plasma
concentration-time profiles of 7,8-benzoflavone, and to perform a preliminary study to
investigate the potential for BCRP-mediated drug interactions by examining the effects of
oral 7,8-benzoflavone on the disposition of the BCRP substrate nitrofurantoin.
146

7,8-benzoflavone and the internal standard 8-methylflavone were purchased from

Indofine (Hillsborough, NJ). Tetraglycol, polyethylene glycol 400 (PEG 400) and
hydroxypropyl--cyclodextrin were purchased from Sigma-Aldrich (St. Louis, MO).
Saline was purchased from Henry-Schein (Melville, NY). All other reagents or solvents
used were either analytical or high-performance liquid chromatography (HPLC) grade
and were purchased from Fisher Scientific (Springfield, NJ).
Animals and Surgery
Female Sprague-Dawley (SD) rats (body weight ~ 250 g) were purchased from Harlan
(Indianapolis, IN). Animals were kept in a temperature and humidity-controlled
environment with a 12-h light-dark cycle and received a standard diet with water ad
libitum. Animals were acclimatized to this environment for at least one week before
experiments. All protocols of animal studies were reviewed and approved by the
University at Buffalo Institutional Animal Care and Use Committee. Two or three days
prior to the study day, rats were anesthetized (ketamine 90 mg/kg and xylazine 10 mg/kg,
i.m. injection) and implanted with a cannula (Micro-Renathane, type MRE-040, 0.040
O.D. 0.025 I.D., Braintree Scientific, Inc., MA) in the right jugular vein. The cannula
was flushed with saline containing 50 IU/ml heparin daily until the study day. The rats
were fasted overnight for the oral pharmacokinetic studies.
147
Pharmacokinetic studies were performed as previously reported 15. The rats were kept in
metabolic cages for blood collection throughout the study. For the intravenous
pharmacokinetic study, 7,8-benzoflavone was dissolved in DMSO as a 25 mg/ml stock
solution and was further diluted with a solution of PEG 400 and hydroxypropyl-cyclodextrin (75%: 25%, v/v) to such concentrations that specified doses (5, 10 and 25
mg/kg) were delivered as 5 l/g body weight drug solution. Saline containing 50 IU/ml
heparin was given immediately after 7,8-benzoflavone administration to flash the drug
remaining in the cannula. For the oral pharmacokinetic study, 7,8-benzoflavone was
dissolved in a solution consisting of tetraglycol, PEG 400 and ethanol (40%: 30%: 30%,
v/v). Oral doses were given via gavage at doses of 12.5, 25, and 50 mg/kg. The dosing
volume was 5.8 l/g body weight. Blood samples (200 l) were collected from the
jugular vein cannula at 0 (predose), 2, 5, 15, 30, 60, 120, 240, 480 and 720 min after 7,8benzoflavone administration. The plasma was separated from the whole blood by
centrifugation at 2,000 g for 5 min at 4oC. All plasma samples were stored at -80oC until
analysis by HPLC.
Oral Interaction Study
The pharmacokinetic interaction study was performed similarly as previously described

11,16
. Briefly, for the oral drug interaction studies, rats were fasted overnight with free
access to tap water. A 50 mg/kg oral dose of 7,8-benzoflavone (dissolved in tetraglycol

as a 5 mg/ml solution) or the vehicle tetraglycol (as control) was administered to rats by
gavage. Five minutes later, the animals were administered 10 mg/kg nitrofurantoin orally
148
(3 mg/ml dissolved in 50% (v/v) ethanol and 50% (v/v) polyethylene glycol 400). The
blood samples (150 l) were taken from the jugular vein cannula at 0 (predose), 5, 15, 30,
60, 120, 180, 240, 480, and 720 min after nitrofurantoin administration. All samples
were stored at 80C until the time of HPLC analysis. Four rats were used for each
group.
HPLC Assay
Sample preparation and HPLC methods were developed and were used to detect the
concentration of 7,8-benzoflavone in plasma samples. Briefly, 80 l of acetonitrile was
added to a 40 l plasma sample to precipitate protein. Four l of 8-methylflavone
solution (100 g /ml) was added as the internal standard. The mixture was vortexed for 1
min and then centrifuged at 22,000 g for 10 min. Sixty l of supernatant was injected
into HPLC for analysis.
The HPLC analysis was conducted on a system consisting of a Waters 1525 pump, a 717
plus autosampler, a 2847 UV detector, and Waters BreezeTM workstation. The separation
was performed using an Alltech Alltima C18 column (125 4.6 mm, 5-m particle size).
The composition of the mobile phase was acetonitrile/water (80%: 20%, v/v). The
mobile phase was delivered isocratically with a flow rate of 1.0 ml/min. UV absorbance
was measured at 280 nm.
The concentration of nitrofurantoin in rat plasma samples was analyzed as previously

described 11,16. Briefly, an aliquot of 50 l sample was spiked with 5 l of 12.5 g/ml
149
furazolidone solution (internal standard), and then 50 l of cold methanol (20oC) was
added. The mixture was vortexed vigorously for 60 sec and incubated at 20oC for 15
min. The organic and water phases were separated by centrifugation at 16,000g for 10
min at 4oC, and 30 l of the supernatant was injected into HPLC system. The HPLC
analysis was performed by measuring UV absorbance of nitrofurantoin and furazolidone
at 366 nm. The composition of the mobile phase was 25 mM potassium phosphate buffer
(pH3)/acetonirile (75%: 25%, v/v). The mobile phase was delivered isocratically with a
flow rate of 1.0 ml/min. Nitrofurantoin appeared on the chromatograph at approximately
8 min with no interference peaks. The standard curve was linear over the concentration
range of 0.01 to 20 g/ml with a regression coefficient greater than 0.99. The lower limit
of quantitation of nitrofurantoin was 10 ng/ml.
The plasma concentration data were analyzed by noncompartmental and compartmental

analysis using WinNonlin version 2.1 (Pharsight, Mountain View, CA) and ADAPT II
(BMSR, University of South California, Los Angeles CA), respectively. For
noncompartmental analysis, the area under the plasma concentration-time curve (AUC)
was calculated using the log-linear trapezoidal rule. The elimination half-life (t1/2) was
calculated by the equation: t1/2 = 0.693/, where was estimated from the terminal slope
of the plasma concentration vs. time curve. The clearance (CL) was determined from
Dose/AUC. The volume of distribution at steady state (Vss) was determined by Vss =
CL (AUMC/AUC), where AUMC is the area under the first moment curve
(Concentration time vs. time curve). Oral bioavailability (F) was calculated by the ratio
150
of dose-normalized AUCs following oral and intravenous administration. The AUC

obtained from the lowest intravenous dose group (5 mg/kg) was used in the calculation of
oral bioavailability since the plasma concentrations after oral administration were similar
to the concentrations at the lowest intravenous dose, where clearance is linear.
For the compartmental analysis, all intravenous and oral plasma concentration data of
7,8-benzoflavone were fitted simultaneously using ADAPT II software. The initial
estimates for model parameters were obtained from noncompartmental analysis.
Different pharmacokinetic models with linear or nonlinear oral absorption and
elimination were constructed to characterize the kinetics of 7,8-benzoflavone. The
appropriate model was selected based on the goodness-of-fit criteria including coefficient
variation of the estimates (CV %), R-square, Akaikes Information Criterion (AIC) and
Schwarz Criterion (SC).
The differential equations derived from the final pharmacokinetic model were as follows:
(1)
dX a
= k a X a
dt
(2)
Vmax_ int X int

dX int
= ka X a
k int X int
dt
K m _ int + X int
(3)
Vmax X C
dX C
= k int X int
k12 X C + k 21 X T
K m VC + X C
dt
( IC = Dose)
(4) dX C = Vmax X C k X + k X
12
C
21
T
dt
K m VC + X C
(5)
dX T
= k12 X C k 21 X T
dt
( IC = 0)
151
( IC = 0)
( IC = 0)
( IC = Dose)
The initial conditions for each equation are shown in parenthesis. Equations (1), (2), (3),
and (5) were used to fit the oral data. Equations (4) and (5) were used to fit the
intravenous data. In the equations, Xa represents the amount of 7,8-benzoflavone at the
absorption site. Xint represents the amount of 7,8-benzoflavone in the intestinal
compartment. XC and XT represent the amount of 7,8-benzoflavone in the central and
peripheral compartment, respectively. ka is the absorption rate constant at the absorption
site. kint is the rate constant of absorption from the intestinal to the central compartment.
k12 and k21 represent the first-order rate constants of distribution between the central and
peripheral compartments. VC represents the volume of distribution of 7,8-benzoflavone
in the central compartment. Km represents the Michaelis-Menten parameter of the
nonlinear elimination from the central compartment and Vmax is the maximal velocity of
the nonlinear elimination from the central compartment. Km_int is the Michaelis-Menten
parameter of the degradation due to the first-pass metabolism in the intestinal
compartment and Vmax_int is the maximum velocity of the degradation due to the first-pass
metabolism in the intestinal compartment.
Statistical analysis was conducted using Students t test or a one-way ANOVA (Prism 3.0
software, GraphPad, San Diego, CA) followed by a Dunnetts post hoc test or KruskalWallis test. p values < 0.05 were considered statistically significant.
152
RESULTS
HPLC Assay for 7,8-benzoflavone in Plasma
A simple HPLC method was developed to detect the concentration of 7,8-benzoflavone

in plasma samples. 8-Methylflavone (internal standard) and 7,8-benzoflavone appeared
on the chromatograph at approximately 7 and 10 min, respectively, with no interfering
peaks. The lower limit of quantitation of 7,8-benzoflavone was 10 ng/ml. The standard
curve was linear over the concentration range of 0.01 to 20 g/ml with a regression
coefficient greater than 0.99. The recovery from sample extraction was 106 %. The interand intra-day accuracy was 97 % to 103 %, and the precision expressed as CV % was 0.7
% to 5.7 %.
Pharmacokinetics of 7,8-benzoflavone Following Intravenous Administration
The plasma concentration vs. time profiles of 7,8-benzoflavone following its intravenous
administration are shown in Figure 2A. At the lowest dose level of 5 mg/kg, AUC, CL,
Vss, and t1/2 values were 287 6.65 mgL-1min, 17.5 0.40 mlmin-1kg-1, 1.47 0.20
L/kg, and 82.0 8.96 min, respectively (Table 1). When the dose was increased to 10
mg/kg, AUC proportionally increased to 560 36.4 mgL-1min. However, at the dose of
25 mg/kg, the increase in AUC (2500 97.1 mgL-1min) was greater than proportional.
The dose-normalized AUC was similar between the 5 and 10 mg/kg groups, but was
significantly higher in the 25 mg/kg group (57.3 1.33, 56.0 3.64 and 100 3.88 kgL1
min for 5, 10 and 25 mg/kg groups, respectively, Table 1). Moreover, the dose-
normalized plasma concentration-time curves did not superimpose at the dose of 25

mg/kg (Figure 2B), suggesting the nonlinearity in 7,8-benzoflavone pharmacokinetics at
153
higher doses. Consistent with the changes in AUC, the systemic clearance was nonlinear
at the highest dose level (17.5 0.40, 17.9 1.13 and 10.0 0.40 mlmin-1kg-1 for 5, 10
and 25 mg/kg groups, respectively, Table 1). In contrast to the changes in AUC and
clearance, the Vss and t1/2 were similar at all dose levels (Table1).
Pharmacokinetics of 7,8-benzoflavone Following Oral Administration
The 7,8-benzoflavone plasma concentration-time profiles after oral administration are

shown in Figure 3A. 7,8-Benzoflavone was rapidly absorbed and the plasma
concentrations peaked within 30 min at doses of 12.5, 25 and 50 mg/kg (Table 1). AUC
was 8.77 2.02, 83.8 33.8 and 379 351 mgL-1min at doses of 12.5, 25 and 50
mg/kg, respectively. The increase in AUC was not proportional to the increase of dose.
Similarly, the dose-nonproportionality was observed in Cmax (0.21 0.05, 0.59 0.52 and
2.02 1.19 g/ml for doses of 12.5, 25 and 50 mg/kg, respectively, Table 1). After
normalization by dose, the plasma concentration-time curves did not superimpose at the
three different dose levels, suggesting again that the pharmacokinetics of 7,8benzoflavone was nonlinear (Figure 3B). Oral bioavailability (F) was estimated by
dividing the AUC/Dose values for the three oral doses by the AUC/Dose value from the
lowest intravenous dose, where clearance was linear. The calculated F significantly
increased with the increase of dose (0.61 0.14, 5.85 2.36, p > 0.05, and 13.2 12.2
%, p < 0.05, for doses of 12.5, 25 and 50 mg/kg, respectively), indicating the nonlinearity
in 7,8-benzoflavone bioavailability.
154
Compartmental Modeling of 7,8-benzoflavone Plasma Concentration-Time Data
To better understand the in vivo disposition of 7,8-benzoflavone, different linear and

nonlinear kinetic models were proposed to simultaneously fit the plasma concentration
data following intravenous and oral administration. Several pharmacokinetic models
were constructed to characterize the kinetics of 7,8-benzoflavone after intravenous
administration, including one-, two-, and three-compartment models with linear or
Michaelis-Menten elimination. Based on the goodness-of-fit criteria (e.g., CV %, Rsquare, AIC and SC), a two-compartment model with Michaelis-Menten elimination
provided the best fitting (data not shown). Using this intravenous model as a base model,
a pharmacokinetic model for all the data was developed by incorporating an oral
absorption compartment and an intestinal compartment (Figure 4). Since the
bioavailability of 7,8-benzoflavone increased with the increase of dose, we proposed
several pharmacokinetic models with linear or nonlinear oral absorption and intestinal
degradation due to the first-pass metabolism (Table 2). Based on the goodness-of-fit
criteria following simultaneous fitting (e.g., CV %, R-square, AIC and SC), a model with
linear oral absorption and Michaelis-Menten intestinal degradation provided the best
fitting (Table 2, Figure 5). The R-square values were 0.978, 0.995, 0.972, 0.926, 0.938,
and 0.976 for the curves following intravenous doses of 5, 10, and 25 mg/kg and oral
doses of 12.5, 25, and 50 mg/kg, respectively. The fitted parameters for this model
obtained using ADAPT II using this model are summarized in Table 3. We also
proposed different models without the oral absorption (Xa) compartment (only the
intestinal compartment with linear or nonlinear oral absorption and intestinal
155
degradation). However, the fitting was less satisfactory compared to the models with the
intestinal compartment (data not shown).
Oral Interaction Study
As shown in Table 4, oral coadministration of 7,8-benzoflavone (50 mg/kg) significantly

increased the AUC of nitrofurantoin (10 mg/kg) (351 21.0 in the 7,8-benzoflavonetreated group versus 277 34.0 gml-1min in the vehicle-treated group, p<0.05). In
contrast, the apparent oral clearance of nitrofurantoin (CL/F) was significantly decreased
from 36.6 4.85 mlmin-1kg-1 in the control group to 28.7 1.77 mlmin-1kg-1 in the
7,8-benzoflavone-treated group (p<0.05). The terminal t1/2, however, was not
significantly changed following the coadministration of 7,8-benzoflavone (Table 4).
156
DISCUSSION
Flavonoids have been recently identified as a class of inhibitors of breast cancer

resistance protein, an efflux transporter important in limiting drug oral absorption and
elimination 8,17. In a structure activity relationship study with 25 flavonoids, 7,8benzoflavone has been identified as the most potent BCRP inhibitor in inhibiting the
efflux of mitoxantrone with an IC50 of 0.07 0.02 M 10. Therefore, 7,8-benzoflavone
represents a potent transport inhibitor with a potential role in altering the in vivo
disposition of drugs that are BCRP substrates. However, whether an interaction may
occur in vivo will be largely dependent on the pharmacokinetics and bioavailability of
7,8-benzoflavone.
Therefore, in the present study, the pharmacokinetics and bioavailability of 7,8benzoflavone were investigated. Our investigation found that, at an intravenous dose of
25 mg/kg, the dose-normalized AUC was significantly increased and the systemic
clearance was substantially decreased when compared with the values at lower doses.
These results suggested that a nonlinear process was possibly involved in the elimination
of this compound. 7,8-Benzoflavone has been previously reported to be metabolized to a
number of hydroxyl- and oxide-metabolites by both rat and human liver microsomes 18,19.
Therefore, saturation of 7,8-benzoflavone metabolic pathways at high doses likely
contributes to the nonlinearity in 7,8-benzoflavone elimination. Alternatively, the
nonlinear elimination of 7,8-benzoflavone might be due to saturation of its excretion into
the bile and urine at high doses. Several flavonoids have been identified as BCRP
substrates in a previous report 20 and studies from our laboratory (unpublished data).
157
BCRP is expressed in many organs important in drug disposition, such as liver (the
canalicular membrane of hepatocytes) and kidney (the apical membrane of renal
proximal tubular cells) 17,21. Therefore, 7,8-benzoflavone may be excreted into the bile
and urine by processes mediated by BCRP, and these processes may be saturated at high
doses; however, this is only speculative at this time.
The Vss for 7,8-benzoflavone ranged from 1.35 to 1.57 L/kg at doses of 5 25 mg/kg.
The Vss value determined at the dose of 25 mg/kg might not be accurate based on
noncompartmental analysis since the elimination of 7,8-benzoflavone is nonlinear at this
intravenous dose. The high values of Vss suggest that 7,8-benzoflavone is likely
extensively bound to tissues, which is consistent with its highly lipophilic nature (Log P
= 4.62). From the plasma concentration vs. time plots (Figure 2), it can be noted that the
initial slopes in the two low dose groups were steeper than that in the high dose group.
However, the terminal slopes were similar among the three intravenous treatment groups,
which suggested that the nonlinear pharmacokinetics of 7,8-benzoflavone might be
attributed to the early phase when the plasma concentrations were substantially higher.
7,8-Benzoflavone was rapidly absorbed after oral administration with an absorption rate
constant ka of 0.33 min-1. The tmax values for the three oral doses were less than 30 min.
The rapid oral absorption is possibly due to the highly lipophilic nature of 7,8benzoflavone. Similar rapid oral absorption profiles have been observed for a flavonoid
analogue, (-)-epicatechin-3-O-gallate, in which peak plasma concentrations of this
compound occurred around 5 min 22. The dose-normalized AUC and Cmax values
158
following oral dosing were not dose-proportional, which further suggested that the
pharmacokinetics of 7,8-benzoflavone was nonlinear. For calculation of bioavailability
of 7,8-benzoflavone, the AUC obtained from the lowest intravenous dose group (5
mg/kg) was used as the reference since the pharmacokinetics of 7,8-benzoflavone are
linear at the dose of 5 and 10 mg/kg (Figure 2B) and the plasma concentrations of 7,8benzoflavone are similar or lower than those observed at the lowest intravenous dose.
The bioavailability of 7,8-benzoflavone was very low, ranging from 0.61 % to 13.2 %.
The increase in bioavailability with the increase of dose again suggested nonlinearity in
7,8-benzoflavone pharmacokinetics. The low bioavailability of 7,8-benzoflavone could
be either due to its substantial metabolic degradation 23 or due to its low solubility (0.014
mg/ml or 50 M) 18. Similar low bioavailability has been reported for the flavonoids
biochanin A and chrysin due to their extensive conjugative metabolism 13 14. Compared
to biochanin A and chrysin, the bioavailability of 7,8-benzoflavone is significantly higher
(13.2 % at a 50 mg/kg dose). Consistent with its higher bioavailability, 7,8benzoflavone, at an oral dose of 50 mg/kg, significantly increased the AUC and
decreased the clearance of the BCRP substrate nitrofurantoin, while chrysin had no
significant effect at a dose of 50 mg/kg 11. The significant interaction is possibly due to
the inhibition of BCRP-mediated intestinal efflux of nitrofurantoin or due to the
inhibition of BCRP-mediated systemic elimination.
To better understand the pharmacokinetics of 7,8-benzoflavone, a pharmacokinetic model

was constructed to describe the in vivo data. First of all, 7,8-benzoflavone
pharmacokinetics following intravenous administration was determined based on the
159
fitting criteria from different models (data not shown). A two-compartment model
provided the best fitting, which is consistent with literature reports of other flavonoid
analogues, such as quercetin 24 and puerarin 25. The capacity-limited elimination of 7,8benzoflavone (Km, Vmax) from the central compartment is consistent with its nonlinear
pharmacokinetic profiles following intravenous administration. The addition of two
capacity-limited elimination terms in the model to characterize both processes of
metabolism and efflux transporter-mediated excretion did not improve the model fitting
(data not shown); a single capacity-limited process well characterized 7,8-benzoflavone
elimination. However, we cannot exclude the possibility that this single capacity-limited
elimination may be composed of the both nonlinear metabolism and saturable efflux
transport of 7,8-benzoflavone. Further studies are needed to differentiate the contribution
of capacity-limited metabolism and efflux transport in the clearance of 7,8-benzoflavone.
Based on the analysis of the intravenous data, a pharmacokinetic model was established
by incorporating an oral absorption compartment and an intestinal compartment. The
inclusion of an intestinal compartment provided better model fitting than the models with
only the oral absorption compartment. This indicated that the intestinal metabolism
might be important in determining 7,8-benzoflavone pharmacokinetics and our model
mechanistically characterized this physiological process. The oral and intravenous data
were simultaneously fitted using models with either linear or nonlinear oral absorption
and intestinal degradation. Based on the fitting criteria from four different models, a
model with linear oral absorption and nonlinear intestinal degradation provided the best
fitting. The linear oral absorption is possibly due to the highly lipophilic nature of 7,8benzoflavone, which enables it to cross enterocytes easily. The nonlinear intestinal
160
degradation of 7,8-benzoflavone is likely attributed to its saturable first-pass metabolism

in the intestine.
In summary, 7,8-benzoflavone demonstrated nonlinear pharmacokinetics following oral

and intravenous administration. A pharmacokinetic model with linear absorption,
capacity-limited intestinal metabolism and capacity-limited elimination best described the
pharmacokinetic profiles of 7,8-benzoflavone in rats. The clearance was dose-dependent,
likely due to the saturation of metabolism and elimination pathways. Consistent with its
lipophilic nature, the Vss of 7,8-benzoflavone was large and oral absorption was rapid.
The bioavailability of 7,8-benzoflavone was low (0.61 % ~ 13.2 %) and dose-dependent,
possibly due to its limited solubility and first-pass metabolism. The significant effects of
7,8-benzoflavone on nitrofurantoin pharmacokinetics demonstrate the potential for
BCRP-mediated drug interactions.
161
REFERENCES:
1.
Stermitz FR, Bais HP, Foderaro TA, Vivanco JM 2003. 7,8-Benzoflavone: a
phytotoxin from root exudates of invasive Russian knapweed. Phytochemistry 64(2):493497.
2.
Lee HS, Jin C, Park J, Kim DH 1994. Modulation of cytochrome P450 activities
by 7,8-benzoflavone and its metabolites. Biochem Mol Biol Int 34(3):483-491.
3.
Koley AP, Buters JT, Robinson RC, Markowitz A, Friedman FK 1997.
Differential mechanisms of cytochrome P450 inhibition and activation by alphanaphthoflavone. J Biol Chem 272(6):3149-3152.
4.
Boek-Dohalska L, Hodek P, Sulc M, Stiborova M 2001. alpha-Naphthoflavone
acts as activator and reversible or irreversible inhibitor of rabbit microsomal CYP3A6.
Chem Biol Interact 138(1):85-106.
5.
Kellis JT, Jr., Vickery LE 1984. Inhibition of human estrogen synthetase
(aromatase) by flavones. Science 225(4666):1032-1034.
6.
Meinhardt U, Mullis PE 2002. The essential role of the aromatase/p450arom.
Semin Reprod Med 20(3):277-284.
7.
Dhawan K, Kumar S, Sharma A 2002. Beneficial effects of chrysin and
benzoflavone on virility in 2-year-old male rats. J Med Food 5(1):43-48.
8.
Zhang S, Yang X, Morris ME 2004. Flavonoids are inhibitors of breast cancer
9.
Mao Q, Unadkat JD 2005. Role of the breast cancer resistance protein (ABCG2)
in drug transport. Aaps J 7(1):E118-133.
10.
Zhang S, Yang X, Coburn RA, Morris ME 2005. Structure activity relationships
and quantitative structure activity relationships for the flavonoid-mediated inhibition of
breast cancer resistance protein. Biochem Pharmacol 70(4):627-639.
11.
Wang X, Morris ME 2007. Effects of the flavonoid chrysin on nitrofurantoin
pharmacokinetics in rats: potential involvement of ABCG2. Drug Metab Dispos
35(2):268-274.
12.
Galijatovic A, Otake Y, Walle UK, Walle T 1999. Extensive metabolism of the
flavonoid chrysin by human Caco-2 and Hep G2 cells. Xenobiotica 29(12):1241-1256.
13.
Moon YJ, Sagawa K, Frederick K, Zhang S, Morris ME 2006. Pharmacokinetics
and bioavailability of the isoflavone biochanin A in rats. Aaps J 8(3):E433-442.
14.
Walle T, Otake Y, Brubaker JA, Walle UK, Halushka PV 2001. Disposition and
metabolism of the flavonoid chrysin in normal volunteers. Br J Clin Pharmacol
51(2):143-146.
15.
Ji Y, Kuo Y, Morris ME 2005. Pharmacokinetics of dietary phenethyl
isothiocyanate in rats. Pharm Res 22(10):1658-1666.
16.
Merino G, Jonker JW, Wagenaar E, van Herwaarden AE, Schinkel AH 2005. The
breast cancer resistance protein (BCRP/ABCG2) affects pharmacokinetics, hepatobiliary
excretion, and milk secretion of the antibiotic nitrofurantoin. Mol Pharmacol 67(5):17581764.
17.
van Herwaarden AE, Schinkel AH 2006. The function of breast cancer resistance
protein in epithelial barriers, stem cells and milk secretion of drugs and xenotoxins.
Trends Pharmacol Sci 27(1):10-16.
162
18.
Lee CA, Manyike PT, Thummel KE, Nelson SD, Slattery JT 1997. Mechanism of
cytochrome P450 activation by caffeine and 7,8-benzoflavone in rat liver microsomes.
Drug Metab Dispos 25(10):1150-1156.
19.
Lee HS, Jin CB, Chong HS, Yun CH, Park JS, Kim DH 1994. Involvement of
P4503A in the metabolism of 7,8-benzoflavone by human liver microsomes. Xenobiotica
24(11):1053-1062.
20.
Imai Y, Tsukahara S, Asada S, Sugimoto Y 2004. Phytoestrogens/flavonoids
reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance. Cancer
Res 64(12):4346-4352.
21.
Tanaka Y, Slitt AL, Leazer TM, Maher JM, Klaassen CD 2005. Tissue
distribution and hormonal regulation of the breast cancer resistance protein (Bcrp/Abcg2)
in rats and mice. Biochem Biophys Res Commun 326(1):181-187.
22.
Takizawa Y, Morota T, Takeda S, Aburada M 2003. Pharmacokinetics of (-)epicatechin-3-O-gallate, an active component of Onpi-to, in rats. Biol Pharm Bull
26(5):608-612.
23.
Nesnow S, Bergman H 1981. Metabolism of alpha-naphthoflavone by rat liver
microsomes. Cancer Res 41(7):2621-2626.
24.
Ferry DR, Smith A, Malkhandi J, Fyfe DW, deTakats PG, Anderson D, Baker J,
Kerr DJ 1996. Phase I clinical trial of the flavonoid quercetin: pharmacokinetics and
evidence for in vivo tyrosine kinase inhibition. Clin Cancer Res 2(4):659-668.
25.
Li Y, Pan WS, Chen SL, Xu HX, Yang DJ, Chan AS 2006. Pharmacokinetic,
tissue distribution, and excretion of puerarin and puerarin-phospholipid complex in rats.
Drug Dev Ind Pharm 32(4):413-422.
163
Table 1. Pharmacokinetic parameters of 7,8-benzoflavone determined by noncompartmental analysis

