Professional Documents
Culture Documents
By
Xiaodong Wang
August 2007
TABLE OF CONTENTS
Acknowledgements
iii
Abstract
iv
Chapter 1
Introduction
Chapter 2
Chapter 3
68
110
143
Rats.
Chapter 6
175
Kaempferol.
Chapter 7
204
Chapter 8
Conclusions
234
Appendix
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
vi
CHAPTER 1
INTRODUCTION
FlavonoidsDrug Interactions: Effects of Flavonoids on
Drug Transporters
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
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.
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
. 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
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.
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
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.
12
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.
2,3
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
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
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22
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
Substrates
Flavones
Apigenin
Daunomycin
Activity
81
Daunomycin
Vincristine
Activity
Activity
81
Luteolin
Daunomycin
Activity
81
Diosmin
Daunomycin
Activity
81
Daunorubicin
Daunomycin
Activity
Activity
50
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
(Substrate-dependent)
Activity
50
50
Chrysin
Flavonols
Fisetin
Galangin
Effect on P-gp
Ref
47
81
48
45
81
47
48
45
Daunorubicin
Daunomycin
Activity
Activity
Daunomycin
Activity
Daunorubicin
Daunomycin
Activity
Activity
50
Rhodamine-123
Daunorubicin
Daunomycin
Adriamycin
Rhodamine-123
Vincristine
Hoechst 33342
Rhodamine123/Doxorubicin
Adriamycin
Activity
Activity
Activity
Activity
50
Biphasic effect
Activity
or Activity
(Substrate-dependent)
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
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
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
EGC
EGCG
Flavanolols
Silibinin/
silymarin
Isoflavones
Biochanin A
Genistein
25
Activity
Activity
Activity
Activity
84
84
84
81
85
84
82,83
87
81
82,83
87
81
81
88
Chrysin
Luteolin
Diosmetin
Diosmin
Flavonols
Fisetin
Galangin
Kaempferol
Substrates
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)
Vesicles from Hela-MRP1 cells
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
DNM/VBL
DNP-SG
Calcein
Calcein
DNM/VBL
Myricetin
MCF7-pMTG5 cells
Human erythrocytes
MDCK-MRP1
MDCK-MRP2
Vesicles from Hela-MRP1 cells
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
GLC4/ADR lung cancer cells (MRP1)
Vesicles from Hela-MRP1 cells
Vesicles from Hela-MRP1 cells
E217G
Glutathione
DNM
VBL
DNP-SG
Vincristine
Calcein
Daunorubicin
Glutathione
LTC4/E217G
DNM/VBL
Quercetin
MCF7-pMTG5 cells
Vesicles from human erythrocytes
HEK293-MRP1
HEK293-MRP5
MDCK-MRP1
MDCK-MRP2
Rutin
Flavanones
Hesperitin
Naringenin
DNM
VBL
DNM/VBL
DNP-SG
cGMP
Calcein
BCECF
Glutathione
LTC4/E217G
HEK293-MRP1
HEK293-MRP5
Vesicles from Hela-MRP1 cells
Vesicles from Hela-MRP1 cells
Panc-1 cells (MRP1)
Vesicles from human erythrocytes
Naringin
Flavanols
Catechin
EGC
Flavanolols
Silibinin/
silymarin
DNP-SG
DNP-SG
cGMP
Calcein
BCECF
Calcein
Calcein
HEK293-MRP1
HEK293-MRP5
Vesicles from Hela-MRP1 cells
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
DNM/VBL
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
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
Daunorubicin
DNM/VBL
Daunorubicin
Adriamycin
LTC4
Glutathione
DNM/VBL
Human erythrocytes
BCPCF
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
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
MCF-7/MR and K562/BCRP
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
MCF-7/MR and K562/BCRP
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
MCF-7/MR and K562/BCRP
K562/BCRP
30
Mitoxantrone/SN38
Topotecan
Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Reversal resistance
Accumulation
Accumulation
Accumulation
Moderate reversal
74
30
28
74
MCT1
MCT1
31
CHAPTER 2
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
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).
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
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
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
I max C
IC50 + C
),
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
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).