IV
IV
IV
PO
PO
PO
5 mg/kg
10 mg/kg
25 mg/kg
12.5 mg/kg
25 mg/kg
50 mg/kg
(n = 4)
(n = 6)
(n = 4)
(n = 4)
(n = 4)
(n = 4)
AUC (mgL-1min)
287 6.65
560 36.4**
2500 97.1**
8.77 2.02
83.8 33.8
379 351
AUC/D (kgL-1min)
57.3 1.33
56.0 3.64
100 3.88**
0.70 0.16
3.35 1.35
7.57 7.02
CL (mlmin-1kg-1)
17.5 0.40
17.9 1.13
10.0 0.40**
Vss (L/kg)
1.47 0.20
1.57 0.13
1.35 0.076
tmax (min)
7.50 2.89
27.5 24.0
10.0 5.77
Cmax (mg/L)
0.21 0.05
0.59 0.52
2.02 1.19*
82.0 8.96
85.5 5.66
104 30.2
0.61 0.14
5.85 2.36
13.2 12.2*
Parameters
t1/2 (min)
F (%)
The pharmacokinetic parameters were obtained by noncompartmental analysis using WinNonlin. Data were presented as
mean SD. The number of animals for each group was specified in parentheses after the corresponding group names (*: p <
0.05, **: p < 0.01 compared with the corresponding low dose group).
164
Table 2. Comparison of four final models proposed to characterize the

pharmacokinetics of 7,8-benzoflavone
Base Model: 2-Compartment with nonlinear elimination
AIC
SC
Linear absorption + Linear intestinal degradation
20.4
41.0
Linear absorption + Nonlinear intestinal degradation
-14.6
7.84
Nonlinear absorption + Linear intestinal degradation
22.5
44.9
Nonlinear absorption + Nonlinear intestinal degradation
4.97
29.3
AIC: Akaikes Information Criterion; SC: Schwarz Criterion.
165
Table 3. Estimated pharmacokinetic parameters of 7,8-benzoflavone in rats following

oral and intravenous administration determined by compartmental analysis
Parameters
Units
Estimated value
CV%
Km
mg/L
3.85
4.69
Vmax
mgmin-1kg-1
0.10
0.02
VC
L/kg
1.25
4.84
k12
min-1
0.011
23.1
k21
min-1
0.014
19.2
ka
min-1
0.32
23.0
kint
min-1
0.50
43.1
Km_int
g/kg
0.011
67.2
Vmax_int
mgmin-1kg-1
1.32
0.02
Km, Michaelis-Menten parameter of 7,8-benzoflavone nonlinear clearance from the

central compartment; Vmax, maximum velocity of 7,8-benzoflavone nonlinear clearance
from the central compartment; VC, volume of distribution of 7,8-benzoflavone in the
central compartment; k12, first-order distribution rate constant from the central to
peripheral compartment; k21, first-order distribution rate constant from the peripheral to
central compartment; ka, absorption rate constant; kint, rate constant of absorption from
the intestinal to the central compartment; Km_int, Michaelis-Menten parameter of 7,8benzoflavone degradation due to the first-pass metabolism in the intestinal compartment;
Vmax_int, maximum velocity of 7,8-benzoflavone degradation due to the first-pass
metabolism in the intestinal compartment.
166
Table 4. Pharmacokinetic parameters of nitrofurantoin in SD female rats after oral

administration of nitrofurantoin with or without the coadministration of 7,8-benzoflavone
Nitrofurantoin (10 mg/kg, oral)
Parameters
Control
(n = 4)
7,8-benzoflavone
(50 mg/kg, oral)
(n = 4)
AUC (gml-1min)
277 34.0
351 21.0 *
CL/F (mlmin-1kg-1)
36.6 4.85
28.7 1.77 *
t1/2 (min)
107 11.3
88.9 19.1
The pharmacokinetic parameters were obtained by noncompartmental analysis using

WinNonlin. Data are expressed as mean SD. n = 4 per each group, * p<0.05 compared
with the control group.
167
FIGURE LEGENDS:
Figure 1. The chemical structures of 7,8-benzoflavone and chrysin.
Figure 2. A) Plasma concentration vs. time profile of 7,8-benzoflavone following

intravenous administration. B) The dose-normalized plasma concentration vs. time
profile of 7,8-benzoflavone following intravenous administration. Rats were dosed
intravenously with 5 (), 10 (V), or 25 (z) mg/kg of 7,8-benzoflavone (n = 4 ~ 6 for
each group). The results are presented as mean SD.
Figure 3. A) Plasma concentration vs. time profile of 7,8-benzoflavone following oral

administration. B) The dose-normalized plasma concentration vs. time profile of 7,8benzoflavone following oral administration. Rats were dosed orally with 12.5 (), 25
(V), or 50 (z) mg/kg of 7,8-benzoflavone (n = 4 for each group). The results are
presented as mean SD.
Figure 4. The proposed compartmental model of 7,8-benzoflavone following oral and

intravenous administration. Xa represents the drug amount at the absorption site; ka is the
absorption rate constant; Xint represents the drug amount in the intestinal compartment;
kint is the rate constant of absorption from the intestinal to the central compartment;
Vmax_int and Km_int are the Michaelis-Menten parameters to characterize 7,8-benzoflavone
degradation due to the first-pass metabolism in the intestinal compartment; XC and XT
represent 7,8-benzoflavone amount in the central and peripheral compartment,
respectively; VC and VT represent the volume of distribution of 7,8-benzoflavone in the
168
central and peripheral compartment, respectively; Vmax and Km are the Michaelis-Menten
parameters to characterize 7,8-benzoflavone elimination from the central compartment;
k12 and k21 represent the first-order rate constants of distribution between the central and
peripheral compartments.
Figure 5. Simultaneous fitting of the intravenous and oral data based on the proposed
model. (A): rats were dosed intravenously with 5 (), 10 (V), or 25 (z) mg/kg of 7,8benzoflavone; (B): rats were dosed orally with 12.5 (), 25 (V), or 50 (z) mg/kg of 7,8benzoflavone. The results are presented as mean SD, n = 4 ~ 6. The lines represent the
predicted plasma concentration of 7,8-benzoflavone.
169
170
171
172
173
174
CHAPTER 6
ABCG2-Mediated Transport of the Flavonoids

Biochanin A and Kaempferol
This chapter has been written for publication in the format of Drug Metab Dispos.
175
ABSTRACT
The flavonoid biochanin A and kaempferol are potent inhibitors of breast cancer
resistance protein (BCRP), and can inhibit the efflux of mitoxantrone in MCF7/MX100
cells with IC50 values of 1.62 and 6.04 M, respectively. However, the mechanisms of
BCRP inhibition by biochanin A and kaempferol are not well understood. The objective
of this study was to further characterize the interaction between BCRP and biochanin A
or kaempferol by examining whether these two flavonoids are BCRP substrates. The
transport of [3H]biochanin A and [3H]kaempferol across MDCK cell monolayers was
determined in both parental and murine Bcrp1-expressing cells. The intracellular
accumulation of [3H]biochanin A and [3H]kaempferol was determined in MDCK cells in
the absence or presence of FTC at the end of the transport study. The parent drug and
metabolite profiles of [3H]biochanin A and [3H]kaempferol in the medium from the
transport study were determined by HPLC followed by radioactivity counting (RadioHPLC). In MDCK-Bcrp1 cells, polarized transport was observed for both [3H]biochanin
A and [3H]kaempferol with efflux ratios (Papp, B-A/Papp, A-B) of 8.97 and 8.35, respectively.
Compared with MDCK-Bcrp1 cells in the absence of FTC, significantly higher
accumulation of [3H]biochanin A and [3H]kaempferol was determined in the parental
MDCK cells and in MDCK-Bcrp1 cells in the presence of FTC. Our results indicated
that murine Bcrp1 transported one major metabolite of biochanin A, and both the parent
and metabolite of kaempferol. The enzymatic hydrolysis results indicated that the
metabolite of kaempferol was likely a sulfate conjugate. In summary, our data
demonstrate that biochanin A and kaempferol and/or their metabolites are substrates of
BCRP.
176
INTRODUCTION
The Breast Cancer Resistance Protein (BCRP/ABCG2) is a newly identified ATPbinding cassette drug transporter with a molecular weight of 72-kDa (Doyle and Ross,
2003). Since its discovery in 1998, BCRP has attracted considerable scientific and
therapeutic interest due to its importance in conferring cellular resistance to many
anticancer agents, including anthracyclines, mitoxantrone, camptothecin derivatives, and
nucleoside analogs (Mao and Unadkat, 2005). In addition, BCRP has been shown to play
a critical role in the disposition of dietary carcinogens, such as PhIP and alfatoxin B1
(Jonker et al., 2005; van Herwaarden et al., 2006), as well as many clinically important
drugs, such as imatinib mesylate (Breedveld et al., 2005), nitrofurantoin (Merino et al.,
2005), topotecan, and cimetidine (Jonker et al., 2005). In addition to transporting parent
drugs, BCRP can also transport a number of sulfate and glucuronide conjugates both in
vitro and in vivo (Suzuki et al., 2003; Adachi et al., 2005; Sesink et al., 2005). In BCRP
knockout mice (bcrp1-/-), it has been reported that the oral absorption and brain
penetration of BCRP substrates are significantly higher, while the clearance and
hepatobiliary elimination are significantly decreased when compared with the wild type
mice (van Herwaarden et al., 2003; Breedveld et al., 2005; Merino et al., 2005). In recent
pre-clinical and clinical studies, inhibition of BCRP by GF120918 has been shown to
significantly increase the bioavailability and decrease the biliary excretion of topotecan
(Jonker et al., 2000; Kruijtzer et al., 2002). Therefore, BCRP represents an important
determinant of in vivo drug disposition.
177
Biochanin A and kaempferol (Figure 1) are two naturally occurring flavonoids;

flavonoids are a class of polyphenols abundant in fruits, vegetables and plant-derived
beverages, and in many herbal products marketed as over-the-counter medicine.
Extensive studies have demonstrated that flavonoids exhibit a variety of biochemical and
pharmacological properties including antioxidant, antiviral, anti-carcinogenic and antiinflammatory activities (Middleton et al., 2000; Havsteen, 2002). As a result, flavonoids
have been implicated to play a protective role in the prevention of cancer, coronary
disease, and other degenerative diseases (Hertog et al., 1993; Potter et al., 1998; Kohno et
al., 2002).
Biochanin A is a major component of the widely consumed herbal product red clover
extracts (Delmonte and Rader, 2006). Red clover extracts are commonly used to prevent
or relieve post-menopausal symptoms such as hot flashes and bone loss, and to maintain
prostate health (Risbridger et al., 2001; Atkinson et al., 2004; Booth et al., 2006).
Commercially available products contain approximately 26 mg of biochanin A per tablet
(Howes et al., 2000). Kaempferol is an active ingredient in onions, green tea and ginkgo
biloba extracts, and contributes up to 30 % of total flavonoid intake (de Vries et al., 1997;
Sloley et al., 2000; Park et al., 2006). It is estimated that the average daily consumption
of kaempferol is approximately 10 mg/day (de Vries et al., 1997). As an antioxidant,
kaempferol exerts strong radical scavenging activity and attenuates hydroxyl peroxideinduced cytotoxicity (Niering et al., 2005). Kaempferol has also been reported to be
neuroprotective (Sloley et al., 2000) and anti-apoptotic (Samhan-Arias et al., 2004).
Among individual ginkgo biloba components tested, kaempferol has been shown to be
one of the major components that provided the maximum neuroprotective effects in an
178
Alzheimers disease model (Smith and Luo, 2003). Due to the above-mentioned healthpromoting effects, biochanin A and kaempferol-containing products have been popularly
used in the general population (Sparreboom et al., 2004; Booth et al., 2006).
Recently, we reported that flavonoids represent a class of inhibitors of BCRP (Zhang et

al., 2004). Among the flavonoids tested, biochanin A and kaempferol are two potent
BCRP inhibitors that inhibit the efflux of mitoxantrone in MCF7/MX100 cells with IC50
values of 1.62 and 6.04 M, respectively (Zhang et al., 2004; Zhang et al., 2005b).
However, the mechanisms of BCRP inhibition by biochanin A and kaempferol have not
been well characterized. In a previous study, the flavonoid genistein has been reported to
inhibit BCRP-mediated drug efflux by acting as a BCRP substrate. In BCRP-transfected
LLC-PK1 cells, genistein has been shown to be transported by BCRP in its parent form
but not following metabolism (Imai et al., 2004).
The objective of this study was to further characterize the interaction between BCRP and
the flavonoid biochanin A and kaempferol by examining whether these two flavonoids
are BCRP substrates. This study will help better characterize the in vivo disposition of
biochanin A and kaempferol, and better understand the potential in vivo flavonoid-drug
interactions mediated by BCRP.
179

[3H]biochanin A (20.8 Ci/mmol) and [3H]kaempferol (5.6 Ci/mmol) were purchased from
Moravek Biochemicals (Brea, CA). Biochanin A, kaempferol, mitoxantrone, sulfatase
(from helix pomatia), and -glucuronidase (from bovine liver) were purchased from
Sigma-Aldrich (St. Louis, MO). Dulbeccos Modified Eagle Medium (DMEM), Hanks
buffered salt solution (HBSS), fetal bovine serum, penicillin, and streptomycin were
purchased from Invitrogen (Carlsbad, CA). Fumitremorgin C (FTC) was a kind gift from
Dr. Susan E. Bates (National Cancer Institute, Bethesda, MD). All other reagents or
solvents used were either analytical or high-performance liquid chromatography (HPLC)
grade and were purchased from Fisher Scientific (Springfield, NJ).
Cell Culture
The polarized Madin-Darby canine kidney cell line (MDCK-II) was used in the transport
study. MDCK II parental (MDCK-II cells transfected with empty vector) and MDCKBcrp1 (MDCK-II cells transfected with murine Bcrp1) cells were kind gifts from Dr.
Alfred Schinkel (Netherlands Cancer Institute, Amsterdam, The Netherlands). The
MDCK cells were cultured in 75-cm2 flasks with DMEM medium supplemented with
10% fetal bovine serum at 37oC in a humidified 5% CO2 /95 % air atmosphere. The
culture medium also contained 100 units/ml penicillin and 100 g/ml streptomycin.
Bidirectional Transport Study in MDCK Cells
180
Transport studies using MDCK cell monolayers were performed as described previously
(Zhang et al., 2005a) with minor modifications. Briefly, MDCK II and MDCK-bcrp1
cells were seeded on Transwell polycarbonate inserts (six-well, 0.4-m pore size;
Transwell 3412, Corning Glassworks, Corning, NY) at a density of approximately 106
cells/well. Cells were grown for 5 ~ 7 days, and medium was replaced every day. On the
day of the transport study, cell monolayers were first washed with HBSS (pH 7.2) twice,
and incubated at 37C for a total of 30 min. Transport buffer (HBSS, pH 7.2) containing
0.1% dimethyl sulfoxide (control) or 10 M FTC was then loaded into both apical (1.5
ml) and basolateral (2.6 ml) chambers. After a 15-min of incubation, [3H]biochanin A or
[3H]kaempferol was added to either the apical or basolateral chamber (donor chamber).
The final concentrations of [3H]biochanin A and [3H]kaempferol in the donor chambers
were 0.42 M and 0.95 M, respectively. Aliquots (100 l) were taken from the opposite
chamber (receiver chamber) at 30, 60, and 90 min after the addition of [3H]biochanin A
or [3H]kaempferol, and replaced with the same volume of fresh transport buffer. The
radioactivity in the aliquots was determined by liquid scintillation counting (1900 CA,
Tri-Carb liquid scintillation analyzer; PerkinElmer Life Sciences). The integrity of the
monolayer was measured by monitoring the flux of [3H]mannitol, a paracellular marker,
across cell monolayers. The apparent permeability coefficients (Papp) of mannitol in both
apical to basolateral (Papp, A-B) and basolateral to apical (Papp, B-A) directions were
measured in triplicate, and these values were all lower than 1.0 x 106 cm/s. The apparent
permeability coefficient was calculated as follows:
Papp =
Q
1
t A C 0
181
where Q/t is the rate of [3H]biochanin A or [3H]kaempferol appearing in the receiver

chamber, which was determined from the slope of the regression lines on the transporttime plots of [3H]biochanin A or [3H]kaempferol; C0 is the initial concentration of
[3H]biochanin A or [3H]kaempferol loaded in the donor chamber; and A is the cell
monolayer surface area (4.71 cm2).
The intracellular accumulation of [3H]biochanin A or [3H]kaempferol following apical or

basolateral dosing was determined at the end of the transport study. Briefly, the cell
monolayers were cut from the transwell inserts and washed three times with ice-cold
HBSS. The monolayers were then solubilized using 1 ml of 0.3N sodium hydroxide
solution containing 1% sodium dodecyl sulfate. The radioactivity in 400 l cell lysates
was determined by liquid scintillation counting (1900 CA, Tri-Carb liquid scintillation
analyzer; PerkinElmer Life Sciences). Results were normalized by protein concentration
determined by bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL).
Intracellular accumulation following apical or basolateral dosing was plotted as the
percentage of corresponding accumulation in MDCK-Bcrp1 cell monolayers in the
absence of FTC.
Radio-HPLC Assay
A radio-HPLC method was applied to determine whether the parent [3H]biochanin A and
[3H]kaempferol or their metabolites were transported by murine Bcrp1. Briefly, at the
end of the transport studies, the medium in the apical chambers following basolateral
182
dosing was collected from the MDCK-Bcrp1 control plate (in the absence of FTC) and
the MDCK-Bcrp1 FTC plate (in the presence of FTC).
The HPLC separation was performed on a system consisting of an Alltech Alltima C18
column (125 4.6 mm, 5-m particle size), a Waters 1525 pump, a 717 plus
autosampler, a 2847 UV detector, and Waters BreezeTM workstation. Ten l of biochanin
A (50 g/ml) or kaempferol (50 g/ml) was added to 90 l medium from the transport
studies to determine the retention time of biochanin A and kaempferol by UV detection.
Sixty l of the mixture was injected onto the HPLC and the radioactivity in the medium
was eluted with a mobile phase consisting of acetonitrile/water (55%: 45%, v/v). The
mobile phase was delivered isocratically with a flow rate of 1.0 ml/min. UV absorbance
was measured at 254 nm to detect both biochanin A and kaempferol. The retention times
of biochanin A and kaempferol were 10.2 and 4.8 min, respectively. The elutes were
collected every 30 seconds for a total of 15 min and every 20 seconds for a total of 7 min
for biochanin A and kaempferol, respectively. The radioactivity in each sample was
determined by liquid scintillation counting (1900 CA, Tri-Carb liquid scintillation
analyzer; PerkinElmer Life Sciences).
Enzymatic Hydrolysis of Kaempferol Metabolites with -Glucuronidase and

Sulfatase
Enzymatic hydrolysis of samples was conducted using a previously published method

(Yodogawa et al., 2003) with minor modification. Briefly, the medium in the apical
compartment following basolateral dosing was collected at the end of transport study
183
from the plate of MDCK-Bcrp1 cells in the absence of FTC. A 200 L aliquot of the
medium was incubated with no enzymes, with 5 units of sulfatase (pH 7.4), or with 50
units of -glucuronidase (pH 5.0) at 37oC for 2 hours. The radioactivity in the medium
was eluted by HPLC as described above.
Statistical analysis was conducted using a Students t-test or ANOVA (Prism 3.0
software, GraphPad, San Diego, CA) followed by Bonferronis post hoc test. p values <
0.05 were considered statistically significant.
184
RESULTS
Transport of Biochanin A and Kaempferol in MDCK Cells
To determine whether biochanin A and kaempferol are BCRP substrates, [3H]biochanin

A and [3H]kaempferol were used as model compounds and their transport in both
parental MDCK II cells and MDCK-Bcrp1 cells was investigated. As shown in Figure 2,
the polarized transport of [3H]biochanin A and [3H]kaempferol was observed in MDCKBcrp1 cells in the absence of FTC, with Papp, B-A values significantly higher than the
corresponding Papp, A-B values (2.89 0.11 versus 0.32 0.02 10-6 cm/s, p < 0.001 and
2.20 0.24 versus 0.26 0.01 10-6 cm/s, p < 0.001 for [3H]biochanin A and
[3H]kaempferol, respectively) (Table 1). The efflux ratios (Papp, B-A / Papp, A-B) of
[3H]biochanin A and [3H]kaempferol in MDCK-Bcrp1 cells were 8.97 and 8.35,
respectively. When the specific murine Bcrp1 and human BCRP inhibitor FTC (10 M)
was present, apically directed transport of [3H]biochanin A and [3H]kaempferol was
significantly decreased and basolaterally directed transport was significantly increased,
resulting in vectorial transport patterns similar to those of the parental MDCK II cells
(Figure 2 and Table 1). In the presence of FTC, the efflux ratios of [3H]biochanin A and
[3H]kaempferol in MDCK-Bcrp1 cells were 0.39 and 0.45, respectively, which were
similar to the values in the parental MDCK II cells (0.55 and 0.62 for biochanin A and
kaempferol, respectively). In the parental MDCK II cells, [3H]biochanin A and
[3H]kaempferol was consistently transported somewhat more efficiently in the basolateral
direction than in the apical direction, indicating low endogenous basolaterally directed
transport.
185
At the end of the transport studies, the accumulation of [3H]biochanin A and

[3H]kaempferol in MDCK II and MDCK-Bcrp1 cell monolayers was determined. In the
absence of FTC, the accumulation of [3H]biochanin A following the apical and
basolateral dosing was 0.079 0.004 and 0.245 0.003 pmol/mg protein, respectively.
Similarly, in the absence of FTC, the accumulation of [3H]kaempferol following the
apical and basolateral dosing was 0.13 0.01 and 0.36 0.02 pmol/mg protein,
respectively. The relatively higher accumulation of [3H]biochanin A and [3H]kaempferol
following the basolateral dosing was consistent with the polarized transport mediated by
murine Bcrp1. In the presence of FTC or in the parental MDCK II cells, the
accumulation of [3H]biochanin A and [3H]kaempferol following both apical and
basolateral dosing was significantly increased when compared to the corresponding
accumulation in MDCK-Bcrp1 cells in the absence of FTC (Figure 3).
Radio-HPLC Assay
Since flavonoids undergo extensive intracellular metabolism, it is important to determine

whether the parent flavonoids or their metabolites are BCRP/Bcrp1 substrates. In the
present study, we characterized the parent/metabolite profiles of [3H]biochanin A and
[3H]kaempferol across the MDCK-Bcrp1 cell monolayers in the absence and presence of
FTC. The medium in the apical chambers following the basolateral dosing was collected
at the end of the transport study from the MDCK-Bcrp1 cell plates. The radioactivity in
the medium (60 l) was eluted and the fractions of [3H]biochanin A and [3H]kaempferol
were collected. The retention times of biochanin A and kaempferol were around 10.2 and
4.8 min, respectively. As shown in Figure 4, at the end of a 2-hour transport study,
186
biochanin A was completely metabolized in MDCK-Bcrp1 cells and no parent biochanin

A was present in the medium. Two metabolite peaks (M1 and M2) of biochanin A were
identified. In the presence of FTC, the total radioactivity was substantially decreased,
mainly due to the decreased transport of M2 (Figure 4A). In contrast, kaempferol was
not completely metabolized. Both the parent kaempferol and one metabolite peak were
present on the chromatograph. In the presence of FTC, the total radioactivity was
significantly decreased, mainly due to the completely inhibited transport of the parent
kaempferol and partially inhibited transport of the metabolite (Figure 4B).
To further identify the metabolites from the Radio-HPLC study, the kaempferol
metabolite(s) was determined using the medium from the apical compartment following
basolateral dosing at the end of transport study from the plate of MDCK-Bcrp1 cells in
the absence of FTC. As shown in Figure 5, at the end of a 2-hour incubation with
sulfatase, the metabolite peak of kaempferol (M) was substantially decreased and the
parent peak was significantly increased when compared with the control group (no
enzyme treatment). However, the treatment with -glucuronidase had no significant
effects on the metabolite profiles of kaempferol.
187
DISCUSSION
Flavonoids have been recently identified as a class of inhibitors of breast cancer

resistance protein, an efflux transporter important in limiting drug oral absorption and
elimination (Zhang et al., 2004; van Herwaarden and Schinkel, 2006). However, the
mechanisms of BCRP inhibition by flavonoids have not been well understood. Given the
potent BCRP inhibitory activity of flavonoids and the popular use of flavonoidcontaining dietary supplements, it is likely that flavonoid-drug interactions mediated by
BCRP might occur in vivo. To better interpret the potential pharmacokinetic interactions
between flavonoids and BCRP substrates, it is important to understand the mechanisms
of inhibition by flavonoids and the in vivo disposition of flavonoids. Therefore, it is of
interest to characterize whether flavonoids themselves can be transported by BCRP.
In the present study, [3H]biochanin A and [3H]kaempferol were selected as two model
flavonoids and their transport in both murine Bcrp1 and human BCRP-overexpressing
cells was determined. In MDCK-Bcrp1 cells, apically directed transport was observed
for both [3H]biochanin A and [3H]kaempferol. The efflux ratios were 8.97 and 8.35 for
[3H]biochanin A and [3H]kaempferol, respectively, suggesting that murine Bcrp1
efficiently transported biochanin A and kaempferol. In the presence of 10 M FTC, a
specific human BCRP and murine Bcrp1 inhibitor, apically directed transport of
[3H]biochanin A and [3H]kaempferol was significantly decreased and basolaterally
directed transport was significantly increased, resulting in vectorial transport patterns
similar to those in MDCK II parental cells. These results suggested that Bcrp1-mediated
transport of biochanin A and kaempferol could be completely inhibited by 10 M FTC.
188
In the parental MDCK II cells, similar to the transport of other BCRP substrates (Merino
et al., 2005), [3H]biochanin A and [3H]kaempferol were consistently transported
somewhat more efficiently in the basolateral direction than in the apical direction (the
efflux ratios were 0.55 and 0.62 for biochanin A and kaempferol, respectively). These
results indicated the presence of low endogenous basolaterally directed transporter(s). In
a previous study, the endogenous drug transporter MRP1 has been identified in the
parental MDCK II cells (Goh et al., 2002), which might mediate the basolaterally
directed transport of biochanin A and kaempferol and/or their metabolites. Other
basolaterally directed transporters might also be present in the parental MDCK II cells as
well. Further studies are needed to identify these unknown endogenous transporters. In
a recent study, the flavonoid quercetin was reported as a good substrate of murine Bcrp1
(Sesink et al., 2005). The reported efflux ratio of quercetin was approximately 160,
which was much higher than those of biochanin A and kaempferol determined in our
study (8.97 and 8.35, respectively). This is likely due to the more efficient transport of
quercetin by murine Bcrp1 or due to differences in experimental design. In the transport
study by Sesink et al., 5 M PSC 833 (a potent inhibitor of P-gp and a moderate inhibitor
of MRP (Leier et al., 1994; Tan et al., 2000)) was added to inhibit the activities of the
endogenous transporters, resulting in no directional transport in the parental MDCK II
cells and higher apically directed transport in MDCK-Bcrp1 cells.
The accumulation of [3H]biochanin A and [3H]kaempferol in MDCK II and MDCKBcrp1 cell monolayers was determined at the end of the transport studies. In the absence
of FTC, the accumulation of [3H]biochanin A and [3H]kaempferol following basolateral
189
dosing was significantly higher than that following apical dosing. The lower
accumulation following the apical dosing is due to the apically directed transport by
murine Bcrp1, which limits the intracellular concentration of [3H]biochanin A and
[3H]kaempferol. In the presence of FTC or in MDCK II cells, the accumulation of
[3H]biochanin A and [3H]kaempferol was significantly increased following both apical
and basolateral dosing. This is due to the inhibition of Bcrp1-mediated transport of
[3H]biochanin A and [3H]kaempferol into the apical compartment.
Since many flavonoids undergo extensive glucuronidation and/or sulfation both in vitro
and in vivo (Sfakianos et al., 1997; Peterson et al., 1998; Yodogawa et al., 2003; Moon et
al., 2006), it is of interest to determine whether the parent drugs or metabolites of
flavonoids are transported by BCRP. We collected the medium in the apical
compartments following basolateral dosing from the MDCK-Bcrp1 cell plates at the end
of the transport study and determined the radioactivity in HPLC fractions. As shown in
Figure 4, at the end of a 2-hour transport study, biochanin A was completely metabolized
in MDCK-Bcrp1 cells and two metabolite peaks (M1 and M2) of biochanin A were
identified. The total radioactivity was significantly decreased by FTC, mainly due to the
abolished transport of metabolite M2 (Figure 4A). These data clearly indicate that the
major biochanin A metabolite (M2) is a good substrate of murine Bcrp1. However, we
cannot exclude the possibility that the parent biochanin A is a substrate of murine Bcrp1
as well. Differently, kaempferol was not completely metabolized in MDCK-Bcrp1 cells
during the course of transport study. Both the parent kaempferol and one metabolite peak
were present on the HPLC chromatograph. In the presence of FTC, the total radioactivity
190
was substantially decreased, mainly due to the completely abolished transport of the
parent kaempferol and the partially inhibited transport of the metabolite (Figure 4B).
These data clearly indicate that both the parent kaempferol and its metabolite are
substrates of murine Bcrp1.
Biochanin A and kaempferol have been reported to undergo glucuronidation and

sulfation (Peterson et al., 1998; Yodogawa et al., 2003; Moon et al., 2006). Both
glucuronide and sulfate conjugates have been shown to be BCRP substrates (Suzuki et
al., 2003; Adachi et al., 2005; Sesink et al., 2005). Therefore, the metabolites of
biochanin A and kaempferol transported by murine Bcrp1 are likely the glucuronide or
sulfate or double conjugates. Our enzymatic hydrolysis studies indicate that the sulfate
conjugate of kaempferol is likely a substrate of BCRP (Figure 5). This is consistent with
the literature data that sulfate conjugation is substantial in MDCK cells (Ng et al., 2003).
Due to the insufficient sample amount, we did not submit the putative metabolic peaks to
LC-MS/MS, which would have been helpful to confirm the metabolite structures. In
Figure 4B, FTC did not completely abolish the transport of the metabolite of kaempferol
(M). This is possibly due to the presence of MRP2 in MDCK II cells (Goh et al., 2002),
which can also transport the sulfate conjugates of some flavonoids, such as chrysin and
epicatechin (Walle et al., 1999; Vaidyanathan and Walle, 2001). Interestingly, in a
previous study, quercetin was efficiently transported by murine Bcrp1 in MDCK II cells,
and no metabolites of quercetin could be detected in the medium during the course of the
study (Sesink et al., 2005). This is possibly due to differences in experimental protocols.
In the study by Sesink et al., 50 M of quercetin was loaded in the donor chamber, which
191
might saturate or inhibit the phase II enzymes for the glucuronidation and sulfation
reactions. In our study, much lower concentrations of [3H]biochanin A and
[3H]kaempferol were added in the donor chambers (0.42 M and 0.95 M, respectively).
These low substrate concentrations are less likely to saturate or inhibit the phase II
enzymes, resulting in relatively higher percentages of the metabolites. Moreover, HPLC
with UV detection was used for quercetin quantitation, which might provide lower
sensitivity compared with radioisotope counting used in our studies.
In summary, our studies clearly demonstrated that BCRP could efficiently transport the
flavonoid biochanin A and kaempferol in their parent and/or metabolized forms.
Therefore, these two flavonoids and/or their metabolites are BCRP substrates. The
information from this study will be useful in characterizing the in vivo disposition of
biochanin A and kaempferol, and to better understand the potential in vivo flavonoid-drug
interactions mediated by BCRP.
192
REFERENCES:
Adachi Y, Suzuki H, Schinkel AH and Sugiyama Y (2005) Role of breast cancer

resistance protein (Bcrp1/Abcg2) in the extrusion of glucuronide and sulfate
conjugates from enterocytes to intestinal lumen. Mol Pharmacol 67:923-928.
Atkinson C, Compston JE, Day NE, Dowsett M and Bingham SA (2004) The effects of
phytoestrogen isoflavones on bone density in women: a double-blind,
randomized, placebo-controlled trial. Am J Clin Nutr 79:326-333.
Booth NL, Piersen CE, Banuvar S, Geller SE, Shulman LP and Farnsworth NR (2006)
Clinical studies of red clover (Trifolium pratense) dietary supplements in
menopause: a literature review. Menopause 13:251-264.
Breedveld P, Pluim D, Cipriani G, Wielinga P, van Tellingen O, Schinkel AH and
Schellens JH (2005) The effect of Bcrp1 (Abcg2) on the in vivo pharmacokinetics
and brain penetration of imatinib mesylate (Gleevec): implications for the use of
breast cancer resistance protein and P-glycoprotein inhibitors to enable the brain
penetration of imatinib in patients. Cancer Res 65:2577-2582.
de Vries JH, Janssen PL, Hollman PC, van Staveren WA and Katan MB (1997)
Consumption of quercetin and kaempferol in free-living subjects eating a variety
of diets. Cancer Lett 114:141-144.
Delmonte P and Rader JI (2006) Analysis of isoflavones in foods and dietary
supplements. J AOAC Int 89:1138-1146.
Doyle LA and Ross DD (2003) Multidrug resistance mediated by the breast cancer
resistance protein BCRP (ABCG2). Oncogene 22:7340-7358.
Goh LB, Spears KJ, Yao D, Ayrton A, Morgan P, Roland Wolf C and Friedberg T (2002)
Endogenous drug transporters in in vitro and in vivo models for the prediction of
drug disposition in man. Biochem Pharmacol 64:1569-1578.
Hertog MG, Feskens EJ, Hollman PC, Katan MB and Kromhout D (1993) Dietary
antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly
Study. Lancet 342:1007-1011.
Howes JB, Sullivan D, Lai N, Nestel P, Pomeroy S, West L, Eden JA and Howes LG
(2000) The effects of dietary supplementation with isoflavones from red clover on
the lipoprotein profiles of post menopausal women with mild to moderate
hypercholesterolaemia. Atherosclerosis 152:143-147.
Imai Y, Tsukahara S, Asada S and Sugimoto Y (2004) Phytoestrogens/flavonoids reverse
breast cancer resistance protein/ABCG2-mediated multidrug resistance. Cancer
Res 64:4346-4352.
Jonker JW, Merino G, Musters S, van Herwaarden AE, Bolscher E, Wagenaar E,
Mesman E, Dale TC and Schinkel AH (2005) The breast cancer resistance protein
BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins into milk. Nat
Med 11:127-129.
Jonker JW, Smit JW, Brinkhuis RF, Maliepaard M, Beijnen JH, Schellens JH and
Schinkel AH (2000) Role of breast cancer resistance protein in the bioavailability
and fetal penetration of topotecan. J Natl Cancer Inst 92:1651-1656.
193
Kohno H, Tanaka T, Kawabata K, Hirose Y, Sugie S, Tsuda H and Mori H (2002)

azoxymethane-induced colon carcinogenesis in male F344 rats. Int J Cancer
101:461-468.
GF120918. J Clin Oncol 20:2943-2950.
Leier I, Jedlitschky G, Buchholz U, Cole SP, Deeley RG and Keppler D (1994) The MRP
gene encodes an ATP-dependent export pump for leukotriene C4 and structurally
related conjugates. J Biol Chem 269:27807-27810.
Mao Q and Unadkat JD (2005) Role of the breast cancer resistance protein (ABCG2) in
drug transport. Aaps J 7:E118-133.
Merino G, Jonker JW, Wagenaar E, van Herwaarden AE and Schinkel AH (2005) The
breast cancer resistance protein (BCRP/ABCG2) affects pharmacokinetics,
hepatobiliary excretion, and milk secretion of the antibiotic nitrofurantoin. Mol
Pharmacol 67:1758-1764.
Moon YJ, Sagawa K, Frederick K, Zhang S and Morris ME (2006) Pharmacokinetics and
bioavailability of the isoflavone biochanin A in rats. Aaps J 8:E433-442.
Ng KH, Lim BG and Wong KP (2003) Sulfate conjugating and transport functions of
MDCK distal tubular cells. Kidney Int 63:976-986.
Niering P, Michels G, Watjen W, Ohler S, Steffan B, Chovolou Y, Kampkotter A,
Proksch P and Kahl R (2005) Protective and detrimental effects of kaempferol in
rat H4IIE cells: implication of oxidative stress and apoptosis. Toxicol Appl
Park JS, Rho HS, Kim DH and Chang IS (2006) Enzymatic preparation of kaempferol
from green tea seed and its antioxidant activity. J Agric Food Chem 54:29512956.
Peterson TG, Ji GP, Kirk M, Coward L, Falany CN and Barnes S (1998) Metabolism of
the isoflavones genistein and biochanin A in human breast cancer cell lines. Am J
Clin Nutr 68:1505S-1511S.
Potter SM, Baum JA, Teng H, Stillman RJ, Shay NF and Erdman JW, Jr. (1998) Soy
protein and isoflavones: their effects on blood lipids and bone density in
postmenopausal women. Am J Clin Nutr 68:1375S-1379S.
Risbridger GP, Wang H, Frydenberg M and Husband A (2001) The in vivo effect of red
clover diet on ventral prostate growth in adult male mice. Reprod Fertil Dev
13:325-329.
Samhan-Arias AK, Martin-Romero FJ and Gutierrez-Merino C (2004) Kaempferol
blocks oxidative stress in cerebellar granule cells and reveals a key role for
reactive oxygen species production at the plasma membrane in the commitment to
apoptosis. Free Radic Biol Med 37:48-61.
Sesink AL, Arts IC, de Boer VC, Breedveld P, Schellens JH, Hollman PC and Russel FG
(2005) Breast cancer resistance protein (Bcrp1/Abcg2) limits net intestinal uptake
194
of quercetin in rats by facilitating apical efflux of glucuronides. Mol Pharmacol

67:1999-2006.
Sfakianos J, Coward L, Kirk M and Barnes S (1997) Intestinal uptake and biliary
excretion of the isoflavone genistein in rats. J Nutr 127:1260-1268.
Sloley BD, Urichuk LJ, Morley P, Durkin J, Shan JJ, Pang PK and Coutts RT (2000)
Identification of kaempferol as a monoamine oxidase inhibitor and potential
Neuroprotectant in extracts of Ginkgo biloba leaves. J Pharm Pharmacol 52:451459.
Smith JV and Luo Y (2003) Elevation of oxidative free radicals in Alzheimer's disease
models can be attenuated by Ginkgo biloba extract EGb 761. J Alzheimers Dis
5:287-300.
22:2489-2503.
Suzuki M, Suzuki H, Sugimoto Y and Sugiyama Y (2003) ABCG2 transports sulfated
conjugates of steroids and xenobiotics. J Biol Chem 278:22644-22649.
Tan B, Piwnica-Worms D and Ratner L (2000) Multidrug resistance transporters and
modulation. Curr Opin Oncol 12:450-458.
Vaidyanathan JB and Walle T (2001) Transport and metabolism of the tea flavonoid (-)epicatechin by the human intestinal cell line Caco-2. Pharm Res 18:1420-1425.
van Herwaarden AE, Jonker JW, Wagenaar E, Brinkhuis RF, Schellens JH, Beijnen JH
and Schinkel AH (2003) The breast cancer resistance protein (Bcrp1/Abcg2)
restricts exposure to the dietary carcinogen 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine. Cancer Res 63:6447-6452.
van Herwaarden AE and Schinkel AH (2006) The function of breast cancer resistance
protein in epithelial barriers, stem cells and milk secretion of drugs and
xenotoxins. Trends Pharmacol Sci 27:10-16.
van Herwaarden AE, Wagenaar E, Karnekamp B, Merino G, Jonker JW and Schinkel AH
(2006) Breast cancer resistance protein (Bcrp1/Abcg2) reduces systemic exposure
of the dietary carcinogens aflatoxin B1, IQ and Trp-P-1 but also mediates their
secretion into breast milk. Carcinogenesis 27:123-130.
Walle UK, Galijatovic A and Walle T (1999) Transport of the flavonoid chrysin and its
conjugated metabolites by the human intestinal cell line Caco-2. Biochem
Yodogawa S, Arakawa T, Sugihara N and Furuno K (2003) Glucurono- and sulfoconjugation of kaempferol in rat liver subcellular preparations and cultured
hepatocytes. Biol Pharm Bull 26:1120-1124.
Zhang S, Wang X, Sagawa K and Morris ME (2005a) Flavonoids chrysin and
benzoflavone, potent breast cancer resistance protein inhibitors, have no
significant effect on topotecan pharmacokinetics in rats or mdr1a/1b (-/-) mice.
Zhang S, Yang X, Coburn RA and Morris ME (2005b) Structure activity relationships
and quantitative structure activity relationships for the flavonoid-mediated
inhibition of breast cancer resistance protein. Biochem Pharmacol 70:627-639.
Zhang S, Yang X and Morris ME (2004) Flavonoids are inhibitors of breast cancer
195
Table 1. Transport of [3H]biochanin A and [3H]kaempferol in MDCK II and MDCKBcrp1 cells in the absence or presence of 10 M FTC
A.
Biochanin A
Papp, A-B
Papp, B-A
(cm/s, 10^6)
(cm/s, 10^6)
Papp, B-A/Papp, A-B
MDCK II
1.63 0.14 ***
0.89 0.06 b, ***
0.55
MDCK-Bcrp1
0.32 0.02
2.89 0.11 b
8.97
MDCK-Bcrp1 + FTC
1.86 0.15 ***
0.73 0.09 b, ***
0.39
B.
B) Kaempferol
Papp, A-B
Papp, B-A
(cm/s, 10^6)
(cm/s, 10^6)
Papp, B-A/Papp, A-B
MDCK II
1.18 0.09 ***
0.73 0.02 a, ***
0.62
MDCK-Bcrp1
0.26 0.01
2.20 0.24 b
8.35
MDCK-Bcrp1 + FTC
1.56 0.07 ***
0.71 0.04 b, ***
0.45
The apparent permeability was calculated as described in the Materials and Methods.
Data are presented as mean SD, n = 3. Papp, A-B, apparent permeability from the
apical to basolateral compartment; Papp, B-A, apparent permeability from the
basolateral to apical compartment. ***: p < 0.001, compared with the corresponding
Papp in MDCK-Bcrp1 cells in the absence of FTC; a: p < 0.01, b: p < 0.001,
comparison between Papp, B-A and Papp, A-B in each group.
196
FIGURE LEGEND:
Figure 1. The chemical structures of biochanin A and kaempferol.
Figure 2. Transport of [3H]biochanin A (left panel) and [3H]kaempferol (right panel)

across monolayers of MDCK II cells (A), MDCK-Bcrp1 cells in the absence of 10 M
FTC (B), and MDCK-Bcrp1 cells in the presence of 10 M FTC (C). Data are presented
as mean SD, n = 3. The error bars are shown in the figure or are within the symbols.
{, transport from the basolateral to apical compartment; z, transport from the apical to
basolateral compartment.
Figure 3. Accumulation of [3H]biochanin A (A) and [3H]kaempferol (B) in MDCK II

and MDCK-Bcrp1 cell monolayers in the absence (control) or presence of 10 M FTC.
Accumulation of [3H]biochanin A (A) and [3H]kaempferol in MDCK cells was
determined at the end of transport study by measuring the radioactivity in cell
monolayers. Intracellular accumulation following apical (solid bar) or basolateral (open
bar) dosing is plotted as the percentage of corresponding accumulation in MDCK-Bcrp1
cells in the absence of FTC. Data are presented as mean SD, n = 3. *: p < 0.05, ***: p
< 0.001, compared with the corresponding accumulation in MDCK- Bcrp1 cells in the
absence of FTC.
Figure 4. Representative Radio-HPLC chromatographs of [3H]biochanin A (A) and

[3H]kaempferol (B) in the samples from the transport study. The medium in the apical
compartment following basolateral dosing was collected at the end of transport study
197
from the plates of MDCK-Bcrp1 cells in the absence of FTC (upper panel) and MDCKBcrp1 cells in the presence of FTC (lower panel). The radioactivity in the medium (60
l) was eluted with a mobile phase consisting of acetonitrile/water (55%: 45%, v/v). The
retention times of biochanin A and kaempferol were around 10.2 and 4.8 min,
respectively. BCA: biochanin A; M1: metabolite peak 1 of biochanin A; M2: metabolite
peak 2 of biochanin A; KMP: kaempferol; M: metabolite peak of kaempferol.
Figure 5. Enzymatic hydrolysis of kaempferol metabolites with -glucuronidase and

sulfatase. The medium in the apical compartment following basolateral dosing was
collected at the end of transport study from the plate of MDCK-Bcrp1 cells in the
absence of FTC. A 200 L aliquot of the medium was incubated with no enzymes (upper
panel), with 5 units of sulfatase (middle panel, pH 7.4), or with 50 units of glucuronidase (lower panel, pH 5.0) at 37oC for 2 hours. The radioactivity in the medium
was eluted by HPLC.
198
Figure 1.
199
Figure 2.
200
Figure 3.
201
Figure 4.
202
Figure 5.
203
CHAPTER 7
Lack of In Vivo Interactions between the Flavonoid Biochanin

A and P-glycoprotein Substrates Doxorubicin and
Cyclosporine A in Rats
This chapter has been written for publication in the format of Drug Metab Dispos.
Drs. Robert Arnold, Shuzhong Zhang and Kazuko Sagawa (Pfizer Inc.) contributed to the
animal studies performed for this chapter.
204
ABSTRACT
The purpose of this study was to investigate the in vitro and in vivo interactions between
flavonoids and P-glycoprotein (P-gp) substrates. The inhibitory effects of flavonoids on
P-gp were determined by accumulation studies in P-gp-overexpressing MCF-7/ADR cells
using daunomycin (DNM) as a model substrate. The results from the accumulation study
indicated that the flavonoids morin, phloretin, biochanin A, chalcone, and silymarin at a
concentration of 100 M, all significantly increased DNM accumulation by more than
2.5 fold, suggesting these flavonoids are P-gp inhibitors. Among all the flavonoids
tested, biochanin A demonstrated the largest effects, resulting in a 4-fold increase in
[3H]DNM accumulation. To further explore the potential in vivo interactions of
flavonoids with P-gp, biochanin A was chosen as a transport inhibitor, and its interaction
with the P-gp substrates doxorubicin and cyclosporine A were investigated. P-gp
substrates were administered either orally or intravenously, and biochanin A was given
by either intraperitoneal or oral administration. In contrast to the in vitro results,
intraperitoneal or oral administration of biochanin A did not significantly change the
pharmacokinetics of doxorubicin and cyclosporine A. The disconnect between the in
vitro and in vivo data suggests that P-gp interactions mediated by biochanin A may be
limited due to its poor bioavailability and rapid clearance. It is also possible that other
transporters or metabolizing enzymes are more important in the in vivo disposition of
doxorubicin and cyclosporine A than P-glycoprotein.
205
INTRODUCTION
Flavonoids have attracted considerable scientific and public interest in recent years due to
their health-promoting and beneficial pharmacological activities including antioxidant,
antiviral, anti-carcinogenic and anti-inflammatory properties (Middleton et al., 2000;
Havsteen, 2002). Biochanin A (Figure 1) is a naturally occurring flavonoid and a major
component of the widely consumed herbal product red clover extracts (Delmonte and
Rader, 2006). Biochanin A has been shown to inhibit carcinogen-induced mammary and
lung tumor growth (Lee et al., 1991; Gotoh et al., 1998) and inhibit the incidence of
prostate tumors in xenograft mice (Rice et al., 2002). Red clover extracts are commonly
used to prevent or relieve post-menopausal symptoms such as hot flashes and bone loss,
and to maintain prostate health (Risbridger et al., 2001; Atkinson et al., 2004; Booth et
al., 2006). Red clover extracts such as Promensil (Novogen, Inc., Samford, CT) are now
commercially available as dietary supplements, which contain approximately 26 mg of
biochanin A per tablet (Howes et al., 2000).
Flavonoid-drug interactions have been increasingly evidenced due to the popular use of
flavonoid-containing dietary supplements. The mechanisms underlying the majority of
these interactions are attributed to the modulation of cytochrome P450 (CYP) enzymes
and phase II (conjugation) enzymes responsible for the detoxification of carcinogens
(Moon et al., 2006b). Recently, P-glycoprotein (P-gp) has also been identified as an
important player responsible for the interactions between flavonoids and clinically
important P-gp substrates. For example, oral coadministration of the flavonoids
quercetin and flavone significantly increased the bioavailability of paclitaxel in a dose-
206
dependent manner (Choi et al., 2004b; Choi et al., 2004c). In some cases, these
flavonoid-drug interactions can be serious or even life threatening. This is especially true
when flavonoids or dietary supplements are used concomitantly with narrow therapeutic
index drugs, which was exemplified by the interaction between quercetin and digoxin in
pigs (Wang et al., 2004). In a recent clinical study, oral administration of quercetin (5
mg/kg, 30 min or 3 days pretreatment) significantly increased the area under the plasma
concentration-time curve (AUC) of cyclosporine by 36% and 47%, respectively (Choi et
al., 2004a). Interestingly, in a preclinical study, the oral administration of quercetin (50
mg/kg) to pigs and rats resulted in significant decreases in the AUC of cyclosporine by
56% and 43%, respectively (Hsiu et al., 2002). All these studies indicated that P-gpmediated flavonoid-drug interactions could occur in vivo. However, the reasons for the
observed contradictory results due to different dosing regimens and different species
remain largely unknown. Since cyclosporine A is also a drug with a narrow therapeutic
index (Beauchesne et al., 2007), it is important to characterize and understand its
interaction with different flavonoid inhibitors abundant in dietary supplements.
The objective of this study was to investigate the interactions between P-gp substrates
and potential flavonoid inhibitors. Two clinically important P-gp substrates, namely
doxorubicin (Figure 1), an anthracycline antibiotic with broad-spectrum antineoplastic
activity (Minotti et al., 2004), and cyclosporine (Figure 1), a widely used
immunosuppressant drug (Beauchesne et al., 2007), were used in this study.
207
MATERIALS AND METHODS:

Biochanin A, cyclosporine A, cremephor and hydroxypropyl--cyclodextrin (HPCD)

were purchased from Sigma-Aldrich (St. Louis, MO). Doxorubicin was a generous gift
from Sicor (Rome, Italy). [3H]daunomycin (DNM, 16 Ci/mmol) was purchased from
Perkin Elmer (Waltham, MA). Saline was purchased from Henry-Schein (Melville, NY).
RPMI 1640, fetal bovine serum, L-glutamine, penicillin, and streptomycin were
purchased from Invitrogen (Carlsbad, CA). All other reagents or solvents used were
either analytical or high-performance liquid chromatography (HPLC) grade and were
purchased from Fisher Scientific (Springfield, NJ).
Cell Culture and Accumulation Studies
P-glycoprotein-overexpressing MCF-7/ADR cells (Tseng et al., 2002) with passage

number between 16-24 were grown in RPMI 1640 supplemented with 10% fetal bovine
serum, 2mM L-glutamine, penicillin (100 units/ml) and streptomycin (100 g/ml). Cells
were cultured in 75-cm2 flasks at 37oC in a humidified 5% CO2 /95 % air atmosphere. At
the 90 % confluence, cells were trypsinized and seeded into 35mm2 Petri dishes for
accumulation studies. Experiments were performed 2-3 days after seeding.
For the accumulation studies, cell culture medium was removed from the Petri dishes and
cells were washed twice with uptake buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2,
1.2mM MgCl2, 10mM HEPES, pH 7.4). One ml of uptake buffer containing 0.05M of
[3H]-DNM and 100 of flavonoid was added to the dish and incubated for 2 hours.
208
Verapamil, a P-gp inhibitor, was used as a positive control in the studies. The uptake of
[3H]-DNM was stopped by aspirating the incubation buffer and washing the cells three
times with ice-cold stopping solution (137 mM NaCl, 14mM Tris-base, pH 7.4). The
cells were then solubilized using 1 ml of 0.3N sodium hydroxide solution containing 1%
sodium dodecyl sulfate. The radioactivity in 150 l aliquots of cell lysates was
determined by liquid scintillation counting (1900 CA, Tri-Carb liquid scintillation
analyzer; PerkinElmer Life Sciences). The accumulation in MCF7/ADR cells was
normalized by the protein concentration determined by bicinchoninic acid protein assay
(Pierce Chemical, Rockford, IL).
Animals and Surgery
Male Sprague-Dawley (SD) rats (body weight ~ 250 g) were purchased from Harlan
(Indianapolis, IN) and Charles River Laboratories (Wilmington, MA). Animals were
kept in a temperature and humidity-controlled environment with a 12-h light-dark cycle
and received a standard diet with water ad libitum. Animals were acclimatized to this
environment for at least one week before experiments. All animal procedures were
performed in accordance with Institutional Animal Care and Use Committee guidelines
and followed approved protocols. Two or three days prior to the study day, rats were
anesthetized (ketamine 90 mg/kg and xylazine 10 mg/kg, i.m. injection) and implanted
with a cannula (Micro-Renathane, type MRE-040, 0.040 O.D. 0.025 I.D., Braintree
Scientific, Inc., MA) in the right jugular vein. The cannula was flushed with saline
containing 50 IU/ml heparin daily until the study day. The rats were fasted overnight for
the oral pharmacokinetic studies.
209
Pharmacokinetic interaction studies

Biochanin A and doxorubicin interaction study
Doxorubicin was dissolved in saline at a concentration of 5 mg/ml. Biochanin A was

prepared in 75% DMSO at a concentration of 20 mg/ml. In the control group, rats were
pretreated with 75% DMSO (Biochanin A vehicle, 5ml/kg, ip) 5 min prior to the
intravenous administration of doxorubicin (7.5 mg/kg). In the treatment group,
Biochanin A in 75% DMSO was given intraperitoneally at 5 ml/kg (i.e., 100 mg/kg) 5
min before the intravenous administration of doxorubicin (7.5 mg/kg). The blood
samples were taken at 5 min, 15, 30, 1 hr, 2, 4, 8, 12, 24, 48, 72, and 96. The plasma was
separated from the whole blood by centrifugation at 2,000 g for 5 min at 4oC. All plasma
samples were stored at -80oC until analysis by LC-MS/MS. Six or seven rats were used
for each group.
Biochanin A and cyclosporine A interaction study
The iv formulation of Cyclosporine A was prepared in ethanol:cremephor (1:1), then

diluted 6-fold with saline to a final concentration of 3 mg/ml. The oral formulation was
made using olive oil at a concentration of 5 mg/ml. The oral formulation of biochanin A
was made in 25% HPCD at a concentration of 125 mg/ml.
For the intravenous interaction study, control rats were pretreated with 25% HPCD
(Biochanin A vehicle, 2ml/kg, po) 1 hour prior to the intravenous administration of
cyclosporine A (3 mg/kg). In the treatment group, biochanin A in HPCD was given
210
orally at 2 ml/kg (i.e., 250 mg/kg) 1 hour before the intravenous administration of
cyclosporine A (3 mg/kg). For the oral interaction study, control rats were pretreated
with 25% HPCD (biochanin A vehicle, 2ml/kg, po) 10 min prior to the oral
administration of cyclosporine A (10 mg/kg). In the treatment group, biochanin A in
HPCD (2 ml/kg or 250 mg/kg) was given orally 10 min before the oral administration of
cyclosporine A (10 mg/kg). Rats were fasted overnight and food returned after the 4 hour
blood sample. Rats had free access to water throughout the study. The blood samples
were taken at 5, 10, 15 and 30 min and 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours for the iv
study, and at 15 and 30 min and 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours for the oral study.
Three rats were used for each group.
Liquid Chromatography-Tandem Mass Spectrometry Analysis

Doxorubicin Analysis
Total doxorubicin was extracted from plasma and quantified by liquid chromatographytandem mass spectroscopy (LC-MS/MS), as described previously (Arnold et al., 2004).
In brief, plasma samples were diluted 5-fold in extraction solvent (60% acetonitrile and
40% 5 mM ammonium acetate pH 3.5). Plasma samples were cooled in an ice water bath
for 10 min, mixed intermittently, and centrifuged for 10 min at 15,000g at 4oC. The
deproteinized supernatant was recovered and analyzed immediately by LC-MS/MS. The
mobile phase consisted of 40% acetonitrile and 60% 5mM ammonium acetate, pH 3.5.
An electrospray ionization source was used on an ABI/Sciex API3000 triple quadruple
mass spectrometer (ABI Inc., Foster City, CA) following chromatographic separation
using an Agilent HPLC system (Model 1100; Palo Alto, CA). The concentration of
211
doxorubicin in plasma samples was calculated from a standard curve prepared using
standard and quality control samples prepared in blank plasma and extracted as described
previously (Arnold et al., 2004). The ions measured for doxorubicin were 544/361. The
assay was linear over the concentration range of 0.125 to 10,000 nM and is selective for
doxorubicin.
Cyclosporine A Analysis
The plasma cyclosporine A concentrations were determined following the addition of 200
l of 50/50 methanol/acetonitrile to 50 l samples. The mixture was centrifuged, and

150 l of the supernatant was removed. After a second centrifugation, 20 l of
supernatant was injected onto the column. The analysis was carried out on a PE Sciex
API3000 triple quadruple mass spectrometer with a turbo ion spray source linked to a
Shimadzu LC10 liquid chromatography system equipped with a Supelcosil LC-1column
(C18, 4.6 x 50 mm i.d., 5 m). The source temperature was set at 500C. The ionization
was set at positive mode. The mobile phase consisted of (A) methanol, and (B) 10 mM
ammonium acetate + 1% 2-propanol. The sample was eluted using a gradient from 0 95% A at 0.75 ml/min. The ions measured for cyclosporine A were 1202/100. The assay
was linear over the concentration range of 1 to 5000 ng/ml.
Biochanin A Analysis
The plasma biochanin A concentrations were determined following the addition of 200 l
of 50/50 methanol/acetonitrile to 25 l samples. The mixture was centrifuged, and 150
l of the supernatant was removed. After a second centrifugation, 20 l of supernatant
212
was injected onto the column. The analysis was carried out on a PE Sciex API3000 triple
quadruple mass spectrometer with a turbo ion spray source linked to a Shimadzu LC10
liquid chromatography system equipped with a Supelcosil LC-1column (C18, 4.6 x 50
mm i.d., 5 m). The source temperature was set at 500C. The ionization was set at
negative mode. The mobile phase consisted of (A) methanol, and (B) 10 mM ammonium
acetate + 1% 2-propanol. The sample was eluted using a gradient from 0 - 95% A at 0.75
ml/min. The ions measured for biochanin A were 283/239. The assay was linear over
the concentration range of 1 to 5000 ng/ml.
The plasma concentration data were analyzed by noncompartmental analysis using