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).
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
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.
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.
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).
52
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Cvetkovic M, Leake B, Fromm MF, Wilkinson GR and Kim RB (1999) OATP and Pglycoprotein transporters mediate the cellular uptake and excretion of
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porcine LLC-PK1 cells that co-express human OATP1B1 (OATP-C) and MRP2.
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55
TABLE 1.
Effects of flavonoids on the uptake of [3H]DHEAS in both OATP1B1-expressing and
OATP1B1-negative cells
OATP1B1-expressing cells
OATP1B1-negative cells
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
OATP1B1-negative 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).
60
epigallocatechin-3-gallate. *, P < 0.05, **, P < 0.01 compared with the control of either
OATP1B1-expressing or OATP1B1-negative cells.
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
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
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).
70
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).
72
73
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).
75
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
( 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 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
(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
(6)
GFR
( IC = Dose)
dX GHB
Vmax X GHB
=
dt
k m VGHB + X GHB
(
GFR
VGHB
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.
Statistical Analysis
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).
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).
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.
85
DISCUSSION
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
89
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
92
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O, Izeki S and Tsuji A (1999) Immunohistochemical and functional
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94
95
Control
Luteolin (4 mg/kg)
Control
(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
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
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
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
-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 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
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.
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
This chapter has been published in Drug Metab Dispos, 35(2): 268-74, 2007.
110
ABSTRACT
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.
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
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
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
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
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
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 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.
Pharmacokinetic Analysis
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.).
Statistical Analysis
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
121
somewhat more efficiently in the basolateral direction than in the apical direction,
indicating low endogenous basolaterally directed transport.
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.
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
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
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
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134
FIGURE LEGEND
Figure 1. Transepithelial transport of 10 M nitrofurantoin in MDCK-mock (parent)
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).
135
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
Control
(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
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
2-4
145
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
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
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.
. Briefly, for the oral drug interaction studies, rats were fasted overnight with free
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.
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.
Pharmacokinetic Analysis
150
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)
(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
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
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-
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).
154
155
degradation). However, the fitting was less satisfactory compared to the models with the
intestinal compartment (data not shown).
156
DISCUSSION
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.
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
161
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van Herwaarden AE, Schinkel AH 2006. The function of breast cancer resistance
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163
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
AIC
SC
20.4
41.0
-14.6
7.84
22.5
44.9
4.97
29.3
165
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
166
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
167
FIGURE LEGENDS:
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
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
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).
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.
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
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).
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
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
185
Radio-HPLC Assay
186
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
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.
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:
193
194
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)
MDCK II
0.55
MDCK-Bcrp1
0.32 0.02
2.89 0.11 b
8.97
MDCK-Bcrp1 + FTC
0.39
B.
B) Kaempferol
Papp, A-B
Papp, B-A
(cm/s, 10^6)
(cm/s, 10^6)
MDCK II
0.62
MDCK-Bcrp1
0.26 0.01
2.20 0.24 b
8.35
MDCK-Bcrp1 + FTC
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:
basolateral compartment.
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.
198
Figure 1.
199
Figure 2.
200
Figure 3.
201
Figure 4.
202
Figure 5.
203
CHAPTER 7
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
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).
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
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.
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
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
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.
Pharmacokinetic Analysis
213
Statistical Analysis
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
min, 45.6 15.4 h, 40.3 18.5 mlmin-1kg-1, and 118 86.2 L/kg, respectively (Table
1).
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).
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.
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.
221
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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
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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
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Wang JC, Liu XY, Lu WL, Chang A, Zhang Q, Goh BC and Lee HS (2006)
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224
Control
(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
225
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:
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.
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.
238
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.
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
241
REFERENCES:
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.
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
resistance protein (ABCG2)-mediated transport. Mol Pharmacol 65:1208-1216.
242
APPENDIX
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
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).
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
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
253
254
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
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
Ginseng
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
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.
. 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
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.
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
271
272
. 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.
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
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
276
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289
In Vitro/
In Vivo
St. Johns
wort
In vitro
Grapefruit
juice
Substrate
Mechanisms
Ref.