WinNonlin version 2.1 (Pharsight, Mountain View, CA). The area under the plasma
concentration-time curve (AUC) was calculated using the log-linear trapezoidal rule. The
elimination half-life (t1/2) was calculated by the equation: t1/2 = 0.693/, where was
estimated from the terminal slope of the plasma concentration vs. time curve. The
clearance (CL) was determined from Dose/AUC. The volume of distribution at steady
state (Vss) was determined by Vss = CL (AUMC/AUC), where AUMC is the area under
the first moment curve (Concentration time vs. time curve). Oral bioavailability (F)
was calculated by the ratio of dose-normalized AUCs following oral and intravenous
administration.
213
Statistical analysis was conducted using a Students t test or one-way ANOVA (Prism 3.0
software, GraphPad, San Diego, CA) followed by a Dunnetts post hoc test. p values <
0.05 were considered statistically significant.
214
RESULTS:
Effect of flavonoids on DNM accumulation in MCF-7/ADR cells
MCF-7/ADR cells were simultaneously incubated with various flavonoids (100M) or

the flavonoid vehicle and [3H]DNM for 2 hours (Figure 2). In the control group, the
uptake of [3H]DNM was determined as 0.52 0.23 pmol/mg protein. Verapamil, a P-gp
inhibitor used as the positive control, significantly increased [3H]DNM accumulation to
1.30 0.39 pmol/mg protein (p < 0.01). The flavonoids morin, phloretin, biochanin A,
chalcone, and silymarin all significantly increased [3H]DNM accumulation by 2-fold or
higher. Biochanin A was the most effective inhibitor, with a 4-fold increase of [3H]DNM
(2.07 0.76 pmol/mg protein, p < 0.01). Concentration-dependent studies demonstrated
P-gp inhibition at concentrations of 10-30 M for biochanin A and silymarin (Zhang and
Morris, 2003a). All other flavonoids such as hesperetin, myricetin, naringenin, galangin,
kaempferol, genistein, chrysin, naringin, and luteolin had no significant effects on the
[3H]DNM accumulation in MCF-7/ADR cells.
Effects of Biochanin A on the Pharmacokinetics of Doxorubicin
To explore the potential in vivo interactions of flavonoid inhibitors with P-glycoprotein,

biochanin A was selected as a model flavonoid inhibitor due to its potent in vitro P-gp
inhibitory activity. The pharmacokinetics of the P-gp substrate doxorubicin was
characterized with and without the coadministration of biochanin A. As shown in Figure
3, doxorubicin pharmacokinetics, after intravenous administration, could be described by
a biexponential profile, exhibiting a rapid decrease at the early time points followed by a
slower decrease beyond 8 hr. In the control rats, the AUC and CL were determined as
215
152 69.6 gml-1min and 43.9 16.3 mlmin-1kg-1, respectively. In addition,

doxorubicin demonstrated a long terminal half-life t1/2 (49.7 15.6 hr) and a large steadystate volume of distribution (Vss) (158 95.3 L/kg). The coadministration of a 100
mg/kg dose of biochanin A by intraperitoneal injection had no significant effects on the
pharmacokinetics of doxorubicin. The AUC, t1/2, CL, and Vss were 184 86.5 gml1
min, 45.6 15.4 h, 40.3 18.5 mlmin-1kg-1, and 118 86.2 L/kg, respectively (Table
1).
Effects of Biochanin A on the Pharmacokinetics of Cyclosporine A
The interaction of biochanin A with P-gp was also explored by using cyclosporine A as a
P-gp substrate. As shown in Figure 4 and Table 2, after intravenous administration, the
AUC and clearance of cyclosporine A in control rats were 4960 983 ngml-1hr and 619
113 mlhr-1kg-1, respectively. The steady-state volume of distribution Vss was large
(5.66 1.90 L/kg) and the terminal half-life t1/2 was determined as 6.92 0.56 hr. The
oral administration of biochanin A at a 250 mg/kg dose had no significant effects on the
pharmacokinetics of cyclosporine A. The AUC, t1/2, CL, and Vss were 7050 1080
ngml-1hr, 9.68 1.69 hr, 432 66.3 mlhr-1kg-1, and 4.91 1.02 L/kg, respectively
(Table 2).
For the oral interaction study, the Cmax and tmax of cyclosporine A after oral administration
in control rats were 206 122 ng/ml and 4.33 0.58 hr, respectively. The AUC and
apparent oral clearance (CL/F) of cyclosporine A were 2280 801 ngml-1hr and 4780
1740 mlhr-1kg-1, respectively. The terminal half-life t1/2 was similar to that determined
216
after intravenous administration (10.5 4.84 hr). The oral coadministration of biochanin
A at 250 mg/kg had no significant effects on the pharmacokinetics of cyclosporine A.
The Cmax and tmax of cyclosporine A in biochanin A-treated rats were 227 133 ng/ml and
4.00 2.00 hr, respectively. The AUC, t1/2, and CL/F were 2290 375 ngml-1hr, 6.29
1.01 hr, and 4440 683 mlhr-1kg-1, respectively (Table 2). There was no significant
difference in cyclosporine A bioavailability between the control and biochanin A
coadministered rats (13.8 4.80 and 13.8 2.30 %, respectively).
The plasma concentrations of biochanin A following oral administration were also

determined. As shown in Figure 5, the Cmax and tmax of biochanin A were 48.7 27.4
ng/ml and 6.50 1.26 hr, respectively. The plasma concentrations of biochanin A
throughout the study were in the range of 10-100 ng/ml (Figure 5).
217
DISCUSSION
In recent years, with the increased public interest in alternative medicines, the use of
herbal preparations and dietary supplements containing high doses of flavonoids for
health maintenance has become very popular (Sparreboom et al., 2004), raising the
potential for interactions with conventional drug therapies. However, to date, very
limited information is available on the pharmacokinetic interactions between flavonoids
and clinically important drugs. Therefore, more studies are needed to better understand
the flavonoid-drug interactions and to utilize these interactions for a therapeutic benefit or
to avoid potential adverse reactions. In the present study, the effects of biochanin A, a
naturally occurring flavonoid, on the pharmacokinetics of doxorubicin and cyclosporine
A were investigated.
Doxorubicin and cyclosporine A have been reported to be P-gp substrates (Tsuji et al.,
1993; Endres et al., 2006). To determine P-gp inhibitory activity of flavonoids, the
effects of 14 flavonoids on [3H]DNM accumulation were investigated in P-gpoverexpressing MCF-7/ADR cells. As shown in Figure 2, the flavonoids morin,
phloretin, biochanin A, chalcone, and silymarin all significantly increased [3H]DNM
accumulation by 2-fold or higher. Among these flavonoids, biochanin A was the most
potent inhibitor, with a 4-fold increase in [3H]DNM accumulation. This is consistent
with our previous report that biochanin A inhibited P-gp mediated efflux of digoxin and
vinblastine in Caco-2 cells (Zhang and Morris, 2003a).
218
To further explore the potential in vivo interactions between biochanin A and P-gp
substrates, the pharmacokinetics of doxorubicin was characterized with and without the
administration of biochanin A (100 mg/kg, ip). As shown in Figure 3, in the control rats,
the pharmacokinetics of doxorubicin was characterized by a rapid decrease in plasma
concentrations, followed by a slow elimination phase. Consistent with the literature
(Camaggi et al., 1988; Robert, 1988; Robert and Gianni, 1993; Gabizon et al., 2003),
doxorubicin exhibited a long terminal half-life t1/2 (49.7 15.6 hr) and a large steadystate volume of distribution Vss (158 95.3 L/kg), indicating a significant tissue
distribution of doxorubicin in rats (Jacquet et al., 1998; Wang et al., 2006). The
coadministration of biochanin A (100 mg/kg, ip) had no significant effects on the
pharmacokinetics of doxorubicin (Table 1). One explanation for this lack of interaction
in vivo might be due to the rapid and extensive metabolism of biochanin A into
glucuronidate/sulfate conjugates (Peterson et al., 1998; Moon et al., 2006a) in the
intestine and liver.
The interaction between biochanin A with cyclosporine A, an immunosuppressant with a

narrow therapeutic index, was also investigated in our study. As shown in Figure 4, in
the control rats, the plasma concentrations of cyclosporine A decreased rapidly at early
times followed by a slower elimination phase. Cyclosporine A had a long terminal halflife t1/2 (6.92 0.56 hr) and a large steady-state volume of distribution Vss (5.66 1.90
L/kg), indicating a significant tissue distribution of cyclosporine A in rats. This is
consistent with the highly lipophilic nature of cyclosporine A (Log P = 4.3) (Cheng et al.,
2006). The oral administration of biochanin A (250 mg/kg) increased the AUC of
219
cyclosporine A (3 mg/kg, iv), but this was not statistically significant. All other
pharmacokinetic parameters were similar to those in the control group (Table 2). The
lack of interaction following administration of biochanin A (po) and cyclosporine A (iv)
is likely due to the low bioavailability of biochanin A. In a previous study, the
bioavailability of biochanin A was shown to be poor (1.2 % at a 50 mg/kg dose) (Moon et
al., 2006a). Consistent with this, the plasma concentrations of biochanin A following a
250 mg/kg oral dose were in the 10-100 ng/ml range (equivalent to 0.035-0.35 M)
(Figure 5). Given the in vitro inhibition potency of biochanin A at high micromolar
range (Ki ~ 30 M in inhibiting P-gp-mediated efflux of digoxin in Caco-2 cells) (Zhang
and Morris, 2003b; Zhang and Morris, 2003a), it is not surprising that oral administration
of biochanin A had minimal effects on P-gp-mediated systemic elimination of
cyclosporine A. Interestingly, oral coadministration of biochanin A also had no
significant effects on the pharmacokinetics of cyclosporine A (10 mg/kg, po). Following
the oral dose of biochanin A at a 250 mg/kg dose, high intestinal concentrations of
biochanin A, in the mM range, would be expected; these concentrations are likely
sufficient to inhibit intestinal P-gp activity. In a recent study, the oral administration of
biochanin A at the dose of 50 mg/kg significantly increased the bioavailability of P-gp
substrates paclitaxel and digoxin in rats (Peng et al., 2006). The mechanisms for the
observed different results remain unknown. However, similar results have been reported
in the quercetin interaction studies. In a previous clinical study, oral administration of
quercetin (5 mg/kg in capsule) significantly increased the AUC of cyclosporine (300 mg
per subject) by 36% (Choi et al., 2004a). In contrast, the oral administration of quercetin
(50 mg/kg in glycofurol) to pigs and rats resulted in significant decreases in the AUC of
220
cyclosporine (10 mg/kg) by 56% and 43%, respectively (Hsiu et al., 2002). The exact
reasons for these different results from different studies are not clear, but it might be due
to the species differences, the dosing vehicle, the methods of administration (concomitant
or separate administration), and the doses used. At a high dose, biochanin A is likely
present as an emulsion, which might result in the adsorption of cyclosporine A, resulting
in a lower bioavailability. An alternative explanation for this might be due to the
involvement of other drug transporters or metabolizing enzymes in the disposition of
cyclosporine A. Many flavonoids have been shown to modulate the CYP P450 system
(Moon et al., 2006b) and cyclosporine A has also been reported to be a CYP3A substrate
(van Herwaarden et al., 2005). Further studies are needed to understand the differences
reported for in vivo flavonoid-drug interactions for P-gp substrates.
In summary, our results demonstrated that the flavonoid biochanin A significantly

increased [3H]DNM accumulation in MCF-7/ADR cells, suggesting it is a P-gp inhibitor.
Different from the in vitro results, biochanin A at doses of 100 mg/kg (ip) or 250 mg/kg
(po) did not significantly alter the pharmacokinetics of the P-gp substrates doxorubicin
and cyclosporine A. The disconnect between the in vitro and in vivo data suggests that Pgp interactions mediated by biochanin A may be limited due to its poor bioavailability
and rapid clearance, or due to the significance of permeability properties of doxorubicin
and cyclosporine A, or the involvement of other transporters or metabolizing enzymes in
the disposition of these P-gp substrates.
221
REFERENCES:
Arnold RD, Slack JE and Straubinger RM (2004) Quantification of Doxorubicin and

metabolites in rat plasma and small volume tissue samples by liquid
chromatography/electrospray tandem mass spectroscopy. J Chromatogr B Analyt
Technol Biomed Life Sci 808:141-152.
Atkinson C, Compston JE, Day NE, Dowsett M and Bingham SA (2004) The effects of
phytoestrogen isoflavones on bone density in women: a double-blind,
randomized, placebo-controlled trial. Am J Clin Nutr 79:326-333.
Beauchesne PR, Chung NS and Wasan KM (2007) Cyclosporine A: a review of current
oral and intravenous delivery systems. Drug Dev Ind Pharm 33:211-220.
Booth NL, Piersen CE, Banuvar S, Geller SE, Shulman LP and Farnsworth NR (2006)
Clinical studies of red clover (Trifolium pratense) dietary supplements in
menopause: a literature review. Menopause 13:251-264.
Camaggi CM, Comparsi R, Strocchi E, Testoni F, Angelelli B and Pannuti F (1988)
Epirubicin and doxorubicin comparative metabolism and pharmacokinetics. A
cross-over study. Cancer Chemother Pharmacol 21:221-228.
Cheng WP, Gray AI, Tetley L, Hang Tle B, Schatzlein AG and Uchegbu IF (2006)
Polyelectrolyte nanoparticles with high drug loading enhance the oral uptake of
hydrophobic compounds. Biomacromolecules 7:1509-1520.
Choi JS, Choi BC and Choi KE (2004a) Effect of quercetin on the pharmacokinetics of
oral cyclosporine. Am J Health Syst Pharm 61:2406-2409.
Choi JS, Choi HK and Shin SC (2004b) Enhanced bioavailability of paclitaxel after oral
Choi JS, Jo BW and Kim YC (2004c) Enhanced paclitaxel bioavailability after oral
administration of paclitaxel or prodrug to rats pretreated with quercetin. Eur J
Pharm Biopharm 57:313-318.
Davies B and Morris T (1993) Physiological parameters in laboratory animals and
humans. Pharm Res 10:1093-1095.
Delmonte P and Rader JI (2006) Analysis of isoflavones in foods and dietary
supplements. J AOAC Int 89:1138-1146.
Endres CJ, Hsiao P, Chung FS and Unadkat JD (2006) The role of transporters in drug
interactions. Eur J Pharm Sci 27:501-517.
Gabizon A, Shmeeda H and Barenholz Y (2003) Pharmacokinetics of pegylated
liposomal Doxorubicin: review of animal and human studies. Clin Pharmacokinet
42:419-436.
Gotoh T, Yamada K, Yin H, Ito A, Kataoka T and Dohi K (1998) Chemoprevention of
N-nitroso-N-methylurea-induced rat mammary carcinogenesis by soy foods or
biochanin A. Jpn J Cancer Res 89:137-142.
Howes JB, Sullivan D, Lai N, Nestel P, Pomeroy S, West L, Eden JA and Howes LG
(2000) The effects of dietary supplementation with isoflavones from red clover on
the lipoprotein profiles of post menopausal women with mild to moderate
hypercholesterolaemia. Atherosclerosis 152:143-147.
222
Hsiu SL, Hou YC, Wang YH, Tsao CW, Su SF and Chao PD (2002) Quercetin
72:227-235.
Jacquet P, Averbach A, Stuart OA, Chang D and Sugarbaker PH (1998) Hyperthermic
intraperitoneal doxorubicin: pharmacokinetics, metabolism, and tissue distribution
in a rat model. Cancer Chemother Pharmacol 41:147-154.
Lee YS, Seo JS, Chung HT and Jang JJ (1991) Inhibitory effects of biochanin A on
mouse lung tumor induced by benzo(a)pyrene. J Korean Med Sci 6:325-328.
Minotti G, Menna P, Salvatorelli E, Cairo G and Gianni L (2004) Anthracyclines:
molecular advances and pharmacologic developments in antitumor activity and
cardiotoxicity. Pharmacol Rev 56:185-229.
Moon YJ, Sagawa K, Frederick K, Zhang S and Morris ME (2006a) Pharmacokinetics
and bioavailability of the isoflavone biochanin A in rats. Aaps J 8:E433-442.
Moon YJ, Wang X and Morris ME (2006b) Dietary flavonoids: effects on xenobiotic and
carcinogen metabolism. Toxicol In Vitro 20:187-210.
Peng SX, Ritchie DM, Cousineau M, Danser E, Dewire R and Floden J (2006) Altered
oral bioavailability and pharmacokinetics of P-glycoprotein substrates by
coadministration of biochanin A. J Pharm Sci 95:1984-1993.
Peterson TG, Ji GP, Kirk M, Coward L, Falany CN and Barnes S (1998) Metabolism of
the isoflavones genistein and biochanin A in human breast cancer cell lines. Am J
Clin Nutr 68:1505S-1511S.
Rice L, Samedi VG, Medrano TA, Sweeney CA, Baker HV, Stenstrom A, Furman J and
Shiverick KT (2002) Mechanisms of the growth inhibitory effects of the
isoflavonoid biochanin A on LNCaP cells and xenografts. Prostate 52:201-212.
Risbridger GP, Wang H, Frydenberg M and Husband A (2001) The in vivo effect of red
clover diet on ventral prostate growth in adult male mice. Reprod Fertil Dev
13:325-329.
Robert J (1988) [Pharmacokinetics of new anthracyclines]. Bull Cancer 75:167-174.
Robert J and Gianni L (1993) Pharmacokinetics and metabolism of anthracyclines.
Cancer Surv 17:219-252.
22:2489-2503.
Tseng E, Kamath A and Morris ME (2002) Effect of organic isothiocyanates on the Pglycoprotein- and MRP1-mediated transport of daunomycin and vinblastine.
Pharm Res 19:1509-1515.
Tsuji A, Tamai I, Sakata A, Tenda Y and Terasaki T (1993) Restricted transport of
cyclosporin A across the blood-brain barrier by a multidrug transporter, Pglycoprotein. Biochem Pharmacol 46:1096-1099.
van Herwaarden AE, Smit JW, Sparidans RW, Wagenaar E, van der Kruijssen CM,
Schellens JH, Beijnen JH and Schinkel AH (2005) Midazolam and cyclosporin a
metabolism in transgenic mice with liver-specific expression of human CYP3A4.
223
Wang JC, Liu XY, Lu WL, Chang A, Zhang Q, Goh BC and Lee HS (2006)
Pharmacokinetics of intravenously administered stealth liposomal doxorubicin
modulated with verapamil in rats. Eur J Pharm Biopharm 62:44-51.
Zhang S and Morris ME (2003a) Effect of the flavonoids biochanin A and silymarin on
the P-glycoprotein-mediated transport of digoxin and vinblastine in human
intestinal Caco-2 cells. Pharm Res 20:1184-1191.
Zhang S and Morris ME (2003b) Effects of the flavonoids biochanin A, morin, phloretin,
and silymarin on P-glycoprotein-mediated transport. J Pharmacol Exp Ther
304:1258-1267.
224
Table 1. Summary of the pharmacokinetic parameters of doxorubicin

Doxorubicin (7.5 mg/kg, iv)
Parameters
Control
Biochanin A, 100 mg/kg, ip
(n = 7)
(n = 6)
AUC (gml-1min)
152 69.6
184 86.5
t1/2 (hr)
49.7 15.6
45.6 15.4
CL (mlmin-1kg-1)
43.9 16.3
40.3 18.5
Vss (L/kg)
158 95.3
118 86.2
The pharmacokinetic parameters were obtained by noncompartmental analysis using

WinNonlin. Data were presented as mean SD. The number of animals for each group
was specified in parentheses after the corresponding group names.
225
Table 2. Summary of the pharmacokinetic parameters of cyclosporine A

Cyclosporine A (3 mg/kg, iv)
Cyclosporine A (10 mg/kg, po)
Control
(n = 3)
Biochanin A
250 mg/kg, po
(n = 3)
Control
(n = 3)
Biochanin A
250 mg/kg, po
(n = 3)
AUC (ngml-1hr)
4960 983
7050 1080
2280 801
2290 375
t1/2 (hr)
6.92 0.56
9.68 1.69
10.5 4.84
6.29 1.01
CL or CL/F (mlhr-1kg-1)
619 113
432 66.3
4780 1740
4440 683
Vss (L/kg)
5.66 1.90
4.91 1.02
Cmax (ng/ml)
208 106
251 112
tmax (hr)
4.33 0.58
4.00 2.00
F (%)
13.8 4.80
13.8 2.30
Parameters
The pharmacokinetic parameters were obtained by noncompartmental analysis using WinNonlin. Data were
presented as mean SD. The number of animals for each group was specified in parentheses after the
corresponding group names.
226
FIGURE LEGEND:
Figure 1. The chemical structures of biochanin A, doxorubicin, and cyclosporine A.
Figure 2. Effect of flavonoids on [3H]DNM accumulation in MCF-7/ADR cells. The

accumulation of [3H]daunomycin (0.05M) in MCF-7/ADR cells was determined at the
end of a 2-hour incubation with flavonoids (100 M). Verapamil is a P-gp inhibitor and
was included as a positive control. The results were normalized by protein concentration
and are presented as mean SD (n 6). *, p < 0.05, **, p < 0.01, compared with the
accumulation in the control group.
Figure 3. Plasma concentration versus time profiles of doxorubicin after an intravenous

bolus (7.5 mg/kg), 5 min following the intraperitoneal administration of control vehicle
(z) or biochanin A (100 mg/kg, {). Plasma levels of doxorubicin were determined by
LC-MS/MS. The data are presented as mean SD, n = 6-7 for each group.
Figure 4. (A) Plasma concentration versus time profiles of cyclosporine A after an

intravenous bolus (3 mg/kg), 1 hour following the oral administration of control vehicle
(z) or biochanin A (250 mg/kg, {). (B) Plasma concentration versus time profiles of
cyclosporine A after oral coadministration of cyclosporine A (10 mg/kg) with control
vehicle (z) or biochanin A (250 mg/kg, {). Plasma levels of cyclosporine A were
determined by LC-MS/MS. The data are presented as mean SD, n = 3 for each group.
227
Figure 5. Plasma concentration versus time profiles of biochanin A following the oral
administration of a dose of 250 mg/kg. The data are presented as mean SD, n = 4.
228
229
230
231
232
233
CHAPTER 8
CONCLUSIONS
234
Flavonoids are a large class of polyphenolic compounds rich in our common diet and in
many herbal products marketed as the over-the-counter medicines. Due to the popular
use of flavonoid-containing products by the public for disease prevention and health
promotion, flavonoid-drug interactions have been increasingly reported. However, our
current understanding of the mechanisms underlying most of these interactions are
related to the modulation of drug metabolizing enzymes. Recently, drug transporters
have been also recognized as important determinants governing the disposition of
xenobiotics. In order to better understand and predict the potential pharmacokinetic
interactions of flavonoids with conventional medicines, the interactions of flavonoids
with drug transporters important in drug disposition were investigated in this dissertation.
The transporters of interest in this dissertation include the uptake transporters OATP1B1
and MCT1, and the efflux transporters BCRP and P-glycoprotein.
Recent in vitro and in vivo fruit juice and fexofenadine interaction studies have indicated
that fruit juices and constituents preferentially inhibit OATPs rather than P-glycoprotein
(Dresser et al., 2002; Kamath et al., 2005). Several flavonoids (quercetin, naringenin and
hesperitin) and their glycosides, at a concentration of 50 M, significantly inhibit
fexofenadine uptake mediated by rat Oatp1a5 (Oatp3) (Dresser et al., 2002), suggesting
that flavonoids could interact with some OATP isoforms. In our studies, many of the
tested flavonoids significantly inhibited the uptake of OATP1B1 substrate
[3H]dehydroepiandrosterone sulfate (DHEAS) in a concentration-dependent manner in
HeLa OATP1B1-expressing cells. Among all the flavonoids tested, biochanin A was
235
shown to be one of the most potent inhibitors with an IC50 of 11.3 3.22 M. Moreover,
flavonoids and their glycosides exhibited distinct effects on [3H]DHEAS uptake. From
the studies with biochanin A, investigating the possible mechanism(s) of this interaction,
we have found that biochanin A is not a substrate for OATP1B1. A kinetic study
revealed that biochanin A inhibited [3H]DHEAS uptake in a noncompetitive manner with
a Ki of 10.2 1.89 M. Taken together, these results indicate that flavonoids are a
novel class of OATP1B1 modulators, suggesting the potential for diet-drug interactions.
The Morris laboratory has previously reported that the flavonoid luteolin is a potent
MCT1 inhibitor in vitro, inhibiting the uptake of GHB with an IC50 of 0.41 M in rat
MCT1-transfected MDA-MB231 cells (Wang and Morris, 2007). We characterized the
effects of luteolin on GHB pharmacokinetics and pharmacodynamics in rats, and
investigated the mechanism of the interaction using model-fitting methods. Luteolin
significantly decreased the plasma concentration and AUC, and increased the total and
renal clearances of GHB in a dose-dependent manner. Moreover, luteolin significantly
shortened the duration of GHB-induced sleep in rats. A pharmacokinetic model
containing capacity-limited renal reabsorption and metabolic clearance was constructed
to characterize the in vivo interaction and revealed that luteolin significantly altered the
pharmacokinetics of GHB by uncompetitively inhibiting its MCT1-mediated transport,
with an inhibition constant of 1.1 M. This study represents the first in vivo investigation
of a flavonoid as a MCT inhibitor, and reports that luteolin is a potent MCT inhibitor
236
both in vitro and in vivo. Our findings may provide a potential clinical detoxification
strategy to treat GHB overdoses.
The flavonoid chrysin is a potent inhibitor of BCRP, inhibiting the efflux of mitoxantrone
with an IC50 of 0.39 M in BCRP-overexpressing human MCF-7 breast cancer cells
(Zhang et al., 2004b; Zhang et al., 2004a). We further characterized the pharmacokinetic
interaction between chrysin and the BCRP substrate nitrofurantoin in rats. In MDCK
cells expressing human BCRP or murine Bcrp1, the polarized transport of nitrofurantoin
was effectively inhibited by chrysin, suggesting that chrysin is an inhibitor of both human
and murine BCRP. Oral and intravenous coadministration of chrysin significantly
increased the AUC of nitrofurantoin. Moreover, the cumulative hepatobiliary excretion
of nitrofurantoin was significantly decreased by approximately 75% at the end of 120
min following the coadministration of chrysin. Taken together, these results indicate that
the flavonoid chrysin significantly inhibits nitrofurantoin transport mediated by human
BCRP and murine Bcrp1. Bcrp1 inhibition by chrysin represents one mechanism for the
observed chrysin-nitrofurantoin pharmacokinetic interaction in rats.
In the chrysin-nitrofurantoin interaction study, chrysin demonstrated significant effects

on nitrofurantoin pharmacokinetics at an oral dose of 200 mg/kg, but not at a lower dose
of 50 mg/kg. This might be possibly due to the extensive metabolism of chrysin in the
intestine, as a result of enzymatic conjugation reactions occurring at the hydroxyl groups
(Galijatovic et al., 1999). Compared with chrysin, 7,8-benzoflavone is a flavonoid with
237
more potent BCRP inhibition activity and with a more stable chemical structure (Zhang
et al., 2004a). Therefore, we further characterized the interaction between 7,8benzoflavone and nitrofurantoin. At a 50 mg/kg oral dose, 7,8-benzoflavone significantly
increased the AUC of nitrofurantoin, indicating the potential for BCRP-mediated drug
interactions. To better understand this in vivo interaction, the pharmacokinetics of 7,8benzoflavone was characterized following its oral and intravenous administration. A
pharmacokinetic model with linear absorption, capacity-limited intestinal metabolism and
capacity-limited elimination best described the pharmacokinetic profiles of 7,8benzoflavone in rats. The clearance was dose-dependent, likely due to the saturation of
metabolism and elimination pathways. Consistent with the compounds lipophilic nature,
the Vss of 7,8-benzoflavone was large and oral absorption was rapid. The bioavailability
of 7,8-benzoflavone was low (0.61 % ~ 13.2 %) and dose-dependent, possibly due to its
limited solubility and first-pass metabolism.
To better understand the BCRP inhibition mechanisms by flavonoids, biochanin A and

kaempferol were used as two model compounds and their transport by BCRP was
investigated. In MDCK-Bcrp1 cells, polarized transport was observed for both
[3H]biochanin A and [3H]kaempferol with efflux ratios of 8.97 and 8.35, respectively.
Radio-HPLC results further indicated that Bcrp1 could efficiently transport the biochanin
A and kaempferol in their parent and/or metabolized forms. Therefore, these two
flavonoids and/or their metabolites are Bcrp1 substrates. This information will be useful
238
in characterizing the in vivo disposition of biochanin A and kaempferol, and to better

understand the potential in vivo flavonoid-drug interactions mediated by BCRP.
In vitro and in vivo studies were performed to examine the effects of the flavonoid
biochanin A on P-glycoprotein-mediated efflux. Biochanin A had significant inhibitory
effects in vitro (IC50 in Caco-2 cells of 30 M) but had minimal effects in vivo on the
pharmacokinetics of P-glycoprotein substrates doxorubicin and cyclosporine A in rats.
Further studies are needed to address this disconnect between the in vitro and in vivo
data. One possible explanation for this disconnect might be due to the poor
bioavailability and/or rapid clearance of biochanin A. Alternatively, this might be due to
high permeability of doxorubicin and cyclosporine A, or the involvement of other
transporters or metabolizing enzymes in the in vivo disposition of these P-gp substrates.
In summary, we demonstrated in this thesis dissertation that many flavonoids could