Rhodamine-123
Accumulation
43
Ritonavir
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
In vivo
In vivo
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
Induction of P-gp/CYP3A4
60,61
In vivo
Tacrolimus
AUC, Clearance
Induction of P-gp/CYP3A4
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
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
96
In vivo
97
In vivo
Saquinavir/
Ritonavir
AUC, Cmax
Induction of P-gp/CYP3A4
98,99
In vitro
In vitro
Rhodamine-123
Doxorubicin
Accumulation
Efflux
Inhibition of P-gp
Inhibition of P-gp
104
In vitro
Methotrexate
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
Inhibition of P-gp
115
Azidopine
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
Photoaffinity labeling
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
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
Substrates
Flavones
Apigenin
Daunomycin
Activity
177
Daunomycin
Vincristine
Activity
Activity
177
Luteolin
Daunomycin
Activity
177
Diosmin
Daunomycin
Activity
177
Daunorubicin
Daunomycin
Activity
Activity
152
Daunomycin
Rhodamine123/Doxorubicin
Adriamycin
Expression
or Activity
(Substrate-dependent)
Activity
177
Rhodamine-123
Daunorubicin
Daunomycin
Vincristine
Rhodamine-123/
Doxorubicin
Adriamycin
Activity
Activity
Activity
Biphasic effect
or Activity
(Substrate-dependent)
Activity
152
152
Chrysin
Flavonols
Fisetin
Galangin
Effect on P-gp
Ref.
149
177
150
147
177
149
150
147
Daunorubicin
Daunomycin
Activity
Activity
Daunomycin
Activity
Daunorubicin
Daunomycin
Activity
Activity
152
Rhodamine-123
Daunorubicin
Daunomycin
Adriamycin
Rhodamine-123
Vincristine
Hoechst 33342
Rhodamine123/Doxorubicin
Adriamycin
Activity
Activity
Activity
Activity
152
Biphasic effect
Activity
or Activity
(Substrate-dependent)
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
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
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
EGC
EGCG
Flavanolols
Silibinin/
silymarin
Isoflavones
Biochanin A
Genistein
294
Activity
Activity
Activity
Activity
104
104
104
177
179
104
120,178
121
177
120,178
121
177
177
181
Chrysin
Luteolin
Diosmetin
Diosmin
Flavonols
Fisetin
Galangin
Kaempferol
Substrates
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)
Vesicles from Hela-MRP1 cells
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
Myricetin
MCF7-pMTG5 cells
Human erythrocytes
MDCK-MRP1
MDCK-MRP2
Vesicles from Hela-MRP1 cells
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
DNM/VBL
MCF7-pMTG5 cells
MDCK-MRP1/2
MDCK-MRP1/2
Quercetin
MCF7-pMTG5 cells
Vesicles from human erythrocytes
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
DNM
VBL
DNM/VBL
DNP-SG
cGMP
Calcein
BCECF
Glutathione
LTC4/E217G
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
161
DNM/VBL
32
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
Daunorubicin
DNM/VBL
Daunorubicin
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
Substrates
Effect on BCRP
Mitoxantrone
SN38
Accumulation
Reverse resistance
34
Mitoxantrone
SN38
Accumulation
Reverse resistance
34
Mitoxantrone
SN38
Accumulation
Reverse resistance
34
Diosmetin
K562/BCRP
SN38
Reverse resistance
164
Diosmin
K562/BCRP
SN38
No effects
164
Mitoxantrone
SN38
Accumulation
No effects
34
Galangin
K562/BCRP
SN38
Reverse resistance
164
Kaempferol
Mitoxantrone
Mitoxantrone/SN38
Accumulation
Reverse resistance
34
Morin
Mitoxantrone
Accumulation
34
Myricetin
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
298
Ref
164
164
164
164
164
164
33
33
164
164
34
33
K562/BCRP
Daidzein
299
Mitoxantrone/SN38
Topotecan
Mitoxantrone
Mitoxantrone/
Prazosin
SN38
Reversal resistance
Accumulation
Accumulation
Accumulation
Moderate reversal
164
34
33
164
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).
301