interact with both uptake and efflux transporters, resulting in significant pharmacokinetic
interactions. The new information provided in this thesis may help us better understand
the pharmacokinetics and pharmacodynamics of this important class of dietary
compounds, and help design clinically relevant therapeutic strategies.
Future Directions
In the luteolin and MCT1 interaction study, GHB and luteolin were administered by
intravenous bolus. The luteolin-GHB interaction following oral coadministration has not
239
been investigated. The expression of MCT1 has been detected in the intestine both in
humans and rats (Tamai et al., 1995; Orsenigo et al., 1999; Gill et al., 2005). Therefore,
luteolin might inhibit intestinal MCT1 and decrease GHB absorption. The oral
interaction study is more clinically relevant since GHB is administered orally to human
subjects. Given the low bioavailability of some flavonoids such as biochanin A and
chrysin, the luteolin-GHB interaction could be likely due to the local intestinal inhibition
of MCT1 by luteolin.
In our and many other studies, flavonoids have been shown to interact with both uptake
and efflux transporters. Therefore, it is interesting to investigate the synergistic, additive
or antagonistic effects of flavonoids, which have different preferentiality in modulating
uptake and efflux transporters. It is particularly important in several biological
membrane systems, such as intestine and liver, where both uptake and efflux transporters
are present.
Based on our rat studies, it is important to translate these results to humans. However,
several important questions need to be addressed for clinically relevant applications. For
example, are the rat and human transporters similar in substrate specificity? Is the
expression of the rat and human transporters similar in major organs? Is the contribution
of transporters and metabolizing enzymes similar in rats and humans?
240
In this thesis, we studied the glycones and aglycones of some flavonoids in vitro.
However, there is little or no information of the potential role that metabolites of the
flavonoids may have on the transporters. This may be especially important for oral
dosing, the most common route of administration, since flavonoids usually have low
bioavailability with extensive first-pass metabolism, often phase II conjugation. Many of
the transporters (e.g., BCRP and MRP) have phase II metabolites as substrates, thus
interactions may be caused by the metabolites. It is important in the future studies to
explore the potential interactions of flavonoid metabolites with drug transporters both in
vitro and in vivo.
241
REFERENCES:
71:11-20.
Galijatovic A, Otake Y, Walle UK and Walle T (1999) Extensive metabolism of the
flavonoid chrysin by human Caco-2 and Hep G2 cells. Xenobiotica 29:12411256.
Gill RK, Saksena S, Alrefai WA, Sarwar Z, Goldstein JL, Carroll RE, Ramaswamy K
and Dudeja PK (2005) Expression and membrane localization of MCT isoforms
along the length of the human intestine. Am J Physiol Cell Physiol 289:C846-852.
Kamath AV, Yao M, Zhang Y and Chong S (2005) Effect of fruit juices on the oral
bioavailability of fexofenadine in rats. J Pharm Sci 94:233-239.
Orsenigo MN, Tosco M, Bazzini C, Laforenza U and Faelli A (1999) A monocarboxylate
transporter MCT1 is located at the basolateral pole of rat jejunum. Exp Physiol
84:1033-1042.
Tamai I, Takanaga H, Maeda H, Sai Y, Ogihara T, Higashida H and Tsuji A (1995)
Participation of a proton-cotransporter, MCT1, in the intestinal transport of
monocarboxylic acids. Biochem Biophys Res Commun 214:482-489.
Wang Q and Morris ME (2007) Flavonoids modulate monocarboxylate transporter-1mediated transport of gamma-hydroxybutyrate in vitro and in vivo. Drug Metab
Dispos 35:201-208.
Zhang S, Yang X and Morris ME (2004a) Combined effects of multiple flavonoids on
breast cancer resistance protein (ABCG2)-mediated transport. Pharm Res
21:1263-1273.
Zhang S, Yang X and Morris ME (2004b) Flavonoids are inhibitors of breast cancer
242
APPENDIX
Diet/nutrient Interactions with Drug Transporters
This is a review article and is being published as a chapter in the book entitled Drug
Transporters. Molecular Characterization and Role in Drug Disposition. Editors: G. You
and M.E. Morris; August 2007; John Wiley & Sons, Inc.
243
Table of Contents
22.1. Introduction
22.2. Diet/nutrient Interactions with Drug Transporters
22.2.1. Interactions of Diet/Dietary Supplements with Drug Transporters
22.2.2.Interactions of Flavonoids with Drug Transporters
22.2.3.Interactions of Organic Isothiocyanates with Drug Transporters
22.3. Conclusions
References
244
Abstract
Dietary effects on drug pharmacokinetics have been long recognized. A number of
mechanisms are responsible for food-drug interactions, including delayed gastric
emptying, alterations in intestinal motility, gastric pH and hepatic blood flow, and
modulation of drug-metabolizing enzymes by dietary components. Recently, food-drug
interactions involving drug transporters have also been identified. Given the growing use
of dietary supplements and significant expression levels of drug transporters in organs
important in drug absorption, distribution, and elimination, it has been increasingly
recognized that diet may have a significant impact on drug disposition via interacting
with drug transporters. In this review, we discuss the interactions of diet/dietary
supplements (e.g., grapefruit juice, St. Johns wort, garlic, green tea, ginseng, milk thistle,
and kava) and some classes of nutrients rich in fruits and vegetables (e.g., flavonoids and
isothiocyanates) with drug transporters. We focus primarily on three major efflux
transporter families (namely P-glycoprotein (P-gp), multidrug resistance-associated
proteins (MRPs), and breast cancer resistance protein (BCRP)) and one family of uptake
transporters (namely organic anion transporting polypeptide (OATP)). In addition, in
vivo diet/nutrient interactions with drug transporters observed in pre-clinical or clinical

studies are discussed.
245
22.1. Introduction
Dietary effects on drug pharmacokinetics have been well documented 1,2. A
number of mechanisms are responsible for food-drug interactions, including alterations in
physiological conditions (e.g., gastric pH, gastric emptying, intestinal motility, and
hepatic blood or bile flow rate), complexation of drugs with dietary components, and
modulation of drug-metabolizing enzymes by dietary constituents 3,4. Over the past
decade, numerous food-drug interaction studies have focused primarily on the effects of
diet on drug-metabolizing enzymes. Alterations in activities of drug-metabolizing
enzymes can subsequently change the pharmacokinetics of drugs that are substrates of
these enzymes 4-6. Foods that contain complex mixtures of phytochemicals, such as
fruits, vegetables, herbs, and teas, have great potential to modulate the activities of both
phase I and phase II enzymes 4. Many clinically significant metabolic food-drug
interactions have been reported, as exemplified by the interactions between cytochrome
P450 (CYP) enzymes and diet/dietary supplements, such as grapefruit juice and St.
Johns wort 4,6,7. In recent years, great advances have been made in elucidating the roles
of drug transporters in drug disposition 8-10. It is now increasingly recognized that drug
transporters can also significantly contribute to food-drug interactions.
Drug transporters can be generally classified into two major groups efflux and
uptake transporters. Most efflux transporters belong to a superfamily of ATP-binding
cassette (ABC) transporters, which utilize energy derived from ATP hydrolysis to export
substrates out of cells against a concentration gradient. Some major members of this
group of transporters include P-glycoprotein (MDR1, ABCB1), multidrug resistance-
246
associated proteins (MRPs, ABCC) and breast cancer resistance protein (BCRP, ABCG2)
11
. In contrast, uptake transporters mediate the translocation of drugs into cells. Included
in this group of transporters are the organic anion transporting polypeptide (OATP,
SLCO) family, the organic anion transporter (OAT, SLC22A) family, and the organic
cation transporter (OCT, SLC22A) family 12-14. Many of these efflux and uptake
transporters are expressed in various major organs, such as intestine, liver, kidney, bloodbrain barrier, and placenta, suggesting their essential roles in governing the oral
absorption, intestinal, hepatobiliary and renal excretion of a variety of endogenous and
exogenous compounds 9-11,15-18. In addition, overexpression of efflux transporters (e.g.,
P-gp, MRP, and BCRP) in tumor cells limits intracellular accumulation of a broad range
of structurally and functionally unrelated anticancer agents and leads to inefficient cell
killing, a phenomenon known as multidrug resistance (MDR), which remains the primary
obstacle to successful cancer chemotherapy 18-20. The transport activities of these efflux
transporters have been shown to confer cellular resistance to many widely-used and
clinically important anticancer agents, such as anthracyclines, vinca alkaloids,
camptothecin derivatives, palitaxel, and mitoxantrone 18,21. Due to the important roles of
these transporters in drug disposition and cancer therapy, modulation of these drug
transporters by inhibitors or inducers may significantly alter the pharmacokinetics and
therapeutic efficacy of many anticancer drugs, resulting in beneficial or adverse drugdrug interactions. The importance of transporter-mediated drug interactions is being
increasingly recognized and has been indicated in a number of pre-clinical and clinical
studies 22-27.
247
In recent years, dietary supplements (e.g., St. Johns wort, ginkgo, garlic, ginseng)
have been widely used in Western countries due to an increased public interest in
alternative medicine and disease prevention 7,28,29. Emerging evidence to date has
suggested that many of these dietary supplements, as well as nutrients contained in these
dietary supplements (e.g., flavonoids), are potent inhibitors or inducers of several major
efflux and uptake drug transporters 30-35. In addition, significant or even life-threatening
pharmacokinetic interactions between diet/nutrients and drug transporters have been
observed in a number of animal and clinical studies 25,37,38. Given the growing
consumption of dietary supplements and the essential roles of drug transporters in drug
disposition, it is important to understand the interactions between diet/nutrients with drug
transporters, as well as the potential clinical consequences of these interactions. In this
review, we focus on the interactions of diet/nutrients with drug transporters arising from
in vitro and in vivo studies, with specific emphasis on several major efflux transporters
(e.g., P-gp, MRP, and BCRP) and uptake transporter (e.g., OATP).
22.2. Diet/nutrient Interactions with Drug Transporters

22.2.1. Interactions of Diet/Dietary Supplements with Drug Transporters
Dietary supplements are products (other than tobacco) intended to supplement the
diet. Many top-selling herbal products, such as St. Johns wort, garlic, green tea,
ginseng, and milk thistle, are classified as dietary supplements 29,39. Since their
marketing does not require FDA approval, the potential interactions of these herbal
products with conventional drugs, in general, have not been carefully evaluated, raising a
248
serious concern about the safety of using these products. Recent in vitro and in vivo
studies have shown that many diet/dietary supplements can modulate the activities of
both efflux and uptake transporters and alter the pharmacokinetics of various therapeutic
agents, resulting in clinically important drug interactions (Table 1).
St. Johns wort
St. John's wort is widely used in the treatment of depression as an over-thecounter herbal medicine 40. As a complex mixture, St. John's wort contains multiple
constituents such as hypericin, pseudohypericin, hyperforin and the flavonoids quercetin
and its methylated form isorhamnetin 41,42. A large number of clinically relevant St.
John's wort-drug interactions have been reported, many of which are drug transportermediated interactions..
A) Effects on Drug Transporters
Several studies have shown that St. John's wort induced P-glycoprotein (P-gp)
both in vitro and in vivo. In LS-180 intestinal carcinoma cells, P-gp expression was
significantly induced after 3 days of exposure to St. John's wort (a 4-fold increase at 300
g/ml) or hypericin (a 7-fold increase at 3 M) in a dose-dependent fashion. The

induction of P-gp in LS-180 cells resulted in enhanced efflux and decreased accumulation
of rhodamine-123, a fluorescent molecule which acts as a probe of P-gp 43. In an in vivo
induction study, 14 days of administration of St. John's wort significantly increased
intestinal P-gp level by 3.8-fold in rats and increased duodenal P-gp level by 1.4-fold in
humans 31. Similarly, chronic treatment with St. John's wort (3 600 mg per day for 16
days) caused a 4.2-fold increase in P-gp expression level in the peripheral blood
249
lymphocytes from healthy volunteers, resulting in reduced accumulation of rhodamine123 44. The underlying mechanism of P-gp induction by St. Johns wort is presumably
due to the activation of pregnane X receptor (PXR), a nuclear receptor that regulates P-gp
expression. It has been shown that hyperforin, a major constituent of St. John's wort, is a
potent agonist for PXR with a Ki value of 27 nM 45,46.
In addition to altering P-gp expression, St. Johns wort was also shown to interact
with P-gp via modulating P-gp-mediated efflux. Using calcein-AM as a fluorescent
marker of P-gp, Weber et al. 47 reported that St. Johns wort extracts, as well as some
constituents such as quercetin and hyperforin, potently modulated the transport by P-gp
in VLB cells (a human lymphocytic leukemia cell line expressing P-gp) and in PBCEC
cells (porcine brain capillary endothelial cells). In Caco-2 cells or canine kidney cells
stably expressing P-gp (MDCK-MDR1), hypericin and quercetin significantly inhibited
P-gp-mediated efflux of ritonavir, resulting in increased intracellular uptake or a
decreased basal-to-apical/apical-to-basal transport ratio of ritonavir 48. Similarly, Wang
et al. 49 showed that hyperforin and hypericin significantly inhibited P-gp activity with
IC50 values of approximately 30 M by using daunorubicin and calcein-AM as
fluorescent substrates.
Recently, several studies suggested that St. Johns wort could also induce MRP2
expression both in vitro and in vivo. In human heptocellular carcinoma HepG2 cells, a
24-hr exposure of St. John's wort or hyperforin to HepG2 cells resulted in 1.55 ~ 1.72fold increases in MRP2 mRNA levels 50. In rats, when St. Johns wort was given at a
dose of 400 mg/kg/day for 10 days, the amount of MRP2 in the liver was significantly
250
increased to 304% of control. The increase in MRP2 was maximal at 10 days after St.
Johns wort treatment and lasted for at least 30 days 51.
B) In Vivo Drug Interactions
In a single-blind, placebo-controlled parallel study, it was shown that the area
under the plasma concentration-time curve (AUC) and peak plasma concentration (Cmax)
of digoxin (a well-known P-gp substrate 52) were decreased by 25% and 26%,
respectively, after the ingestion of St. Johns wort for 10 days (3 300 mg per day) by
healthy volunteers 24. Similarly, administration of St. Johns wort to 8 healthy male
volunteers at a dose of 300 mg 3 times per day for 2 weeks decreased the bioavailability
of digoxin 31. In another study, co-administration of the hyperforin-rich extract to healthy
volunteers for 14 days significantly reduced the AUC and Cmax of digoxin by 24.8% and
37%, respectively 53. Given the fact that digoxin undergoes limited metabolic
transformation, it is likely that induction of P-gp might be the underlying mechanism
responsible for the altered pharmacokinetic profiles of digoxin 24,54.
Interestingly, the effects of St. Johns wort on P-gp activity appear to be exposure
duration-dependent. Using fexofenadine, a P-gp substrate with minimal metabolic
transformation 55,56, Wang et al. 57 reported that a single oral dose of St. Johns wort (900
mg) to healthy volunteers significantly increased the Cmax of fexofenadine by 45% and
significantly decreased the oral clearance by 20%, indicating an inhibition of intestinal Pgp. However, long-term administration of St. Johns wort (3 300 mg per day for 14
days) reversed the changes in fexofenadine disposition observed with single-dose
administration. The mechanism underlying this biphasic effect of St. Johns wort is
251
possibly due to the initial inhibition of P-gp after acute exposure, followed by a
significant induction of intestinal P-gp after long-term use 58.
In addition, co-administration of St. Johns wort (3 300 mg per day for 14 days)
significantly decreased indinavir (a protease inhibitor) AUC by 54% and the plasma
trough concentration (Ctrough) by 81% in healthy subjects 59. Moreover, significant
reductions in AUC and plasma concentration of cyclosporine, an immunosuppressant,
were observed in renal transplant patients after prolonged administration of St. Johns
wort 60,61. It was suggested in several case studies that the decreased cyclosporine
concentration during treatment with St. Johns wort was the possible cause of acute heart
and kidney transplant rejection 62,63. A similar pharmacokinetic interaction was observed
between St. Johns wort and another immunosuppressant, tacrolimus. Co-administration
of St. Johns wort (3 300 mg per day for 18 days) to 10 healthy volunteers significantly
decreased tacrolimus AUC and increased its apparent oral clearance 64. Since indinavir,
cyclosporine, and tacrolimus are dual substrates for both P-gp and CYP3A4, it is likely
that these interactions might involve induction of P-gp or CYP3A4, or both 44,64,65.
Grapefruit Juice
The interaction of grapefruit juice with some drugs was accidentally discovered
when grapefruit juice was used to mask the taste of ethanol in a study using the calcium
channel blocker felodipine. Co-administration of grapefruit juice significantly increased
the bioavailability of felodipine by three-fold 66. Since then, a number of studies have
shown that grapefruit juice can enhance the bioavailability of many clinically important
drugs 67. The predominant mechanism for grapefruit juice-drug interactions appears to be
252
the inhibition of CYP3A4 in the small intestine, resulting in reduced pre-systemic

metabolism and enhanced drug oral bioavailability 68. Another important mechanism of
grapefruit juice-drug interactions, as revealed in many recent studies, is through the
modulation of drug transporters, especially P-gp and OATP 25,69-75.
A) Effects on Drug Transporters
The major constituents in grapefruit juice include flavonoids (e.g., naringin,
naringenin, quercetin and kaempferol) and furanocoumarins (e.g., bergamottin and 6, 7dihydroxybergamottin) . Many studies have shown that both grapefruit juice and its
major constituents can modulate the activities of several drug transporters.
An early report of the grapefruit juice-drug interaction indicated that grapefruit
juice activated the transport of several P-gp substrates across MDCK-MDR1 cells 76.
However, the majority of the more recent studies have indicated that grapefruit juice has
an inhibitory activity on P-gp-mediated transport. In Caco-2 cells using talinolol and
digoxin as specific, yet metabolically stable, P-gp substrates, the apical-to-basolateral
(absorptive) transport of talinolol and digoxin was significantly increased 72,74. Similarly,
in Caco-2 cells and a rat everted sac model, grapefruit juice extracts or some major
components such as naringin and 6, 7-dihydroxybergamottin significantly decreased the
efflux or increased intracellular accumulation of several P-gp substrates, such as
rhodamine-123, fexofenadine, and saquinavir 73,75,77,78. Using vinblastine as a P-gp
substrate, a number of studies indicated that several extracts of grapefruit juice inhibited
the activity of P-gp in Caco-2 cells with different potency. The ethyl acetate extracts of
grapefruit juice, followed by diethyl ethyl and methylene chloride extracts, exhibited the
253
greatest potency in increasing the permeability coefficient of the apical-to-basolateral

transport of vinblastine 71,79.
In addition to modulating P-gp activity, grapefruit juice has been shown to
interact with P-gp via other mechanisms. Using an ATP-hydrolysis assay, Wang et al. 70
reported that bergamottin significantly increased ATP hydrolysis by approximately 2.3fold with a Km value of 8 M, suggesting that grapefruit juice components might also
modulate P-gp ATPase activity. In an in vitro study, exposure of grapefruit juice as well
as kaempferol and naringenin to human proximal tubular HK-2 cells for four days
significantly decreased P-gp mRNA and protein levels in a dose-dependent manner 80.
Recently, it was shown that grapefruit juice could also interact with other drug
transporters. In human MRP2 transfected porcine kidney epithelial cells (LLC-MRP2),
ethyl acetate extracts of grapefruit juice or its components (bergamottin and 6, 7dihydroxybergamottin) significantly inhibited MRP-2-mediated transport of vinblastine
and saquinavir 75. Interestingly, recent interaction studies on grapefruit juice and
fexofenadine (a substrate for both P-gp and human OATP or rat oatp 55) indicated that
grapefruit juice preferentially inhibited human OATP and rat oatp rather than P-gp in cell
culture studies at a concentration of 5% of normal strength. Several constituents,
including bergamottin, 6, 7-dihydroxybergamottin, naringenin and hesperidin, at a
concentration of 50 M, significantly inhibited fexofenadine uptake mediated by rat
Oatp3. Moreover, 6, 7-dihydroxybergamottin potently inhibited rat oatp1 with an IC50
value of 0.28 M, whereas no significant inhibitory effect on P-gp was observed 25.
Moreover, in human embryonic kidney 293 cells stably expressing OATP-B, grapefruit
254
juice at a concentration of 5% significantly inhibited OATP-B-mediated uptake of

estrone-3-sulfate by 82%. Major grapefruit juice constituents, including naringin,
naringenin, quercetin, bergamottin, and 6, 7-dihydroxybergamottin, at a concentration
of 10 M, also significantly inhibited OATP-B-mediated uptake of estrone-3-sulfate by
39%, 28%, 21%, 60%, and 43%, respectively 81.
B) In Vivo Drug Interactions
Several studies have indicated that grapefruit juice can produce significant
increases in the AUC (45 ~ 60%) and Cmax (35 ~ 43%) of cyclosporine in healthy subjects
82-84
. Compared with CYP3A4, intestinal P-gp was suggested to be a more important
determinant of cyclosporine bioavailability, presumably by being a rate-limiting step in

drug absorption 85.
In rats, grapefruit juice caused a nearly 2-fold increase in Cmax and more than a
35% increase in AUC of talinolol, indicating an important role of P-gp in talinolol
disposition 72. In a recent clinical study, however, intake of grapefruit juice significantly
decreased the AUC, Cmax, and urinary excretion of talinolol to 56%, 57%, 56%,
respectively 86. One possible explanation for this discrepancy is the difference in
experimental protocols (adjusted and un-adjusted pH for grapefruit juice) since talinolol
has a pH-dependent intermediate lipid solubility and low water solubility. Another
explanation suggested by the authors is that other intestinal uptake transporters might be
responsible for talinolol uptake and grapefruit juice may have species-different effects on
these transporters 86.
255
On the other hand, inhibition of P-gp by grapefruit juice had no significant effects
on digoxin pharmacokinetics. In two clinical studies, grapefruit juice did not affect AUC,
Cmax, elimination half-life, or renal clearance of digoxin, although it significantly
decreased the digoxin absorption rate constant 87,88. The lack of in vivo effect of
grapefruit juice on digoxin is probably due to the fact that digoxin has a high
bioavailability after oral administration, and therefore P-gp does not have a significant
role in determining its absorption and bioavailability after oral administration 89,90.
Consistent with the in vitro data of OATP inhibition, grapefruit juice significantly
decreased the AUC, Cmax, and urinary excretion values of fexofenadine to 30 ~ 40% of
those with water, with no change in the time to Cmax, elimination half-life (t1/2), renal
clearance, or urinary volume in humans 25. The mechanism underlying this interaction is
presumably related to the reduced intestinal fexofenadine absorption due to the inhibition
of the uptake transporter OATP. A recent clinical study indicated that the volume of
grapefruit juice also had a significant effect on fexofenadine bioavailability. A 300-mL
volume of grapefruit juice decreased the AUC and Cmax of fexofenadine to 58% and 53%,
respectively, to that following ingestion of the same volume of water. With the total dose
being the same, a 1200-mL volume of grapefruit juice had a more pronounced effect and
decreased these parameters to 36% and 33%, respectively, of those with the
corresponding volume of water. It was concluded that grapefruit juice at a commonly
consumed volume decreased the oral bioavailability of fexofenadine, likely due to direct
inhibition of uptake by intestinal OATP-A. A much higher volume caused an additional
256
modest effect, possibly from reduced intestinal concentration and transit time of
fexofenadine 91.
Garlic
Garlic is one of the best-selling herbal supplements in the United States, and has
long been used as an herb medicine for its lipid-lowering, cardioprotective, antioxidant,
antineoplastic, antimicrobial, and antiplatelet effects 29,92. Allicin (diallyl thiosulfinate) is
believed to be one of the major active ingredients in garlic, which can be further
converted to a number of organic sulfur compounds, such as allyl, diallyl, and methyl
sulfides 93.
Recent studies indicated that garlic and its components could interact with drug
transporters via multiple mechanisms. In an in vitro study, allicin significantly inhibited
the P-gp-mediated efflux of ritonavir, increasing uptake of ritonavir in MDCK-MDR1
cells with an IC50 value of 119 M. Moreover, allicin (50 M) remarkably inhibited Pglycoprotein-mediated ritonavir transport across Caco-2 cell monolayers, resulting in an
efflux ratio of ritonavir close to unity 48. Using purified P-gp cell membranes and a
colorimetric ATPase assay, Foster et al. 94 showed that garlic extracts significantly
inhibited P-gp activity, although the potency was low to moderate compared with
verapamil, a positive control. However, in P-gp-overexpressing human carcinoma KBC2 cells, diallyl sulfide or diallyl trisulfide (50 M) had no significant effects on
daunorubicin intracellular accumulation 95. Interestingly, in P-gp-overexpressing K562
resistant cells, exposure of a non-toxic concentration of diallyl sulfide (8.75 mM) resulted
257
in a time-dependent reduction of P-gp expression levels, with a maximum effect observed

at 72 hours. In addition, oral administration of diallyl sulfide (5 mg/kg BW) effectively
reduced vinca alkaloid-induced P-gp overexpression in mouse hepatocyte 96. In contrast,
oral administration of diallyl disulfide (200 mg/kg for 3 days) to rats significantly
induced Mrp2 expression by 7-fold in renal brush-border membranes, while no P-gp
induction was observed 97.
Two clinical trials have indicated that administration of garlic supplements
decreased AUC and Cmax of HIV protease inhibitor saquinavir and ritonavir, substrates
for both CYP3A4 and P-gp 98,99. One mechanism, as suggested by the authors, is
probably due to the induction of CYP3A4 in the gut mucosa. However, the contribution
from induction of P-gp cannot be excluded since saquinavir and ritonavir are also P-gp
substrates 98,100,101.
Green Tea
Green tea is widely consumed as a beverage. Tea polyphenols, known as

catechins, are the major constituents in green tea. The most abundant catechins in a
typical brewed green tea include 10 ~ 15% (-)-epigallocatechin gallate (EGCG), 6 ~ 10%
(-)-epigallocatechin (EGC), 2 ~ 3% (-)-epicatechin gallate (ECG), and 2% (-)-epicatechin
(EC) 102. The extracts of green tea were reported to exhibt a variety of beneficial health
effects, especially chemopreventive, anticarcinogenic, and antioxidant effects 103.
Several recent studies indicated that green tea components could interact with Pgp and inhibit its transport activity. In a multidrug-resistant cell line CHRC5, green tea
258
polyphenols (30 g/ml) inhibited the photolabeling of P-gp by 75% and increased the
accumulation of rhodamine-123 by 3-fold. Among the catechins present in green tea,
EGCG, ECG and (-)-catechin gallate (CG) were the major determinants responsible for
inhibiting P-gp. In addition, EGCG was able to potentiate the cytotoxicity of vinblastine
in CHRC5 cells 104. In carcinoma KB-A1 cells, green tea polyphenols (40 g/ml) and
EGCG (10 g/ml) significantly enhanced doxorubicin cytotoxicity by 5.2- and 2.5-fold,
respectively. More detailed studies revealed that green tea polyphenols were able to
inhibit P-gp ATPase activity and down-regulate P-gp expression 105.
The effects of green tea extracts on MRP2 were also investigated. In human
gastrointestinal epithelial LS-180 cells, green tea extracts, at the concentration of 0.01
mg/ml, had no effects on either activity or expression of MRP2. However, at the
concentration of 0.1 mg/ml, green tea extracts significantly inhibited MRP2-mediated
efflux of methotrexate (a MRP2 substrate) in MRP2 overexpressing MDCK (MDCKMRP2) cells, resulting in the increased cellular accumulation of methotrexate. In contrast
to the inhibitory effects on P-gp, the green tea components EGCG and EGC did not
contribute to the MRP2 inhibition activity 106. Using Caco-2 and MDCK-MRP cells,
green tea polyphenols, such as EGCG, ECG, EGC, and EC, were shown to be potential
substrates of MRPs, suggesting the importance of MRPs in determining cellular
concentrations of green tea components 107-109.
In a recent study involving 15 herbal extracts, green tea extracts were shown to
potently inhibit OATP-B-mediated uptake of estrone-3-sulfate at the putative
gastrointestinal concentration (400 g/ml). Moreover, the OATP-B inhibition by green
259
tea extracts exhibited concentration dependence with an IC50 value of 22.1 4.9 g/ml
110
.
In in vivo xenograft studies, green tea extracts were shown to be effective in
reversal of cancer multidrug resistance 105,111,112. For example, in mice bearing

doxorubicin-resistant human carcinoma KB-A-1 cells, the combination of doxorubicin
with EGCG (40 mg/kg) significantly increased the doxorubicin concentration in the
tumors by 51%, and potentiated doxorubicin -induced apoptosis of tumor cells.
Compared with the mice given either doxorubicin alone or EGCG alone, there was a
considerable reduction of tumor weights in mice given both EGCG and doxorubicin 112.
Ginseng
Ginseng is a well-known traditional Oriental herbal medicine and has gained

popularity as a dietary supplement in the United States during the last decade.
Ginsenosides are considered the major active constituents and contribute to the many
pharmacological activities of ginseng, including cognition-enhancing, neuroprotective,
antioxidant, antineoplastic, and immunomodulatory effects 113.
The effects of ginseng constituents on P-gp-mediated multidrug resistance were
investigated both in vitro and in vivo. In drug-resistant KBV20C cells, ginsenoside Rg(3)
significantly increased cellular accumulation of rhodamine-123 and vinblastine in a dosedependent manner. In addition, Rg(3), at the concentration of 20 M, greatly enhanced
cytotoxicity and reversed multidrug resistance to several anticancer drugs, such as
doxorubicin, colchicine, vincristine, and etoposide. However, Rg(3) did not affect either
260
mRNA or protein levels of P-gp in KBV20C cells. A photo-affinity labeling study with
[3H]azidopine revealed that 100 M Rg(3) completely inhibited [3H]azidopine binding to
P-gp indicating that inhibition of drug efflux by Rg(3) was likely due to the competition
for the common substrate binding sites on P-gp. In mice implanted with doxorubicinresistant murine leukemia P388 cells, combining 4 mg/kg doxorubicin with10 mg/kg
Rg(3) enhanced the cytotoxicity and efficacy of doxorubicin, resulting in a significant
increase in life span and suppression in tumor growth in the mice 114.
Similarly, in acute myelogenous leukemia cell sublines AML-2/D100
(overexpressing P-gp) and AML-2/DX100 (overexpressing MRP), protopanaxatriol
ginsenosides (PTG) were able to reverse P-gp (but not MRP)-mediated resistance in a
concentration-dependent manner. PTG (100 g/mL) increased daunorubicin
accumulation in the AML-2/D100 subline 2-fold higher than that observed in the
presence of verapamil (5 g/mL), but 1.5 -fold less than that by cyclosporin A (3 g/mL).
In contrast to verapamil and cyclosporin A, the maximum non-cytotoxic concentrations
of PTG had no effects on P-gp expression. Moreover, PTG, at a concentration of 200
g/mL or more, completely inhibited the [3H]azidopine binding to P-gp 115. These results
indicated that PTG reversed P-gp-mediated multidrug resistance likely via direct
interaction with P-gp at the azidopine site, resulting in increased intracellular
accumulation of other P-gp substrates.
Although ginseng constituents are potent multidrug resistance chemosensitizers,
no studies to date have been conducted to determine the potential in vivo drug
interactions.
261
Milk Thistle
Milk Thistle is one of the most popular herbal medicines, used as a

hepatoprotectant in the therapy of various liver diseases including hepatitis and alcoholic
cirrhosis 116,117. The major constituent of milk thistle is silymarin, a mixture of
flavonolignans such as silybin, isosilybin, silychristin, silydianin 118,119.
In a P-gp-overexpressing human breast cancer cell line (MDA435/LCC6MDR1),
silymarin significantly increased daunomycin cellular accumulation in a concentrationdependent manner. At the concentration of 50 M, silymarin enhanced daunomycin
accumulation by approximately 4-fold. Moreover, silymarin (100 M) potentiated
doxorubicin cytotoxicity by 3-fold in the resistant cells. In the presence of 100 M
silymarin, the verapamil-induced P-gp ATPase activity was completely inhibited, and
[3H]azidopine photoaffinity labeling of P-gp was significantly decreased. However, preincubation cells with 50 M silymarin did not change P-gp protein levels 120. In Caco-2
cells, silymarin significantly inhibited P-gp-mediated transport of digoxin across the cell
monolayers in a concentration-dependent manner, resulting in decreased efflux and
increased cellular accumulation of digoxin 121. These results indicated that silymarin had
inhibitory effects on P-gp-mediated drug efflux and the mechanism of the interaction
involved, at least in part, a direct interaction.
In addition to interacting with P-gp, silymarin also modulates MRP1 activity. In
MRP1 overexpressing human pancreatic adenocarcinoma Panc-1 cells, silymarin (50
M) significantly increased the cellular accumulation of vinblastine and daunomycin by
262
approximately 3-fold. However, the inhibition of MRP1 by silymarin was not due to the
inhibition of glutathione S-transferase activity or depletion of intracellular glutathione 32.
In vivo drug interactions with milk thistle have been reported recently. However,
the results are less compelling compared with the in vitro results. Using digoxin as a
selective probe of P-gp, the effects of milk thistle on digoxin pharmacokinetics were
studied in sixteen healthy subjects. However, no statistically significant changes in AUC,
Cmax, and oral clearance (CL/F) were observed after administration of milk thistle (900
mg daily) for 2 weeks 122. Similar to the case of grapefruit juice, this lack of in vivo
effect of milk thistle on digoxin is probably due to the high inherent bioavailability of
digoxin 90. In addition, the administration of milk thistle for 14 days 123, 21 days 124, and
28 days 125 to healthy subjects had no significant effects on the pharmacoknetics of
indinavir. In cancer patients, short-term (4 days) or more prolonged intake of milk thistle
(12 days) did not change the pharmacokinetics of irinotecan, a substrate for CY3A4, Pgp, and BCRP 126,127. Given the fact that indinavir and irinotecan are substrates for both
drug metabolizing enzymes and transporters, the lack of drug interactions with milk
thistle is probably due to the complicated in vivo regulation of metabolizing enzymes and
drug transporters, which are highly susceptible to both induction and inhibition by
xenobiotics. Therefore, further studies with better substrate candidates are necessary to
characterize in vivo drug interactions with milk thistle.
263
Kava
Kava has been used for many centuries as a traditional intoxicating beverage in
the Pacific Islands. During the past few decades, kava has gained popularity in the
Western countries as an herbal supplement for its anxiolytic, antistress, and sedative
properties 128. The major constituents in kava are kavalactones, a mixture of more than
18 different -pyrones, including kawain, methysticin, yangonin, dihydrokawain,
desmethoxyyangonin, and dihydromethysticin 129,130. However, in 2002, due to the
reported cases of liver toxicity, kava extracts were withdrawn from the market in several
countries, which prompted wide discussion on kava relative benefits and risks as an
herbal remedy 29,131.
In a P-gp-overexpressing cell line P388/dx, a kava crude extract and 6 main
kavalactones (kawain, dihydrokawain, methysticin, dihydromethysticin, yangonin, and
desmethoxyyangonin) exhibited moderate to potent inhibitory activities on P-gpmediated efflux of calcein-AM. The crude extract and the kavalactones significantly
increased cellular accumulation of calcein-AM, causing increased intracellular
fluorescence intensity. The concentrations needed to double baseline fluorescence were
170 g/ml and 17 to 90 M for crude extract and 6 kavalactones, respectively 132.
A recent in vivo study indicated that oral coadministration of kava extract (256
mg/kg) significantly changed the pharmacokinetcs of kawain, resulting in a tripling of
kawain AUC and a doubling of Cmax. Moreover, kava extract and kavalactones
significantly inhibited CYP enzymes and modestly modulated P-gp ATPase activities in
264
vitro. It was concluded that mechanisms by which kava extract altered the
pharmacokinetics of kawain might include inhibition of CYP450 and/or P-gp 133.
22.2.2. Interactions of Flavonoids with Drug Transporters
Flavonoids are a class of polyphenolic compounds widely present in fruits,

vegetables, and plant-derived beverages, and in many herbal products marketed as overthe-counter dietary supplements, such as St. Johns wort, green tea, and milk thistle. The
concentrations of some flavonoids such as naringin and hesperidin, abundant in fruit
juices, have been reported to be as high as 145 638 mg/L 134 and 200 450 mg/L 135,136,
respectively. The average daily intake of total flavonoids from the U.S. diet was
estimated to be 200 mg to 1 g 137-141. The structures of a number of flavonoid subclasses
are shown in Table 2. Flavonoids have long been associated with a variety of
biochemical and pharmacological properties including antioxidant, antiviral,
anticarcinogenic, and anti-inflammatory activities, with no or low toxicity 142,143. These
health-promoting activities indicate that flavonoids may play a protective role in cancer
prevention and cardiovascular diseases, as well as other age-related degenerative diseases
143-145
. Recently, numerous studies have indicated that flavonoids could interact with
several efflux and uptake transporters such as P-glycoprotein, MRP1, BCRP, and OATP,
suggesting the potential roles of flavonoids for in vivo drug interactions 30,34,120,146.
265
Interactions with P-gp
The effects of flavonoids on P-gp have been extensively studied during the past
decade (Table 3). It was clearly shown from these studies that many of these flavonoids
demonstrated P-gp-modulating activities. However, the effects of flavonoids, especially
for some flavonols, were cell line-dependent, concentration-dependent, and substratedependent. For example, in P-gp-expressing HCT-15 colon cells, flavonols such as
quercetin, kaempferol and galangin were shown to stimulate adriamycin efflux 147. In
contrast, in MCF-7-ADR resistant breast cancer cells, quercetin was able to restore
sensitivity to adriamycin and inhibit rhodamine-123 efflux 148. Interestingly, in mouse
brain capillary endothelial cells (MBEC4), quercetin and kaempferol exhibited biphasic
effects by activating P-gp at a low concentration (10 M) and inhibiting P-gp at a high
concentration (50 M) 149. In rat hepatocytes, the effects of quercetin, kaempferol and
galangin on rhodamine-123 and doxorubicin efflux were shown to be substrate-dependent
150
. Using a purified and reconstituted P-gp system, Shapiro and Ling reported that
quercetin inhibited P-gp-mediated Hoechst 33342 efflux and enhanced its accumulation
in resistant CHRC5 cells. This effect was, at least partly, caused by the inhibition of P-gp
ATPase activity by quercetin 151. More recently, in P-gp-overexpressing KB-C2
carcinoma cells, quercetin and kaempferol significantly inhibited P-gp activity and
increased cellular accumulation of rhodamine-123 and daunorubicin 152. The reason(s)
for these observed disparate results is still unclear. One possible explanation might be
the existence of multiple drug binding sites on P-gp and different allosteric effects of
flavonoids on these sites. It has been shown that quercetin could preferentially bind to
266
the Hoechst 33342-binding site on P-gp and inhibited Hoechst 33342 transport,
presumably via competitive inhibition. In contrast, binding of quercetin to the Hoechst
33342 site resulted in increased binding of rhodamine-123 to another site on P-gp and
stimulated rhodamine-123 transport 153. Alternatively, the difference in experimental
conditions, such as the different cell lines, the different substrates and their
concentrations, and the concentrations of flavonoids used, might contribute to the
discrepancies as well. As shown in the MBEC4 study, low concentrations of quercetin
and kaempferol activated P-gp possibly via enhancing the phosphorylation (and hence
activity) of P-gp, whereas high concentrations of quercetin and kaempferol directly
inhibited P-gp 149. Despite these earlier controversial observations, the majority of recent
studies have shown that many flavonoids aglycones have an inhibitory activity on P-gpmediated drug transport (Table 3).
Using purified C-terminal nucleotide-binding domain (NBD2) from mouse P-gp,
Conseil et al. studied the structure-activity relationship of flavonoids interacting with Pgp based on their binding affinity. As a suitable tool for the rapid screening of P-gp
modulators, NBD2 contains an ATP-binding site as well as a close but distinct
hydrophobic steroid RU486-binding site 30. It was shown in this study that flavones
(apigenin) and flavonols (quercetin) had higher binding affinity than flavanones
(naringenin), isoflavones (genistein), or glycosylated derivatives (rutin). Interestingly,
the flavonol kaempferide exibited bifunctional interactions with both the ATP-binding
and hydrophobic steroid-binding sites 30. It was shown in further studies that, among a
total of 29 flavonoids tested, only flavonols were able to bind to the ATP-binding site but
267
flavones did not, suggesting the essential roles of hydroxyl at position 3 on ring-C in
interacting with ATP-binding site 154. Moreover, hydrophobic substitution by prenylation
at either position 6 or 8 on the A ring significantly increased the binding affinity of
flavonoids for P-gp NBD2 and abolished the interactions of flavonols with the ATPbinding site 154. Based on these findings, a tentative mechanism for the interaction of
flavonoids with P-gp was proposed by Di Pietro et al. 154. In this model, flavonols appear
to interact with the ATP-binding site via hydroxyl groups at position 3 and 5 on the A or
C rings, whereas the rest of the molecule would interact with the hydrophobic steroidbinding region. Increasing hydrophobicity by prenylation will shift flavonol binding
from the ATP-binding site to the vicinal steroid-binding and transmembrane domain
(TMD) (Figure 1).
Additional structure-activity relationship studies using NBD2 and cell lines
suggest that high P-gp modulating activities are associated with molecules containing a 23 double bond (planar structure), 3- and 5-hydroxyl groups, and hydrophobic groups on
the A or B rings. Moreover, glycosylation would dramatically decrease flavonoid P-gpmodulating activity, as exemplified by rutin, heperidin, and naringin (Table 3). With
regards to flavanols (e.g., catechin, catechin gallate, EGC, and EGCG), the presence of a
galloyl moiety on the C ring markedly increases their activities 152,154-156.
The pharmacokinetic interactions of flavonoids with drug transporters have been
reported in a number of studies, most of which were focused on the interactions between
P-gp and quercetin. Using paclitaxel as a P-gp substrate, Choi et al. 157 investigated the
effects of quercetin on paclitaxel pharmacokinetics in rats. It was shown in this study
268
that the oral administration of quercetin increased the AUC and Cmax of paclitaxel (p.o.
dose, 40 mg/kg) in a dose-dependent manner. At a dose of 20 mg/kg, quercetin
administration resulted in a 3.1-fold and 2.7-fold increase in paclitaxel AUC and Cmax,
respectively. Moreover, the absolute bioavailability of paclitaxel was significantly
improved from 2% (control group) to 6.2% (20 mg/kg quercetin treatment group). The
t1/2 and MRT of paclitaxel were also greatly prolonged compared to those of the control
group. Similarly, the oral administration of 40 mg/kg quercetin to pigs significantly
increased the AUC of digoxin (p.o. dose, 0.02 mg/kg) by 170% and increased the Cmax by
413%. Unexpectedly, increasing the dose of quercetin to 50 mg/kg resulted in sudden
death of two out of three pigs within 30 min after digoxin administration 37. The adverse
interaction observed in this study raised a serious safety concern of concomitant use of
dietary supplements and clinically important drugs, especially those with a narrow
therapeutic index. In another study, quercetin (100 M) significantly inhibited P-gpmediated transport of moxidectin and enhanced cellular accumulation of moxidectin in
rat hepatocytes. In addition, subcutaneous administration of quercetin (10 mg/kg)
significantly increased the AUC of moxidectin (p.o. dose, 0.2 mg/kg) by 83.8% 158.
Interestingly, in a study by Hsiu et al. 159, the oral administration of quercetin (50 mg/kg)
to pigs and rats resulted in significant decreases in the AUC of cyclosporine by 56% and
43%, respectively. However, in a recent clinical study, oral administration of quercetin
(5 mg/kg, 30 min or 3 days pretreatment) significantly increased cyclosporine AUC by
36% and 47%, respectively 38. The possible explanations for the contradictory results of
cyclosporine might be due to the species differences, the methods of administration
269
(concomitant or separate administration), and the doses (low or high doses). All these
studies indicated that flavonoid-P-gp interaction could occur in vivo. However, the
observed pharmacokinetic interactions might result from the interactions with P-gp
and/or CYP3A or both since most of these P-gp substrates are also CYP3A substrates.
Interactions with MRPs
A growing number of reports have been published regarding the modulating

effects of flavonoids on MRPs. Many flavonoids have been shown to interact with MRPs
and the potential mechanisms of MRPs reversal might involve: 1) decreasing intracellular
glutathione (GSH) concentrations via stimulating GSH transport; 2) altering the
expression of MRPs; 3) influencing drug transport possibly via direct or indirect binding
interactions with MRPs at substrate or allosteric binding sites; and 4) affecting ATPase
activity, ATP binding, and ADP release (Table 4). Using recombinant nucleotidebinding domain (NBD1) from human MRP1, Trompier et al. 160 studied the direct
interactions of flavonoids with NBD1 and revealed the presence of multiple flavonoidbinding sites. In this study, dehydrosilybin was found to bind to ATP binding site and
inhibit leukotriene C4 (LTC4) transport. Similar to the case of P-gp, hydrophobic Cisoprenylation of dehydrosilybin increased the binding affinity for NBD1, but shifted the
flavonoid binding outside the ATP site, and decreased the inhibition of LTC4 transport.
A number of structure-activity relationship studies indicated that flavones and
flavonols were more potent in modulating MRP1 activities than isoflavones, flavanols,
flavanones, and flavanolols. Glycosylation of flavonoids resulted in a decrease in the
270
inhibitory activity 146,161,162. The structural features necessary for high MRP1 inhibitory
potency include: 1) a planar molecular structure due to the presence of a 2-3 double bond;
2) the presence of both 3- and 4-hydroxyl groups on the B ring; and 3) hydrophobic
substitution of 4-hydroxyl group on the B ring 161-163. In a recent study including 29
flavonoids, diosmetin (3, 5, 7-trihydroxy-4-methoxyflavone ) was identified as the most
potent MRP1 inhibitor with an IC50 value of 2.7 0.6 M 162. In contrast to the wide
variety of flavonoids that can inhibit MRP1, MRP2 displays higher selectivity for
flavonoid type inhibition. Among 29 flavonoids tested, only robinetin and myricetin
inhibited MRP2-mediated calcein efflux with IC50 values of 15.0 3.5 M and 22.2 3.9
M, respectively. The presence of a pyrogallol group on the B ring of flavonols was an

important structural characteristic of flavonoids for MRP2 inhibition 162.
Interactions with BCRP
Several recent studies have demonstrated that many naturally occurring

flavonoids can inhibit BCRP. Among all the subclasses of flavonoids tested, flavones
seemed to be the most potent BCRP inhibitors 164,165 (Table 5). The EC50 values of the
flavones chrysin and apigenin for BCRP inhibition (measured as the concentration of
flavonoids for producing 50% of the maximal increase in mitoxantrone accumulation in
BCRP-overexpressing MCF-7 MX100 cells) were shown to be within the sub- or low
micromolar range (0.39 0.13 M and 1.66 0.55 M, respectively) 165. Glycosylation
dramatically decreased the BCRP-inhibiting activities of some flavonoids 34,164. Recent
structure-activity relationship studies indicated that high BCRP inhibitory potency was
271
associated with flavonoids with the following characteristics: 1) a planar molecular

structure due to the presence of a 2-3 double bond; 2) hydroxylation at position 5; 3) lack
of hydroxylation at position 3; 4) ring-B attached at position 2; and 4) hydrophobic
substitution of 6, 7, 8, or 4-hydroxyl group 165,166. Interestingly, it was shown in a recent
study that prenylation at position 6 strongly enhanced both inhibitory potency and
specificity of flavones. Compared with chrysin (IC50 = 4.6 0.5 M), the inhibitory
potency of 6-prenylchrysin was significantly enhanced (IC50 = 0.29 0.06 M).
Moreover, 6-prenylchrysin seemed to represent a specific BCRP inhibitor since no
interaction was detected with either P-gp or MRP1 166. It should be noted that the
different IC50 values of chrysin reported in two studies might be due to the different
experimental conditions, such as cell lines and substrate concentration used in the
accumulation study 165,166.
Interactions with OATP
In a recent study, 20 naturally occurring flavonoids and some of their

corresponding glycosides were investigated for their modulatory effects on OATP-C by
using [3H]dehydroepiandrosterone sulfate (DHEAS) as a probe substrate. Many of the
tested flavonoids (including biochanin A, genistein, and epigallocatechin-3-gallate)
significantly inhibited [3H]DHEAS uptake in a concentration-dependent manner, with
biochanin A being one of the most potent inhibitors with an IC50 of 11.3 3.22 M. A
kinetic study revealed that biochanin A inhibited [3H]DHEAS uptake in a noncompetitive
manner with a Ki of 10.2 1.89 M. Four of the eight pairs of tested flavonoids and
272
their glycosides, namely, genistein/genistin, diosmetin/diosmin, epigallocatechin

(EGC)/epigallocatechin-3-gallate (EGCG), and quercetin/rutin, exhibited distinct effects
on [3H]DHEAS uptake. For example, genistin did not inhibit DHEAS uptake while
genistein did and rutin stimulated uptake while quercetin had no effect 36 (Table 6). In
another study using HEK293 cells stably transfected with OATP-B, Fuchikami et al.
identified that some flavanols from green tea extracts (e.g., catechin, EC, EGC, ECG, and
EGCG) were potent inhibitors of OATP-B. At a concentration as low as 10 M, EC,
ECG, and EGCG significantly inhibited OATP-B-mediated uptake of estrone-3 sulfate
110
. Similar to P-gp inhibition, the presence of a galloyl moiety on the C-ring of green tea
catechins (e.g., EGCG vs. EGC and ECG vs. EC) markedly enhanced their potency in
both OATP-B and OATP-C inhibition 36,110.
22.2.3. Interactions of Organic Isothiocyanates (ITCs) with Drug Transporters
Organic isothiocyanates (ITCs, R-N=C=S), also known as mustard oils, are

widely present in cruciferous vegetables, such as broccoli, watercress, cabbage, and
cauliflower. Human consumption of glucosinolates, the precursors of ITCs in plants, has
been estimated as high as 300 mg/day and milligram quantities of ITCs can be released
from consumption of normal amounts of vegetables 167,168. The dietary supplements
containing ITCs have been increasingly used recently due to the beneficial health effects
of ITCs, especially cancer chemopreventive effects 169,170. However, the mechanisms of
ITCs in cancer chemoprevention have not been fully characterized.
273
Recently, a number of studies have indicated that ITCs might reverse resistance to
anticancer drugs via interacting with drug transporters. Using P-gp-overexpressing
MCF-7/ADR and MRP1-overexpressing Panc-1 cells, Tseng et al. 171 evaluated the
effects of ITCs on P-gp- and MRP1-mediated transport of chemotherapeutic agents.
Among all the ITCs tested, 1-naphthyl-isothiocyanate (NITC) significantly increased the
accumulation of daunomycin (DNM) and vinblastine (VBL) in both resistant cell lines.
Benzyl-isothiocyanate (BITC) and phenylhexyl- isothiocyanate (PHITC) significantly
increased the accumulation of DNM and/or VBL in MCF-7/ADR cells whereas
phenethyl- isothiocyanate (PEITC), erysolin, PHITC, and phenylbutyl- isothiocyanate
(PBITC) increased the accumulation of DNM and/or VBL in Panc-1 cells. In another
study, Hu et al. 172 reported that BITC and PEITC were able to deplete cellular
concentrations of glutathione (GSH) in Panc-1 and Caco-2 cells after 2- and 24-h ITC
treatments. However, no significant changes in glutathione-S-transferase activity were
found in the presence of BITC, PEITC, or NITC. In addition, PEITC and/or its
metabolites was shown to be transported by MRP1 and MRP2, but not by P-gp 172,173.
These results indicate that certain dietary ITCs inhibit the P-gp- and the MRP1-mediated
efflux of anticancer drugs in MDR cancer cells and the interactions likely involve
multiple mechanisms.
In a recent study, the effects of 12 ITCs on the cellular accumulation of
mitoxantrone (MX) were measured in BCRP-overexpressing human breast cancer (MCF7) and large cell lung carcinoma (NCI-H460) cells. At a concentration of 10 or 30 M, 7
274
ITCs significantly increased MX accumulation in both cell lines and reversed MX

cytotoxicity, indicating that ITCs could also modulate BCRP activities 174.
22.3. Conclusions
In recent years, a wealth of evidence has been generated from in vitro and in vivo
studies showing that many diet/nutrients interact extensively with drug transporters and
play critical roles in multidrug resistance reversal and drug disposition. The importance
of transporter-mediated diet-drug interactions is increasingly recognized and has been
reported in a number of pre-clinical and clinical studies. Some nutrients rich in fruits and
vegetables, such as flavonoids and isothiocyanates, have been identified as potent
inhibitors/inducers of major efflux or uptake transporters. The structural preferences of
flavonoids for some efflux transporters have been described in several structure-activity
relationship studies. However, diet/nutrient interactions with drug transporters still
remain largely unknown. Most in vivo interaction studies reported to date are focused
primarily on the interactions between dietary supplements and P-gp. Given the facts that
MRPs and BCRP, and OATP are also essential in drug disposition, it is important to
appreciate pharmacokinetic interactions of diet/nutrient with these transporters. The
concentrations of many flavonoids required to produce significant modulation on
activities of these transporters appear to be, in general, within the micromolar range,
which is achievable in the intestine after intake of food and, especially, dietary
supplements. Therefore, altered disposition of MRP, BCRP, and OATP substrates
following ingestion of a regular diet is likely to occur and needs further characterization.
275
Moreover, structure-activity relationship (QSAR) studies will be crucial in order to better

understand the potential of dietary components for inhibiting or inducing drug transport
and the potential for significant in vivo diet/dietary supplement-drug interactions.
276
Reference:
1. Singh BN 1999. Effects of food on clinical pharmacokinetics. Clin Pharmacokinet

37(3):213-255.
2.Anderson KE 1988. Influences of diet and nutrition on clinical pharmacokinetics. Clin
Pharmacokinet 14(6):325-346.
3. Evans AM 2000. Influence of dietary components on the gastrointestinal metabolism
and transport of drugs. Ther Drug Monit 22(1):131-136.
4.
Harris RZ, Jang GR, Tsunoda S 2003. Dietary effects on drug metabolism and
transport. Clin Pharmacokinet 42(13):1071-1088.
5.
Walter-Sack I, Klotz U 1996. Influence of diet and nutritional status on drug
metabolism. Clin Pharmacokinet 31(1):47-64.
6.
Ioannides C 2002. Pharmacokinetic interactions between herbal remedies and
medicinal drugs. Xenobiotica 32(6):451-478.
7.Ohnishi N, Yokoyama T 2004. Interactions between medicines and functional foods or
dietary supplements. Keio J Med 53(3):137-150.
8.Kim RB 2002. Transporters and xenobiotic disposition. Toxicology 181-182:291-297.
9.Ho RH, Kim RB 2005. Transporters and drug therapy: implications for drug disposition
and disease. Clin Pharmacol Ther 78(3):260-277.
10.Beringer PM, Slaughter RL 2005. Transporters and their impact on drug disposition.
Ann Pharmacother 39(6):1097-1108.
11. Leslie EM, Deeley RG, Cole SP 2005. Multidrug resistance proteins: role of Pglycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicol Appl
Pharmacol 204(3):216-237.
12.Hagenbuch B, Meier PJ 2004. Organic anion transporting polypeptides of the OATP/
nomenclature and molecular/functional properties. Pflugers Arch 447(5):653-665.
13. You G 2004. Towards an understanding of organic anion transporters: structurefunction relationships. Med Res Rev 24(6):762-774.
14.Koepsell H, Schmitt BM, Gorboulev V 2003. Organic cation transporters. Rev Physiol
15. Allen JD, Schinkel AH 2002. Multidrug resistance and pharmacological protection
mediated by the breast cancer resistance protein (BCRP/ABCG2). Mol Cancer Ther
1(6):427-434.
16. Borst P, Evers R, Kool M, Wijnholds J 2000. A family of drug transporters: the
multidrug resistance-associated proteins. J Natl Cancer Inst 92(16):1295-1302.
17. Konig J, Nies AT, Cui Y, Leier I, Keppler D 1999. Conjugate export pumps of the
multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2mediated drug resistance. Biochim Biophys Acta 1461(2):377-394.
18.
Litman T, Druley TE, Stein WD, Bates SE 2001. From MDR to MXR: new
understanding of multidrug resistance systems, their properties and clinical significance.
Cell Mol Life Sci 58(7):931-959.
19.Gottesman MM, Pastan I 1993. Biochemistry of multidrug resistance mediated by the
multidrug transporter. Annu Rev Biochem 62:385-427.
277
20.Leonard GD, Fojo T, Bates SE 2003. The role of ABC transporters in clinical practice.
Oncologist 8(5):411-424.
21. Doyle LA, Ross DD 2003. Multidrug resistance mediated by the breast cancer
resistance protein BCRP (ABCG2). Oncogene 22(47):7340-7358.
22.Westphal K, Weinbrenner A, Giessmann T, Stuhr M, Franke G, Zschiesche M, Oertel
R, Terhaag B, Kroemer HK, Siegmund W 2000. Oral bioavailability of digoxin is
enhanced by talinolol: evidence for involvement of intestinal P-glycoprotein. Clin
Pharmacol Ther 68(1):6-12.
23. Westphal K, Weinbrenner A, Zschiesche M, Franke G, Knoke M, Oertel R, Fritz P,
von Richter O, Warzok R, Hachenberg T, Kauffmann HM, Schrenk D, Terhaag B,
Kroemer HK, Siegmund W 2000. Induction of P-glycoprotein by rifampin increases
intestinal secretion of talinolol in human beings: a new type of drug/drug interaction. Clin
24.
Johne A, Brockmoller J, Bauer S, Maurer A, Langheinrich M, Roots I 1999.
Pharmacokinetic interaction of digoxin with an herbal extract from St John's wort
(Hypericum perforatum). Clin Pharmacol Ther 66(4):338-345.
25.Dresser GK, Bailey DG, Leake BF, Schwarz UI, Dawson PA, Freeman DJ, Kim RB
2002. Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to
decrease the oral availability of fexofenadine. Clin Pharmacol Ther 71(1):11-20.
26.Kruijtzer CM, Beijnen JH, Rosing H, ten Bokkel Huinink WW, Schot M, Jewell RC,
Paul EM, Schellens JH 2002. Increased oral bioavailability of topotecan in combination
with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918. J Clin
Oncol 20(13):2943-2950.
27. Jonker JW, Smit JW, Brinkhuis RF, Maliepaard M, Beijnen JH, Schellens JH,
Schinkel AH 2000. Role of breast cancer resistance protein in the bioavailability and fetal
penetration of topotecan. J Natl Cancer Inst 92(20):1651-1656.
28.Izzo AA 2005. Herb-drug interactions: an overview of the clinical evidence. Fundam
Clin Pharmacol 19(1):1-16.
29.Sparreboom A, Cox MC, Acharya MR, Figg WD 2004. Herbal remedies in the United
States: potential adverse interactions with anticancer agents. J Clin Oncol 22(12):24892503.
30. Conseil G, Baubichon-Cortay H, Dayan G, Jault JM, Barron D, Di Pietro A 1998.
Flavonoids: a class of modulators with bifunctional interactions at vicinal ATP- and
steroid-binding sites on mouse P-glycoprotein. Proc Natl Acad Sci U S A 95(17):98319836.
31.Durr D, Stieger B, Kullak-Ublick GA, Rentsch KM, Steinert HC, Meier PJ, Fattinger
K 2000. St John's Wort induces intestinal P-glycoprotein/MDR1 and intestinal and
hepatic CYP3A4. Clin Pharmacol Ther 68(6):598-604.
32. Nguyen H, Zhang S, Morris ME 2003. Effect of flavonoids on MRP1-mediated
transport in Panc-1 cells. J Pharm Sci 92(2):250-257.
33.Cooray HC, Janvilisri T, van Veen HW, Hladky SB, Barrand MA 2004. Interaction of
the breast cancer resistance protein with plant polyphenols. Biochem Biophys Res
Commun 317(1):269-275.
278
34. Zhang S, Yang X, Morris ME 2004. Flavonoids are inhibitors of breast cancer
35. Zhou S, Lim LY, Chowbay B 2004. Herbal modulation of P-glycoprotein. Drug
Metab Rev 36(1):57-104.
36. Wang X, Wolkoff AW, Morris ME 2005. Flavonoids as a novel class of human
organic anion-transporting polypeptide OATP1B1 (OATP-C) modulators. Drug Metab
Dispos 33(11):1666-1672.
37. Wang YH, Chao PD, Hsiu SL, Wen KC, Hou YC 2004. Lethal quercetin-digoxin
interaction in pigs. Life Sci 74(10):1191-1197.
38.Choi JS, Choi BC, Choi KE 2004. Effect of quercetin on the pharmacokinetics of oral
cyclosporine. Am J Health Syst Pharm 61(22):2406-2409.
39.Huang SM, Hall SD, Watkins P, Love LA, Serabjit-Singh C, Betz JM, Hoffman FA,
Honig P, Coates PM, Bull J, Chen ST, Kearns GL, Murray MD 2004. Drug interactions
with herbal products and grapefruit juice: a conference report. Clin Pharmacol Ther
75(1):1-12.
40.Bilia AR, Gallori S, Vincieri FF 2002. St. John's wort and depression: efficacy, safety
and tolerability-an update. Life Sci 70(26):3077-3096.
41.Schulz HU, Schurer M, Bassler D, Weiser D 2005. Investigation of the bioavailability
of hypericin, pseudohypericin, hyperforin and the flavonoids quercetin and isorhamnetin
following single and multiple oral dosing of a hypericum extract containing tablet.
Arzneimittelforschung 55(1):15-22.
42.Obach RS 2000. Inhibition of human cytochrome P450 enzymes by constituents of St.
John's Wort, an herbal preparation used in the treatment of depression. J Pharmacol Exp
Ther 294(1):88-95.
43.Perloff MD, von Moltke LL, Stormer E, Shader RI, Greenblatt DJ 2001. Saint John's
wort: an in vitro analysis of P-glycoprotein induction due to extended exposure. Br J
Pharmacol 134(8):1601-1608.
44.Hennessy M, Kelleher D, Spiers JP, Barry M, Kavanagh P, Back D, Mulcahy F, Feely
J 2002. St Johns wort increases expression of P-glycoprotein: implications for drug
interactions. Br J Clin Pharmacol 53(1):75-82.
45.Geick A, Eichelbaum M, Burk O 2001. Nuclear receptor response elements mediate
induction of intestinal MDR1 by rifampin. J Biol Chem 276(18):14581-14587.
46. Moore LB, Goodwin B, Jones SA, Wisely GB, Serabjit-Singh CJ, Willson TM,
Collins JL, Kliewer SA 2000. St. John's wort induces hepatic drug metabolism through
activation of the pregnane X receptor. Proc Natl Acad Sci U S A 97(13):7500-7502.
47.Weber CC, Kressmann S, Fricker G, Muller WE 2004. Modulation of P-glycoprotein
function by St John's wort extract and its major constituents. Pharmacopsychiatry
37(6):292-298.
48. Patel J, Buddha B, Dey S, Pal D, Mitra AK 2004. In vitro interaction of the HIV
protease inhibitor ritonavir with herbal constituents: changes in P-gp and CYP3A4
activity. Am J Ther 11(4):262-277.
49. Wang EJ, Barecki-Roach M, Johnson WW 2004. Quantitative characterization of
direct P-glycoprotein inhibition by St John's wort constituents hypericin and hyperforin. J
Pharm Pharmacol 56(1):123-128.
279
50.Krusekopf S, Roots I 2005. St. John's wort and its constituent hyperforin concordantly
regulate expression of genes encoding enzymes involved in basic cellular pathways.
Pharmacogenet Genomics 15(11):817-829.
51. Shibayama Y, Ikeda R, Motoya T, Yamada K 2004. St. John's Wort (Hypericum
perforatum) induces overexpression of multidrug resistance protein 2 (MRP2) in rats: a
30-day ingestion study. Food Chem Toxicol 42(6):995-1002.
52.Tanigawara Y, Okamura N, Hirai M, Yasuhara M, Ueda K, Kioka N, Komano T, Hori
R 1992. Transport of digoxin by human P-glycoprotein expressed in a porcine kidney
epithelial cell line (LLC-PK1). J Pharmacol Exp Ther 263(2):840-845.
53. Mueller SC, Uehleke B, Woehling H, Petzsch M, Majcher-Peszynska J, Hehl EM,
Sievers H, Frank B, Riethling AK, Drewelow B 2004. Effect of St John's wort dose and
preparations on the pharmacokinetics of digoxin. Clin Pharmacol Ther 75(6):546-557.
54.
Lacarelle B, Rahmani R, de Sousa G, Durand A, Placidi M, Cano JP 1991.
Metabolism of digoxin, digoxigenin digitoxosides and digoxigenin in human hepatocytes
and liver microsomes. Fundam Clin Pharmacol 5(7):567-582.
55. Cvetkovic M, Leake B, Fromm MF, Wilkinson GR, Kim RB 1999. OATP and Pglycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. Drug
Metab Dispos 27(8):866-871.
56. Molimard M, Diquet B, Benedetti MS 2004. Comparison of pharmacokinetics and
metabolism of desloratadine, fexofenadine, levocetirizine and mizolastine in humans.
Fundam Clin Pharmacol 18(4):399-411.
57.Wang Z, Hamman MA, Huang SM, Lesko LJ, Hall SD 2002. Effect of St John's wort
on the pharmacokinetics of fexofenadine. Clin Pharmacol Ther 71(6):414-420.
58. Xie HG, Kim RB 2005. St John's wort-associated drug interactions: short-term
inhibition and long-term induction? Clin Pharmacol Ther 78(1):19-24.
59.
Piscitelli SC, Burstein AH, Chaitt D, Alfaro RM, Falloon J 2000. Indinavir
concentrations and St John's wort. Lancet 355(9203):547-548.
60. Bauer S, Stormer E, Johne A, Kruger H, Budde K, Neumayer HH, Roots I, Mai I
2003. Alterations in cyclosporin A pharmacokinetics and metabolism during treatment
with St John's wort in renal transplant patients. Br J Clin Pharmacol 55(2):203-211.
61. Mai I, Bauer S, Perloff ES, Johne A, Uehleke B, Frank B, Budde K, Roots I 2004.
Hyperforin content determines the magnitude of the St John's wort-cyclosporine drug
interaction. Clin Pharmacol Ther 76(4):330-340.
62.Ruschitzka F, Meier PJ, Turina M, Luscher TF, Noll G 2000. Acute heart transplant
rejection due to Saint John's wort. Lancet 355(9203):548-549.
63.Barone GW, Gurley BJ, Ketel BL, Lightfoot ML, Abul-Ezz SR 2000. Drug interaction
between St. John's wort and cyclosporine. Ann Pharmacother 34(9):1013-1016.
64.Hebert MF, Park JM, Chen YL, Akhtar S, Larson AM 2004. Effects of St. John's wort
(Hypericum perforatum) on tacrolimus pharmacokinetics in healthy volunteers. J Clin
Pharmacol 44(1):89-94.
65.Dresser GK, Schwarz UI, Wilkinson GR, Kim RB 2003. Coordinate induction of both
cytochrome P4503A and MDR1 by St John's wort in healthy subjects. Clin Pharmacol
Ther 73(1):41-50.
280
66. Bailey DG, Spence JD, Munoz C, Arnold JM 1991. Interaction of citrus juices with
felodipine and nifedipine. Lancet 337(8736):268-269.
67.Bailey DG, Malcolm J, Arnold O, Spence JD 1998. Grapefruit juice-drug interactions.
Br J Clin Pharmacol 46(2):101-110.
68. Dahan A, Altman H 2004. Food-drug interaction: grapefruit juice augments drug
bioavailability--mechanism, extent and relevance. Eur J Clin Nutr 58(1):1-9.
69. Kane GC, Lipsky JJ 2000. Drug-grapefruit juice interactions. Mayo Clin Proc
75(9):933-942.
70.Wang EJ, Casciano CN, Clement RP, Johnson WW 2001. Inhibition of P-glycoprotein
transport function by grapefruit juice psoralen. Pharm Res 18(4):432-438.
71.Ohnishi A, Matsuo H, Yamada S, Takanaga H, Morimoto S, Shoyama Y, Ohtani H,
Sawada Y 2000. Effect of furanocoumarin derivatives in grapefruit juice on the uptake of
vinblastine by Caco-2 cells and on the activity of cytochrome P450 3A4. Br J Pharmacol
130(6):1369-1377.
72.Spahn-Langguth H, Langguth P 2001. Grapefruit juice enhances intestinal absorption
of the P-glycoprotein substrate talinolol. Eur J Pharm Sci 12(4):361-367.
73. Tian R, Koyabu N, Takanaga H, Matsuo H, Ohtani H, Sawada Y 2002. Effects of
grapefruit juice and orange juice on the intestinal efflux of P-glycoprotein substrates.
Pharm Res 19(6):802-809.
74. Xu J, Go ML, Lim LY 2003. Modulation of digoxin transport across Caco-2 cell
monolayers by citrus fruit juices: lime, lemon, grapefruit, and pummelo. Pharm Res
20(2):169-176.
75.Honda Y, Ushigome F, Koyabu N, Morimoto S, Shoyama Y, Uchiumi T, Kuwano M,
Ohtani H, Sawada Y 2004. Effects of grapefruit juice and orange juice components on Pglycoprotein- and MRP2-mediated drug efflux. Br J Pharmacol 143(7):856-864.
76. Soldner A, Christians U, Susanto M, Wacher VJ, Silverman JA, Benet LZ 1999.
Grapefruit juice activates P-glycoprotein-mediated drug transport. Pharm Res 16(4):478485.
77.Lim SL, Lim LY 2006. Effects of citrus fruit juices on cytotoxicity and drug transport
pathways of Caco-2 cell monolayers. Int J Pharm 307(1):42-50.
78.Eagling VA, Profit L, Back DJ 1999. Inhibition of the CYP3A4-mediated metabolism
and P-glycoprotein-mediated transport of the HIV-1 protease inhibitor saquinavir by
grapefruit juice components. Br J Clin Pharmacol 48(4):543-552.
79.Takanaga H, Ohnishi A, Matsuo H, Sawada Y 1998. Inhibition of vinblastine efflux
mediated by P-glycoprotein by grapefruit juice components in caco-2 cells. Biol Pharm
Bull 21(10):1062-1066.
80. Romiti N, Tramonti G, Donati A, Chieli E 2004. Effects of grapefruit juice on the
multidrug transporter P-glycoprotein in the human proximal tubular cell line HK-2. Life
Sci 76(3):293-302.
81.Satoh H, Yamashita F, Tsujimoto M, Murakami H, Koyabu N, Ohtani H, Sawada Y
2005. Citrus juices inhibit the function of human organic anion-transporting polypeptide
OATP-B. Drug Metab Dispos 33(4):518-523.
82.Yee GC, Stanley DL, Pessa LJ, Dalla Costa T, Beltz SE, Ruiz J, Lowenthal DT 1995.
Effect of grapefruit juice on blood cyclosporin concentration. Lancet 345(8955):955-956.
281
83.Ducharme MP, Warbasse LH, Edwards DJ 1995. Disposition of intravenous and oral
cyclosporine after administration with grapefruit juice. Clin Pharmacol Ther 57(5):485491.
84.Edwards DJ, Fitzsimmons ME, Schuetz EG, Yasuda K, Ducharme MP, Warbasse LH,
Woster PM, Schuetz JD, Watkins P 1999. 6',7'-Dihydroxybergamottin in grapefruit juice
and Seville orange juice: effects on cyclosporine disposition, enterocyte CYP3A4, and Pglycoprotein. Clin Pharmacol Ther 65(3):237-244.
85. Lown KS, Mayo RR, Leichtman AB, Hsiao HL, Turgeon DK, Schmiedlin-Ren P,
Brown MB, Guo W, Rossi SJ, Benet LZ, Watkins PB 1997. Role of intestinal Pglycoprotein (mdr1) in interpatient variation in the oral bioavailability of cyclosporine.
Clin Pharmacol Ther 62(3):248-260.
86. Schwarz UI, Seemann D, Oertel R, Miehlke S, Kuhlisch E, Fromm MF, Kim RB,
Bailey DG, Kirch W 2005. Grapefruit juice ingestion significantly reduces talinolol
bioavailability. Clin Pharmacol Ther 77(4):291-301.
87.Parker RB, Yates CR, Soberman JE, Laizure SC 2003. Effects of grapefruit juice on
intestinal P-glycoprotein: evaluation using digoxin in humans. Pharmacotherapy
23(8):979-987.
88. Becquemont L, Verstuyft C, Kerb R, Brinkmann U, Lebot M, Jaillon P, FunckBrentano C 2001. Effect of grapefruit juice on digoxin pharmacokinetics in humans. Clin
89. Dresser GK, Bailey DG 2003. The effects of fruit juices on drug disposition: a new
model for drug interactions. Eur J Clin Invest 33 Suppl 2:10-16.
90. Magnani B, Malini PL 1995. Cardiac glycosides. Drug interactions of clinical
significance. Drug Saf 12(2):97-109.
91. Dresser GK, Kim RB, Bailey DG 2005. Effect of grapefruit juice volume on the
reduction of fexofenadine bioavailability: possible role of organic anion transporting
polypeptides. Clin Pharmacol Ther 77(3):170-177.
92. Tattelman E 2005. Health effects of garlic. Am Fam Physician 72(1):103-106.
93. Lawson LD, Gardner CD 2005. Composition, stability, and bioavailability of garlic
products used in a clinical trial. J Agric Food Chem 53(16):6254-6261.
94. Foster BC, Foster MS, Vandenhoek S, Krantis A, Budzinski JW, Arnason JT,
Gallicano KD, Choudri S 2001. An in vitro evaluation of human cytochrome P450 3A4
and P-glycoprotein inhibition by garlic. J Pharm Pharm Sci 4(2):176-184.
95. Nabekura T, Kamiyama S, Kitagawa S 2005. Effects of dietary chemopreventive
phytochemicals on P-glycoprotein function. Biochem Biophys Res Commun 327(3):866870.
96. Arora A, Seth K, Shukla Y 2004. Reversal of P-glycoprotein-mediated multidrug
resistance by diallyl sulfide in K562 leukemic cells and in mouse liver. Carcinogenesis
25(6):941-949.
97. Demeule M, Brossard M, Turcotte S, Regina A, Jodoin J, Beliveau R 2004. Diallyl
disulfide, a chemopreventive agent in garlic, induces multidrug resistance-associated
protein 2 expression. Biochem Biophys Res Commun 324(2):937-945.
98. Piscitelli SC, Burstein AH, Welden N, Gallicano KD, Falloon J 2002. The effect of
garlic supplements on the pharmacokinetics of saquinavir. Clin Infect Dis 34(2):234-238.
282
99.Gallicano K, Foster B, Choudhri S 2003. Effect of short-term administration of garlic

supplements on single-dose ritonavir pharmacokinetics in healthy volunteers. Br J Clin
Pharmacol 55(2):199-202.
100. Kim AE, Dintaman JM, Waddell DS, Silverman JA 1998. Saquinavir, an HIV
protease inhibitor, is transported by P-glycoprotein. J Pharmacol Exp Ther 286(3):14391445.
101.Lee CG, Gottesman MM, Cardarelli CO, Ramachandra M, Jeang KT, Ambudkar SV,
Pastan I, Dey S 1998. HIV-1 protease inhibitors are substrates for the MDR1 multidrug
transporter. Biochemistry 37(11):3594-3601.
102.Fujiki H 2005. Green tea: Health benefits as cancer preventive for humans. Chem
Rec 5(3):119-132.
103.Yang CS, Maliakal P, Meng X 2002. Inhibition of carcinogenesis by tea. Annu Rev
Pharmacol Toxicol 42:25-54.
104. Jodoin J, Demeule M, Beliveau R 2002. Inhibition of the multidrug resistance Pglycoprotein activity by green tea polyphenols. Biochim Biophys Acta 1542(1-3):149159.
105.Mei Y, Qian F, Wei D, Liu J 2004. Reversal of cancer multidrug resistance by green
tea polyphenols. J Pharm Pharmacol 56(10):1307-1314.
106.Netsch MI, Gutmann H, Luescher S, Brill S, Schmidlin CB, Kreuter MH, Drewe J
2005. Inhibitory activity of a green tea extract and some of its constituents on multidrug
resistance-associated protein 2 functionality. Planta Med 71(2):135-141.
107.Zhang L, Zheng Y, Chow MS, Zuo Z 2004. Investigation of intestinal absorption and
disposition of green tea catechins by Caco-2 monolayer model. Int J Pharm 287(1-2):112.
108.Hong J, Lambert JD, Lee SH, Sinko PJ, Yang CS 2003. Involvement of multidrug
resistance-associated proteins in regulating cellular levels of (-)-epigallocatechin-3gallate and its methyl metabolites. Biochem Biophys Res Commun 310(1):222-227.
109.Vaidyanathan JB, Walle T 2003. Cellular uptake and efflux of the tea flavonoid ()epicatechin-3-gallate in the human intestinal cell line Caco-2. J Pharmacol Exp Ther
307(2):745-752.
110.Fuchikami H, Satoh H, Tsujimoto M, Ohdo S, Ohtani H, Sawada Y 2006. Effects of
Herbal Extracts on the Function of Human Organic Anion Transporting Polypeptide,
OATP-B. Drug Metab Dispos.
111.Sartippour MR, Heber D, Ma J, Lu Q, Go VL, Nguyen M 2001. Green tea and its
catechins inhibit breast cancer xenografts. Nutr Cancer 40(2):149-156.
112. Zhang Q, Wei D, Liu J 2004. In vivo reversal of doxorubicin resistance by (-)epigallocatechin gallate in a solid human carcinoma xenograft. Cancer Lett 208(2):179186.
113.Attele AS, Wu JA, Yuan CS 1999. Ginseng pharmacology: multiple constituents and
multiple actions. Biochem Pharmacol 58(11):1685-1693.
114.Kim SW, Kwon HY, Chi DW, Shim JH, Park JD, Lee YH, Pyo S, Rhee DK 2003.
Reversal of P-glycoprotein-mediated multidrug resistance by ginsenoside Rg(3).
Biochem Pharmacol 65(1):75-82.
283
115.Choi CH, Kang G, Min YD 2003. Reversal of P-glycoprotein-mediated multidrug

resistance by protopanaxatriol ginsenosides from Korean red ginseng. Planta Med
69(3):235-240.
116.Jacobs BP, Dennehy C, Ramirez G, Sapp J, Lawrence VA 2002. Milk thistle for the
treatment of liver disease: a systematic review and meta-analysis. Am J Med 113(6):506515.
117.Rambaldi A, Jacobs BP, Iaquinto G, Gluud C 2005. Milk thistle for alcoholic and/or
hepatitis B or C liver diseases--a systematic cochrane hepato-biliary group review with
meta-analyses of randomized clinical trials. Am J Gastroenterol 100(11):2583-2591.
118. Bilia AR, Bergonzi MC, Gallori S, Mazzi G, Vincieri FF 2002. Stability of the
constituents of Calendula, milk-thistle and passionflower tinctures by LC-DAD and LCMS. J Pharm Biomed Anal 30(3):613-624.
119.Kvasnicka F, Biba B, Sevcik R, Voldrich M, Kratka J 2003. Analysis of the active
components of silymarin. J Chromatogr A 990(1-2):239-245.
120.Zhang S, Morris ME 2003. Effects of the flavonoids biochanin A, morin, phloretin,
and silymarin on P-glycoprotein-mediated transport. J Pharmacol Exp Ther 304(3):12581267.
121.Zhang S, Morris ME 2003. Effect of the flavonoids biochanin A and silymarin on the
P-glycoprotein-mediated transport of digoxin and vinblastine in human intestinal Caco-2
cells. Pharm Res 20(8):1184-1191.
122. Gurley BJ, Barone GW, Williams DK, Carrier J, Breen P, Yates CR, Song PF,
Hubbard MA, Tong Y, Cheboyina S 2006. Effect of milk thistle (Silybum marianum) and
black cohosh (Cimicifuga racemosa) supplementation on digoxin pharmacokinetics in
humans. Drug Metab Dispos 34(1):69-74.
123.DiCenzo R, Shelton M, Jordan K, Koval C, Forrest A, Reichman R, Morse G 2003.
Coadministration of milk thistle and indinavir in healthy subjects. Pharmacotherapy
23(7):866-870.
124.Piscitelli SC, Formentini E, Burstein AH, Alfaro R, Jagannatha S, Falloon J 2002.
Effect of milk thistle on the pharmacokinetics of indinavir in healthy volunteers.
Pharmacotherapy 22(5):551-556.
125.Mills E, Wilson K, Clarke M, Foster B, Walker S, Rachlis B, DeGroot N, Montori
VM, Gold W, Phillips E, Myers S, Gallicano K 2005. Milk thistle and indinavir: a
randomized controlled pharmacokinetics study and meta-analysis. Eur J Clin Pharmacol
61(1):1-7.
126. Smith NF, Figg WD, Sparreboom A 2006. Pharmacogenetics of irinotecan
metabolism and transport: An update. Toxicol In Vitro 20(2):163-175.
127.van Erp NP, Baker SD, Zhao M, Rudek MA, Guchelaar HJ, Nortier JW, Sparreboom
A, Gelderblom H 2005. Effect of milk thistle (Silybum marianum) on the
pharmacokinetics of irinotecan. Clin Cancer Res 11(21):7800-7806.
128.Singh YN 2005. Potential for interaction of kava and St. John's wort with drugs. J
Ethnopharmacol 100(1-2):108-113.
129.Bilia AR, Scalise L, Bergonzi MC, Vincieri FF 2004. Analysis of kavalactones from
Piper methysticum (kava-kava). J Chromatogr B Analyt Technol Biomed Life Sci 812(12):203-214.
284
130.Bilia AR, Bergonzi MC, Lazari D, Vincieri FF 2002. Characterization of commercial

kava-kava herbal drug and herbal drug preparations by means of nuclear magnetic
resonance spectroscopy. J Agric Food Chem 50(18):5016-5025.
131. Ulbricht C, Basch E, Boon H, Ernst E, Hammerness P, Sollars D, Tsourounis C,
Woods J, Bent S 2005. Safety review of kava (Piper methysticum) by the Natural
Standard Research Collaboration. Expert Opin Drug Saf 4(4):779-794.
132. Weiss J, Sauer A, Frank A, Unger M 2005. Extracts and kavalactones of Piper
methysticum G. Forst (kava-kava) inhibit P-glycoprotein in vitro. Drug Metab Dispos
33(11):1580-1583.
133.Mathews JM, Etheridge AS, Valentine JL, Black SR, Coleman DP, Patel P, So J,
Burka LT 2005. Pharmacokinetics and disposition of the kavalactone kawain: interaction
with kava extract and kavalactones in vivo and in vitro. Drug Metab Dispos 33(10):15551563.
134.Ross SA, Ziska DS, Zhao K, ElSohly MA 2000. Variance of common flavonoids by
brand of grapefruit juice. Fitoterapia 71(2):154-161.
135.Erlund I, Meririnne E, Alfthan G, Aro A 2001. Plasma kinetics and urinary excretion
of the flavanones naringenin and hesperetin in humans after ingestion of orange juice and
grapefruit juice. J Nutr 131(2):235-241.
136.Manach C, Morand C, Gil-Izquierdo A, Bouteloup-Demange C, Remesy C 2003.
Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of
two doses of orange juice. Eur J Clin Nutr 57(2):235-242.
137.Kuhnau J 1976. The flavonoids. A class of semi-essential food components: their
role in human nutrition. World Rev Nutr Diet 24:117-191.
138. Harborne JB, Williams CA 2000. Advances in flavonoid research since 1992.
Phytochemistry 55(6):481-504.
139.Scalbert A, Williamson G 2000. Dietary intake and bioavailability of polyphenols. J
Nutr 130(8S Suppl):2073S-2085S.
140.Vinson JA, Hao Y, Su X, Zubik L 1998. Phenol antioxidant quantity and quality in
foods: vegetables. J Agric Food Chem 46(9):3630-3634.
141.Vinson JA, Su X, Zubik L, Bose P 2001. Phenol antioxidant quantity and quality in
foods: fruits. J Agric Food Chem 49(11):5315-5321.
142. Middleton E, Jr., Kandaswami C, Theoharides TC 2000. The effects of plant
flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer.
Pharmacol Rev 52(4):673-751.
143.Havsteen BH 2002. The biochemistry and medical significance of the flavonoids.
Pharmacol Ther 96(2-3):67-202.
144. Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D 1993. Dietary
antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study.
Lancet 342(8878):1007-1011.
145. Kohno H, Tanaka T, Kawabata K, Hirose Y, Sugie S, Tsuda H, Mori H 2002.
azoxymethane-induced colon carcinogenesis in male F344 rats. Int J Cancer 101(5):461468.
285
146. Leslie EM, Mao Q, Oleschuk CJ, Deeley RG, Cole SP 2001. Modulation of
multidrug resistance protein 1 (MRP1/ABCC1) transport and atpase activities by
interaction with dietary flavonoids. Mol Pharmacol 59(5):1171-1180.
147. Critchfield JW, Welsh CJ, Phang JM, Yeh GC 1994. Modulation of adriamycin
accumulation and efflux by flavonoids in HCT-15 colon cells. Activation of Pglycoprotein as a putative mechanism. Biochem Pharmacol 48(7):1437-1445.
148.Scambia G, Ranelletti FO, Panici PB, De Vincenzo R, Bonanno G, Ferrandina G,
Piantelli M, Bussa S, Rumi C, Cianfriglia M, et al. 1994. Quercetin potentiates the effect
of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: Pglycoprotein as a possible target. Cancer Chemother Pharmacol 34(6):459-464.
149. Mitsunaga Y, Takanaga H, Matsuo H, Naito M, Tsuruo T, Ohtani H, Sawada Y
2000. Effect of bioflavonoids on vincristine transport across blood-brain barrier. Eur J
Pharmacol 395(3):193-201.
150. Chieli E, Romiti N, Cervelli F, Tongiani R 1995. Effects of flavonols on Pglycoprotein activity in cultured rat hepatocytes. Life Sci 57(19):1741-1751.
151. Shapiro AB, Ling V 1997. Effect of quercetin on Hoechst 33342 transport by
purified and reconstituted P-glycoprotein. Biochem Pharmacol 53(4):587-596.
152.Kitagawa S, Nabekura T, Takahashi T, Nakamura Y, Sakamoto H, Tano H, Hirai M,
Tsukahara G 2005. Structure-activity relationships of the inhibitory effects of flavonoids
on P-glycoprotein-mediated transport in KB-C2 cells. Biol Pharm Bull 28(12):22742278.
153. Shapiro AB, Ling V 1997. Positively cooperative sites for drug transport by Pglycoprotein with distinct drug specificities. Eur J Biochem 250(1):130-137.
154.Di Pietro A, Conseil G, Perez-Victoria JM, Dayan G, Baubichon-Cortay H, Trompier
D, Steinfels E, Jault JM, de Wet H, Maitrejean M, Comte G, Boumendjel A, Mariotte
AM, Dumontet C, McIntosh DB, Goffeau A, Castanys S, Gamarro F, Barron D 2002.
Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and
related ABC transporters. Cell Mol Life Sci 59(2):307-322.
155.Boumendjel A, Di Pietro A, Dumontet C, Barron D 2002. Recent advances in the
discovery of flavonoids and analogs with high-affinity binding to P-glycoprotein
responsible for cancer cell multidrug resistance. Med Res Rev 22(5):512-529.
156. Kitagawa S 2006. Inhibitory effects of polyphenols on p-glycoprotein-mediated
transport. Biol Pharm Bull 29(1):1-6.
157. Choi JS, Jo BW, Kim YC 2004. Enhanced paclitaxel bioavailability after oral
administration of paclitaxel or prodrug to rats pretreated with quercetin. Eur J Pharm
Biopharm 57(2):313-318.
158. Dupuy J, Larrieu G, Sutra JF, Lespine A, Alvinerie M 2003. Enhancement of
moxidectin bioavailability in lamb by a natural flavonoid: quercetin. Vet Parasitol
112(4):337-347.
159. Hsiu SL, Hou YC, Wang YH, Tsao CW, Su SF, Chao PD 2002. Quercetin
72(3):227-235.
286
160.Trompier D, Baubichon-Cortay H, Chang XB, Maitrejean M, Barron D, Riordon JR,

Di Pietro A 2003. Multiple flavonoid-binding sites within multidrug resistance protein
MRP1. Cell Mol Life Sci 60(10):2164-2177.
161.van Zanden JJ, Geraets L, Wortelboer HM, van Bladeren PJ, Rietjens IM, Cnubben
NH 2004. Structural requirements for the flavonoid-mediated modulation of glutathione
S-transferase P1-1 and GS-X pump activity in MCF7 breast cancer cells. Biochem
Pharmacol 67(8):1607-1617.
162.van Zanden JJ, Wortelboer HM, Bijlsma S, Punt A, Usta M, Bladeren PJ, Rietjens
IM, Cnubben NH 2005. Quantitative structure activity relationship studies on the
flavonoid mediated inhibition of multidrug resistance proteins 1 and 2. Biochem
Pharmacol 69(4):699-708.
163.Lania-Pietrzak B, Michalak K, Hendrich AB, Mosiadz D, Grynkiewicz G, Motohashi
N, Shirataki Y 2005. Modulation of MRP1 protein transport by plant, and synthetically
modified flavonoids. Life Sci 77(15):1879-1891.
164.Imai Y, Tsukahara S, Asada S, Sugimoto Y 2004. Phytoestrogens/flavonoids reverse
breast cancer resistance protein/ABCG2-mediated multidrug resistance. Cancer Res
64(12):4346-4352.
165.Zhang S, Yang X, Coburn RA, Morris ME 2005. Structure activity relationships and
quantitative structure activity relationships for the flavonoid-mediated inhibition of breast
cancer resistance protein. Biochem Pharmacol 70(4):627-639.
166.Ahmed-Belkacem A, Pozza A, Munoz-Martinez F, Bates SE, Castanys S, Gamarro
F, Di Pietro A, Perez-Victoria JM 2005. Flavonoid structure-activity studies identify 6prenylchrysin and tectochrysin as potent and specific inhibitors of breast cancer
resistance protein ABCG2. Cancer Res 65(11):4852-4860.
167.Hecht SS 1995. Chemoprevention by isothiocyanates. J Cell Biochem Suppl 22:195209.
168.Talalay P, Fahey JW 2001. Phytochemicals from cruciferous plants protect against
cancer by modulating carcinogen metabolism. J Nutr 131(11 Suppl):3027S-3033S.
169.Hecht SS 2000. Inhibition of carcinogenesis by isothiocyanates. Drug Metab Rev
32(3-4):395-411.
170.Kelloff GJ, Crowell JA, Steele VE, Lubet RA, Malone WA, Boone CW, Kopelovich
L, Hawk ET, Lieberman R, Lawrence JA, Ali I, Viner JL, Sigman CC 2000. Progress in
cancer chemoprevention: development of diet-derived chemopreventive agents. J Nutr
130(2S Suppl):467S-471S.
171.Tseng E, Kamath A, Morris ME 2002. Effect of organic isothiocyanates on the Pglycoprotein- and MRP1-mediated transport of daunomycin and vinblastine. Pharm Res
19(10):1509-1515.
172. Hu K, Morris ME 2004. Effects of benzyl-, phenethyl-, and alpha-naphthyl
isothiocyanates on P-glycoprotein- and MRP1-mediated transport. J Pharm Sci
93(7):1901-1911.
173.Ji Y, Morris ME 2005. Transport of dietary phenethyl isothiocyanate is mediated by
multidrug resistance protein 2 but not P-glycoprotein. Biochem Pharmacol 70(4):640647.
287
174.Ji Y, Morris ME 2004. Effect of organic isothiocyanates on breast cancer resistance

protein (ABCG2)-mediated transport. Pharm Res 21(12):2261-2269.
175.Ikegawa T, Ushigome F, Koyabu N, Morimoto S, Shoyama Y, Naito M, Tsuruo T,
Ohtani H, Sawada Y 2000. Inhibition of P-glycoprotein by orange juice components,
polymethoxyflavones in adriamycin-resistant human myelogenous leukemia
(K562/ADM) cells. Cancer Lett 160(1):21-28.
176. Sadzuka Y, Sugiyama T, Sonobe T 2000. Efficacies of tea components on
doxorubicin induced antitumor activity and reversal of multidrug resistance. Toxicol Lett
114(1-3):155-162.
177. Tseng E, Liang W, Wallen C, Morris ME 2001. Effect of flavonoids on P-gp
mediated transport in a human breast cancer cell line. AAPS PharmSci 3:S2081.
178. Chung SY, Sung MK, Kim NH, Jang JO, Go EJ, Lee HJ 2005. Inhibition of Pglycoprotein by natural products in human breast cancer cells. Arch Pharm Res
28(7):823-828.
179.Kitagawa S, Nabekura T, Kamiyama S 2004. Inhibition of P-glycoprotein function
by tea catechins in KB-C2 cells. J Pharm Pharmacol 56(8):1001-1005.
180.Qian F, Wei D, Zhang Q, Yang S 2005. Modulation of P-glycoprotein function and
reversal of multidrug resistance by (-)-epigallocatechin gallate in human cancer cells.
Biomed Pharmacother 59(3):64-69.
181. Castro AF, Altenberg GA 1997. Inhibition of drug transport by genistein in
multidrug-resistant cells expressing P-glycoprotein. Biochem Pharmacol 53(1):89-93.
182.Versantvoort CH, Schuurhuis GJ, Pinedo HM, Eekman CA, Kuiper CM, Lankelma J,
Broxterman HJ 1993. Genistein modulates the decreased drug accumulation in non-Pglycoprotein mediated multidrug resistant tumour cells. Br J Cancer 68(5):939-946.
183. Hooijberg JH, Broxterman HJ, Scheffer GL, Vrasdonk C, Heijn M, de Jong MC,
Scheper RJ, Lankelma J, Pinedo HM 1999. Potent interaction of flavopiridol with MRP1.
Br J Cancer 81(2):269-276.
184. Leslie EM, Deeley RG, Cole SP 2003. Bioflavonoid stimulation of glutathione
transport by the 190-kDa multidrug resistance protein 1 (MRP1). Drug Metab Dispos
31(1):11-15.
185.Hooijberg JH, Broxterman HJ, Heijn M, Fles DL, Lankelma J, Pinedo HM 1997.
Modulation by (iso)flavonoids of the ATPase activity of the multidrug resistance protein.
FEBS Lett 413(2):344-348.
186.van Zanden JJ, de Mul A, Wortelboer HM, Usta M, van Bladeren PJ, Rietjens IM,
Cnubben NH 2005. Reversal of in vitro cellular MRP1 and MRP2 mediated vincristine
resistance by the flavonoid myricetin. Biochem Pharmacol 69(11):1657-1665.
187.Kauffmann HM, Pfannschmidt S, Zoller H, Benz A, Vorderstemann B, Webster JI,
Schrenk D 2002. Influence of redox-active compounds and PXR-activators on human
MRP1 and MRP2 gene expression. Toxicology 171(2-3):137-146.
188.Wu CP, Calcagno AM, Hladky SB, Ambudkar SV, Barrand MA 2005. Modulatory
effects of plant phenols on human multidrug-resistance proteins 1, 4 and 5 (ABCC1, 4
and 5). Febs J 272(18):4725-4740.
189. Versantvoort CH, Broxterman HJ, Lankelma J, Feller N, Pinedo HM 1994.
Competitive inhibition by genistein and ATP dependence of daunorubicin transport in
288
intact MRP overexpressing human small cell lung cancer cells. Biochem Pharmacol
48(6):1129-1136.
190.Takeda Y, Nishio K, Niitani H, Saijo N 1994. Reversal of multidrug resistance by
tyrosine-kinase inhibitors in a non-P-glycoprotein-mediated multidrug-resistant cell line.
Int J Cancer 57(2):229-239.
289
Table 1. Effects of dietary supplements on drug transporters

Dietary
Supplements
In Vitro/
In Vivo
St. Johns
wort
In vitro
Grapefruit
juice
Substrate
Mechanisms
Ref.
Rhodamine-123
Accumulation
Inhibition of P-gp after acute

exposure
43
Ritonavir
Induction of P-gp after chronic

exposure
Inhibition of P-gp
43,44
In vitro
Accumulation due to
P-gp protein level
Efflux
In vitro
Daunorubicin/
Calcein-AM
Efflux
Inhibition of P-gp
49
In vitro
MRP2 mRNA level
In vivo
P-gp protein level
In vivo
MRP2 protein level
48
50
Activation of PXR
31
51
In vivo
Digoxin
AUC, Cmax
Induction of P-gp
24,31,53
In vivo
Fexofenadine
Cmax, Clearance
Inhibition of P-gp
(single dose)
57
Cmax, AUC
59
In vivo
Indinavir
AUC, Ctrough
Induction of P-gp
(chronic treatment)
Induction of P-gp/CYP3A4
In vivo
Cyclosporine
AUC, Cplasma
60,61
In vivo
Tacrolimus
AUC, Clearance
64
In vitro
Talinolol/Digoxin
Absorptive Transport
Inhibition of P-gp
72,74
In vitro
Efflux
Inhibition of P-gp
73,77,78
In vitro
Rhodamine-123/
Fexofenadine/
Saquinavir
Vinblastine
Efflux or Uptake
Inhibition of P-gp
71,79
In vitro
Vincristine
Uptake
Inhibition of P-gp
175
In vitro
In vitro
Garlic
Effects
P-gp protein and

mRNA levels
Efflux
Inhibition of MRP2/P-gp
75
Uptake
Inhibition of OATP/oatp
25
80
In vitro
Vinblastine/
Saquinavir
Fexofenadine
In vitro
Estrone-3-sulfate
Uptake
Inhibition of OATP-B
81
In vivo
Cyclosporine
AUC, Cmax
Inhibition of P-gp/CYP3A4
82-84
In vivo
Talinolol
Inhibition of P-gp
See text
72
Inhibition of OATP
25,91
In vivo
Fexofenadine
AUC, Cmax
AUC, Cmax
AUC, Cmax
In vivo
Digoxin
Cmax, AUC
See text
87,88
In vitro
In vitro
Ritonavir
Efflux
P-gp protein level
Inhibition of P-gp
48
290
86
96
Green tea
In vivo
P-gp protein level
96
In vivo
Mrp2 protein level
97
In vivo
Saquinavir/
Ritonavir
AUC, Cmax
98,99
In vitro
In vitro
Rhodamine-123
Doxorubicin
Accumulation
Efflux
Inhibition of P-gp
Inhibition of P-gp
104
In vitro
Methotrexate
P-gp gene expression

ATPase activity
Efflux
Inhibition of MRP2
106
In vitro
Estrone-3-sulfate
Uptake
Inhibition of OATP-B
110
In vitro
Rhodamine-123/
Vinblastine
Accumulation
Inhibition of P-gp
114
Azidopine
Daunorubicin
P-gp mRNA or protein

levels
Photoaffinity labeling
Accumulation
Inhibition of P-gp
115
Azidopine
P-gp protein level

Inhibition of P-gp
120
Inhibition of P-gp
121
Inhibition of MRP1
32
In vitro
Ginseng
In vitro
Milk Thistle
In vitro
In vivo
Azidopine
Digoxin/
Vinblastine
Daunomycin/
Vinblastine
Digoxin
In vivo
In vitro
In vitro
Kava
Daunomycin
Accumulation
ATPase activity
Efflux/
Accumulation
Accumulation
176
105
Cmax, AUC
122
Indinavir
Cmax, AUC
123-125
In vivo
Irinotecan
Cmax, AUC
127
In vitro
In vivo
Calcein-AM
Kawain
Accumulation
AUC, Cmax
291
Inhibition of P-gp
Inhibition of P-gp/CYP3A4
132
133
Table 2. Chemical structures of subclasses of flavonoids

Structural
formula
Representative
flavonoids
Substitutions
5
Apigenin
Chrysin
Luteolin
Diosmetin
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
OH
OH
OH
H
OH
O-Me
H
H
H
H
Fisetin
Galangin
Kaempferol
Morin
Myricetin
Quercetin
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
OH
H
H
OH
H
H
H
OH
OH
OH
H
OH
OH
OH
OH
H
H
H
H
OH
H
Hesperitin
Naringenin
OH
OH
OH
OH
H
H
OH
H
O-Me
OH
H
H
Epicatechin
Epigallocatechin
OH
OH
OH
OH
H
H
OH
OH
OH
OH
H
OH
Silibinin
OH
OH
Selane
Biochanin A
Genistein
Daidzein
OH
OH
H
OH
OH
OH
H
H
H
H
H
H
O-Me
OH
OH
H
H
H
Flavones
3'
4'
2'
O
7
6
5'
5
O
Flavonols
O
OH
O
Flavanones
O
Flavanols
O
OH
Flavanolols
O
OH
O
Isoflavones
O
O-Me = Methoxy
292
Table 3. Interactions of flavonoids with P-gp

Flavonoid
Cell Line Used
Substrates
Flavones
Apigenin
Daunomycin
Activity
177

Daunomycin
Vincristine
Activity
Activity
177
Luteolin
Daunomycin
Activity
177
Diosmin
Daunomycin
Activity
177

Daunorubicin
Daunomycin
Activity
Activity
152

Rat hepatocytes
Daunomycin
Adriamycin
Expression
or Activity
Activity
177
Rhodamine-123
Daunorubicin
Daunomycin
Vincristine
Rhodamine-123/
Doxorubicin
Adriamycin
Activity
Activity
Activity
Biphasic effect
or Activity
Activity
152
152
Chrysin
Flavonols
Fisetin
Galangin
HCT-15 colon cells

Kaempferol

Rat hepatocytes
HCT-15 colon cells
Effect on P-gp
Ref.
149
177
150
147
177
149
150
147

cancer cells
Daunorubicin
Daunomycin
Activity
Activity
Daunomycin
Activity

Daunorubicin
Daunomycin
Activity
Activity
152
Rhodamine-123
Daunorubicin
Daunomycin
Adriamycin
Rhodamine-123
Vincristine
Hoechst 33342
Adriamycin
Activity
Activity
Activity
Activity
152
Biphasic effect
Activity
or Activity
Activity
149
Activity
Activity
Activity
Activity
152

Rhodamine-123
Daunorubicin
Daunomycin
Vincristine

Vincristine
Daunomycin
Activity
Activity
177
Hesperidin
Vincristine
Activity
149
Naringenin

Daunorubicin
Daunomycin
Activity
Activity
152
Morin
Myricetin
Quercetin

CHRC5
Rat hepatocytes
HCT-15 colon cells
Rutin
Flavanones
Hesperitin
293
120
177
177
177,178
148
151
150
147
177
149
149
178
Naringin
Flavanols
Catechin
Vincristine
Daunomycin
Vincristine
Activity
Activity
Activity
Activity
177

CHRC5 cells
Rhodamine-123
Activity
104
149
177
149
CG
CH C5 cells
Rhodamine-123
Activity
104
Epicatechin
Rhodamine-123
Daunorubicin
Rhodamine-123
Activity
Activity
Activity
179
Rhodamine-123
Daunorubicin
Rhodamine-123
Activity
Activity
Activity
179
Rhodamine-123
Daunorubicin
Rhodamine-123
Daunomycin
Activity
Activity
Activity
Activity
179
Doxorubicin
Rhodamine-123
Daunorubicin
Rhodamine-123
180
Daunomycin
Activity
Digoxin
Vinblastine
Daunomycin
Activity
Activity
Activity
Daunomycin
Activity
Digoxin
Vinblastine
Daunomycin
Activity
Activity
Activity
Daunomycin
Rhodamine-123
Daunorubicin
Activity
Activity
CHRC5 cells
ECG

CHRC5 cells
EGC

CHRC5 cells
EGCG
KB-A1 carcinoma cells

CHRC5 cells
Flavanolols
Silibinin/
silymarin

cancer cells
Caco-2 cells
Isoflavones
Biochanin A

cancer cells
Caco-2 cells
Genistein

BC19/3 breast cancer cells
294
Activity
Activity
Activity
Activity
104
104
104
177
179
104
120,178
121
177
120,178
121
177
177
181
Table 4. Interactions of flavonoids with MRPs

Flavonoid
Flavones
Apigenin
Chrysin
Luteolin
Diosmetin
Diosmin
Flavonols
Fisetin
Galangin
Kaempferol
Cell Line Used
Substrates

GLC4/ADR lung cancer cells
/membrane vesicles (MRP1)
Panc-1 cells (MRP1)
Daunorubicin
Daunorubicin
Human erythrocytes
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
MCF7-pMTG5 cells
MDCK-MRP1
MDCK-MRP2
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
Panc-1 cells (MRP1)
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
MCF7-pMTG5 cells
MDCK-MRP1
MDCK-MRP2
GLC4/ADR lung cancer cells/
membrane vesicles (MRP1)
Panc-1 cells (MRP1)

MCF7-pMTG5 cells
MDCK-MRP1
MDCK-MRP2
Glutathione
LTC4/E217G
DNM
VBL
BCPCF
Calcein
Calcein
DNM/VBL
Calcein
Calcein
DNM
VBL
DNP-SG
Calcein
Calcein
Calcein
Calcein
DNM/VBL
DNM/VBL
Calcein
Calcein
DNM
VBL
DNP-SG
Calcein
Calcein
Daunorubicin
LTC4/E217G
Glutathione
DNM/VBL
DNP-SG
Calcein
Calcein
DNM/VBL
Morin
Panc-1 cells (MRP1)
Myricetin
MCF7-pMTG5 cells
Human erythrocytes
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
295
DNP-SG
BCPCF
Calcein
Calcein
LTC4
E217G
Glutathione
Effect on MRPs
Ref.
Accumulation
Accumulation
ATPase Activity
GSH transport
Transport
Accumulation
Accumulation
Efflux via MRP1
Efflux
Efflux
Accumulation
Efflux
Efflux
Accumulation
Accumulation
Efflux via MRP1
Efflux
Efflux
Efflux
Efflux
Accumulation
182
Accumulation
Efflux
Efflux
Accumulation
Accumulation
Efflux via MRP1
Efflux
Efflux
ATPase Activity
Accumulation
ATPase Activity
Transport
GSH transport
ATPase Activity
ADP trapping
Accumulation
Efflux via MRP1
Efflux
Efflux
Accumulation
GSH level
Efflux via MRP1
Efflux via MRP1
Efflux
Efflux
Transport
Transport
GSH transport
32
183
146,184
146
32
163
162
32
162
32
161
162
162
32
162
32
161
162
185
183
146
32
161
162
32
161
163
162
146

DNM
VBL
DNP-SG
Vincristine
Calcein
Daunorubicin
Glutathione
LTC4/E217G

Panc-1 cells (MRP1)
DNM/VBL
MCF7-pMTG5 cells
MDCK-MRP1/2
MDCK-MRP1/2
Quercetin
MCF7-pMTG5 cells
HEK293-MRP1
HEK293-MRP5
MDCK-MRP1
MDCK-MRP2
Rutin
Flavanones
Hesperitin
Naringenin
Naringin
Flavanols
Catechin
EGC
Flavanolols
Silibinin/
silymarin
DNP-SG
DNP-SG
cGMP
Calcein
BCECF
Calcein
Calcein
Accumulation
Accumulation
Efflux via MRP1
Efflux
Efflux
Accumulation
GSH transport
Transport
ATPase Activity
ADP trapping
MRP1 mRNA level
Accumulation
GSH level
Efflux via MRP1
Transport via
MRP1
Transport via
MRP4
Accumulation
Accumulation
Efflux
Efflux
Accumulation
Accumulation
32
161
186
162
182
146,184
146
187
32
161
188
162
Panc-1 cells (MRP1)
DNM
VBL
Panc-1 cells (MRP1)

HEK293-MRP1
HEK293-MRP5
DNM/VBL
DNP-SG
cGMP
Calcein
BCECF

Glutathione
LTC4/E217G
Panc-1 cells (MRP1)

HEK293-MRP1
HEK293-MRP5
DNM/VBL
DNP-SG
cGMP
Calcein
BCECF

Panc-1 cells (MRP1)
LTC4
DNM/VBL
MCF7-pMTG5 cells
MDCK-MRP1
MDCK-MRP2
Panc-1 cells (MRP1)
DNP-SG
Calcein
Calcein
DNM
VBL
Efflux via MRP1

Efflux
Efflux
Accumulation
Accumulation
161
Panc-1 cells (MRP1)
DNM/VBL
32
Vesicles from BHK-21-MRP1 cells

Human erythrocytes
LTC4
BCPCF
Accumulation
GSH level
Transport
Efflux via MRP1
296
Accumulation
Transport via
MRP1
Transport via
MRP4
Accumulation
Accumulation
GSH transport
Transport
ATPase Activity
ADP trapping
Accumulation
Transport via
MRP1
Transport via
MRP4
Accumulation
Accumulation
Transport
Accumulation
32
32
188
146,184
146
32
188
146
32
162
32
160
Isoflavones
Biochanin A
Genistein
Genistin
HEK293-MRP1
HEK293-MRP5
DNP-SG
cGMP
Calcein
BCECF

Panc-1 cells (MRP1)
Daunorubicin
DNM/VBL
Daunorubicin
K562/TPA leukemia cells (MRP1)

Panc-1 cells (MRP1)
Adriamycin
LTC4
Glutathione
DNM/VBL
Human erythrocytes
BCPCF

297
Daunorubicin
Daunorubicin
LTC4
Transport via
MRP1
Transport via
MRP4
Accumulation
Accumulation
163
Accumulation
Accumulation
GSH level
Accumulation
Transport
Accumulation
ATPase Activity
Accumulation
ATPase Activity
Transport
GSH transport
Accumulation
GSH level
Efflux via MRP1
182
ATPase Activity
Accumulation
ATPase Activity
Transport
185
188
32
182
189
190
185
183
146
184
32
163
183
146
Table 5. Interactions of flavonoids with BCRP

Flavonoid
Cell Line Used
Substrates
Effect on BCRP
MCF-7/MX100 and NCI-H460 MX20

K562/BCRP
Mitoxantrone
SN38
Accumulation
Reverse resistance
34

K562/BCRP
Mitoxantrone
SN38
Accumulation
Reverse resistance
34

K562/BCRP
Mitoxantrone
SN38
Accumulation
Reverse resistance
34
Diosmetin
K562/BCRP
SN38
Reverse resistance
164
Diosmin
K562/BCRP
SN38
No effects
164

K562/BCRP
Mitoxantrone
SN38
Accumulation
No effects
34
Galangin
K562/BCRP
SN38
Reverse resistance
164
Kaempferol

K562/BCRP
Mitoxantrone
Mitoxantrone/SN38
Accumulation
Reverse resistance
34
Morin
Mitoxantrone
Accumulation
34
Myricetin

K562/BCRP
Mitoxantrone
SN38
Accumulation
No effects
34

Accumulation
Accumulation
34
K562/BCRP
Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Moderate reversal
164
K562/BCRP
SN38
No effects
164

Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Accumulation
Accumulation
34
Reverse resistance
Accumulation
Reverse resistance
Accumulation
Accumulation
34
Mitoxantrone
Mitoxantrone/SN38
Topotecan
Mitoxantrone
K562/BCRP
SN38
No effects
164
EGC
Mitoxantrone
Accumulation
34
EGCG
Mitoxantrone
Accumulation
34
Flavanolols
Silibinin/
silymarin

Accumulation
Accumulation
34
K562/BCRP
Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Moderate reversal
164
Isoflavones
Biochanin A
Mitoxantrone
Accumulation
34
Genistein
Mitoxantrone
Accumulation
34
Flavones
Apigenin
Chrysin
Luteolin
Flavonols
Fisetin
Quercetin
Rutin
Flavanones
Hesperitin
K562/BCRP
Naringenin
Naringin
Flavanols
Catechin

K562/BCRP
298
Ref
164
164
164
164
164
164
33
33
164
164
34
33
K562/BCRP
Daidzein

K562/BCRP
299
Mitoxantrone/SN38
Topotecan
Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Reversal resistance
Accumulation
Accumulation
Accumulation
Moderate reversal
164
34
33
164
Table 6. Interactions of flavonoids with OATPs

Flavonoid
Cell Line Used
Substrates
Effect on OATPs
Ref.
Flavones
Apigenin
Hela/OATP-C
DHEAS
Uptake
36
Chrysin
Hela/OATP-C
DHEAS
Uptake
36
Luteolin
Hela/OATP-C
DHEAS
Uptake
36
Diosmetin
Hela/OATP-C
DHEAS
Uptake
36
Diosmin
Hela/OATP-C
DHEAS
Uptake
36
Flavonols
Fisetin
Hela/OATP-C
DHEAS
Uptake
36
Galangin
Hela/OATP-C
DHEAS
Uptake
36
Kaempferol
Hela/OATP-C
DHEAS
Uptake
36
Morin
Hela/OATP-C
DHEAS
Uptake
36
Myricetin
Hela/OATP-C
DHEAS
Uptake
36
Quercetin
Hela/OATP-C
DHEAS
Uptake
36
Rutin
Hela/OATP-C
HEK/OATP-B
DHEAS
Estrone-3-sulfate
Uptake
Uptake
36
Flavanones
Hesperitin
Hela/OATP-C
DHEAS
Uptake
36
Hesperidin
Hela/OATP-C
DHEAS
Uptake
36
Naringenin
Hela/OATP-C
DHEAS
Uptake
36
Naringin
Hela/OATP-C
DHEAS
Uptake
36
Flavanols
Catechin
HEK/OATP-B
Estrone-3-sulfate
Uptake
110
Epicatechin
HEK/OATP-B
Estrone-3-sulfate
Uptake
110
EGC
Hela/OATP-C
HEK/OATP-B
DHEAS
Estrone-3-sulfate
Uptake
Uptake
36
ECG
HEK/OATP-B
Estrone-3-sulfate
Uptake
110
EGCG
Hela/OATP-C
HEK/OATP-B
DHEAS
Estrone-3-sulfate
Uptake
Uptake
36
110
110
110
Flavanolols
Silibinin/
silymarin
Isoflavones
Biochanin A
Hela/OATP-C
DHEAS
Uptake
36
Hela/OATP-C
DHEAS
Uptake
36
Genistein
Hela/OATP-C
DHEAS
Uptake
36
Genistin
Hela/OATP-C
DHEAS
Uptake
36
Daidzein
Hela/OATP-C
DHEAS
Uptake
36
Daidzin
Hela/OATP-C
DHEAS
Uptake
36
300
Figure 1. Schematic interactions of flavonoids with Pglycoprotein and related multidrug resistance transporters
(Reproduced from DiPietro et al. 154 with permission from
Birkhauser Verlag AG).
Flavonols, such as kaempferide, quercetin, and galangin, display

bifunctional interactions with NBDs, at both the ATP-binding site and the
hydrophobic steroid-binding region. Prenylation of flavonoids would
greatly increase the hydrophobicity of flavonoids, shifting the flavonol
binding outside the ATP-binding site to vicinal steroid-binding region and
TMD drug binding site.
301

(Desertasi) Transporter-Mediated Flavonoid-Drug Interactions - in Vitro and in Vivo PDF

Hochgeladen von

Dokumentinformationen

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

(Desertasi) Transporter-Mediated Flavonoid-Drug Interactions - in Vitro and in Vivo PDF

Hochgeladen von

Copyright:

Verfügbare Formate

Transporter-mediated Flavonoid-Drug

Interactions: In vitro and In vivo

A dissertation submitted to the Faculty of the Graduate School of

Department of Pharmaceutical Sciences

UMI Number: 3277779

UMI Microform 3277779

ProQuest Information and Learning Company

Flavonoids as a Novel Class of Human Organic Anion Transporting 32

Monocarboxylate Transporter 1 (MCT1)-Mediated

Pharmacokinetic Interaction for the Flavonoid Luteolin: Model

Effects of the Flavonoid Chrysin on Nitrofurantoin

Pharmacokinetics in Rats: Potential Involvement of ABCG2.

Pharmacokinetics and Bioavailability of 7,8-Benzoflavone in

ABCG2-Mediated Transport of the Flavonoids Biochanin A and

Lack of Interactions between the Flavonoid Biochanin A and P-

glycoprotein Substrates Doxorubicin and Cyclosporine A in Rats.

Diet/nutrient Interactions with Drug Transporters.

pharmacokinetics of P-glycoprotein substrates doxorubicin and cyclosporine A,

In summary, this dissertation characterized the flavonoid-drug interactions mediated by

Flavonoids are a class of polyphenolic compounds widely present in fruits, vegetables,

expected, raising a potential for flavonoid-drug interactions. Recently, flavonoid-drug

P-gp-Mediated Flavonoid-Drug Interactions

demonstrated P-gp-modulating activities. However, the effects of flavonoids, especially

mg/kg) in a dose-dependent manner. At a dose of 20 mg/kg, quercetin administration

MRP-Mediated Flavonoid-Drug Interactions

potential mechanisms of MRPs reversal might involve: 1) decreasing intracellular

A number of structure-activity relationship studies indicated that flavones and flavonols

BCRP-Mediated Flavonoid-Drug Interactions

clinical studies, inhibition of BCRP by GF120918 has been shown to significantly

Physiological Relevance of Flavonoid Interactions

(equivalent to 330-740 M), respectively.

many flavonoids required to produce significant modulation on activities of these

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and

81.Tseng E, Liang W, Wallen C, Morris ME 2001. Effect of flavonoids on P-gp mediated

Table 1. Chemical structures of subclasses of flavonoids

Table 2. Interactions of flavonoids with P-gp

Cell Line Used

MCF-7 breast cancer cells

MCF-7 breast cancer cells

MCF-7 breast cancer cells

MCF-7 breast cancer cells

KB-C2 carinoma cells

MCF-7 breast cancer cells

HCT-15 colon cells

KB-C2 carinoma cells

KB-C2 carinoma cells

KB-C2 carinoma cells

KB-C2 carinoma cells

MCF-7 breast cancer cells

MBEC4 endothelial cells

MBEC4 endothelial cells

KB-C2 carinoma cells

MCF-7 breast cancer cells

MBEC4 endothelial cells

KB-C2 carinoma cells

MBEC4 endothelial cells

KB-C2 carinoma cells

KB-C2 carinoma cells

KB-C2 carinoma cells

KB-A1 carcinoma cells

MCF-7 and MDA435/LCC6 breast

MCF-7 and MDA435/LCC6 breast

MCF-7 breast cancer cells