Flavonoid-Drug Interactions: Effects of Flavonoids on ABC Transporters

Marilyn E. Morris, Ph.D.

Department of Pharmaceutical Sciences Flavonoids

Basic Structure: ¾The most abundant 8 1 2’ 3’ polyphenols present in 7 O 2 human diet A C B 4’ 6 (vegetables, fruits, red 3 5’ 5 4 6’ wine and tea) Subclasses:

O O O

OH O O O flavones flavonols flavanones O O

O isoflavones chalcones Flavonoids

¾ Epidemiological studies: reduced risk of cancer, coronary heart disease, and osteoporosis ¾ Biochemical and Pharmacological activities: anti-oxidant; anti-viral; anti-carcinogenic; anti-inflammatory; anti-angiogenic; anti-estrogenic (estrogenic). Flavonoids Have Little Toxicity ¾ Toxicity:

• Long history of consumption with exceptional safety record; • Extremely large doses used in animal studies;

Acute LD50 for rats: 2 g / kg BW by direct injection into blood.

“The margin of safety for the therapeutic use of flavonoids in humans, therefore, is very large and probably not surpassed by any other drug in current use” Havsteen, (2002), Pharmacology and Therapeutics 96:67-202. Flavonoid Products Herbal Use in Select Populations • HIV infected patients (Fairfield etal Arch Intern Med 158:2257-2264, 1998) – 68% of patients used herbs, vitamin, dietary supplements – Consumed herbal remedies to boost immunity, prevent nausea, diarrhea, or weight loss, relieve stress or depression. • Post-menopausal women (Mahady et al. Menopause 10:65-72, 2003) – Botanical dietary supplements used by 79% (395/500) of post-menopausal women within the last year. • Commonly used supplements include Soy (42%), green tea (35%), Chamomile (21%), Ginkgo (20%), Ginseng (18%), Echinacea (15%), & SJW (7%). Flavonoid Products

Do not need FDA approval

Drug interactions with conventional drugs have not been evaluated ABC Proteins

• ATP binding cassette (ABC) superfamily P-glycoprotein (MDR1, ABCB1) Multidrug Resistance Associated Proteins (MRP, ABCC) Breast Cancer Resistance Protein (BCRP, ABCG2) • Efflux molecules out of cells out • Tumor → multi-drug resistance • Present in the liver, kidney, BBB, gastrointestinal tract where important for drug disposition in Hepatocyte SMALL ORGANIC MONOVALENT ORGANIC BULKY ORGANIC ANIONS CATIONS BILE SALTS COMPOUNDS OCT1 OAT NTCP OATP

GSH E R

MDR1 MRP 2 MRP 2 MDR3 BSEP/SPGP BCRP PHOSPHATIDYL - CHOLINE MRP 6 98

MRP 3 MM © MRP1

NTCP MONOVALENT BILE SALTS Michael Müller Intestine Transporters apical OATP3 MDR1 • absorption OCT1 MRP2 • efflux MCT1 CNT BCRP PEPT1

CYP Metabolism UGT GST PST

Transporters MRP3 MCT1 MRP1 basolateral MRP3 Sandy K Pang ABC Transporter Expression in the Liver and Gastrointestinal Tract ¾High expression of MDR1, MRP2 and BCRP in the liver ¾Expression throughout the gastrointestinal tract

Dietrich et al, Gut 2003, 52:1788 P-glycoprotein

¾ Two homologous halves ¾ Each consists of: 6 TM 1 ATP binding site ¾ Substrate binding sites are located in TMs

Broad substrate specificity: anthracyclines, Vinca alkaloids, epipodophyllotoxins and taxol cyclosporine, digoxin, verapamil etc. One of the major mechanisms for cancer MDR and drug-drug, drug-food interactions. General Study Design

¾ In vitro studies- sensitive (no expression) and overexpressing cell lines (characterized) examining accumulation or flux ¾In vitro studies- effects on the cytotoxicity of chemotherapeutic drugs ¾In vitro studies- mechanism of interaction; additive effects; SAR/QSAR ¾In vivo studies in animals 3H-DNM Accumulation in MCF-7 cells

MCF-7/sensitive MCF-7/ADR

200 n 400 o

i *** ***

t ***

)

l o u 300

150 r

m n

u ***

c c

c ***

200 f

100 a

N (

(% of control) 50 100

- H

H-DNM accumulation accumulation H-DNM 3 3 0 l 0 l l A n n i l A n n i o n i i o n n ti i tr in ri t m tr i ri e m n n o e ar a n n o r ar a o a r p o a o p h M lo a h M l ym a C c lym r C c h l r h i e o i e io P S i P S B V B V Flavonoid concentration: 50 µM, Verapamil concentration: 100 µM Data expressed as mean ±SD, N= 9-12; ***: p < 0.001 Increase of 3H-DNM accumulation in P-gp Positive Cells Is Flavonoid Concentration Dependent

1400 Biochanin A 1200 Morin Phloretin 1000 Silymarin 800 600 Minimal Conc. for significant change:

(% of control) 400 200 Biochanin A and morin: 20-30 µM H-DNM accumulation 3 Phloretin and silymarin: 30-50 µM 0 0 20 40 60 80 100 120 Flavonoid concentration (µM)

DNM accumulation was determined in MDA435/LCC6MDR1 cells Increase of Doxorubicin Cytotoxicity by Biochanin A

Control 1 10 µM biochanin A IC =30.2 µM 50 30 µM biochanin A 50 µM biochanin A 100 µM biochanin A IC50=0.80µM

Doxorubicin cytotoxicity

Fraction of cell growth was determined in 0 MDA435/LCC6MDR1 cells 0.001 0.01 0.1 1 10 100 1000 Concentration of doxorubicin (µM) Flavonoid-P-gp Interactions

Mechanisms: ¾Biochanin A is not a substrate ¾Flavonoids affect P-gp ATPase activity or the P-gp ATPase activity induced by verapamil ¾ Some flavonoids can inhibit P-gp ATP and/or substrate binding Bifunctional binding interactions with nucleotide binding domains at ATP and vicinal substrate binding site (DiPietro et al., 2002) ¾No effect on P-gp expression in MCF-7/ADR cells or human hepatocytes following longer incubations for the flavonoids and concentrations examined Flavonoid-Drug Interactions

flavone + paclitaxel in rats quercetin + paclitaxel in rats

20 mg/kg flavone 10 mg/kg quercetin 10 mg/kg flavone 2 mg/kg quercetin 2 mg/kg flavone control control

Choi et al. (2004) Int J Pharm 275: 165-170 Choi et al. (2004) Eur J Pharm Biopharm 57: 313-8 Flavonoid-Drug Interactions

quercetin + cyclosporine in pigs phellamurin + cyclosporine in rats

control

lood control lasma b

p +quercetin

+phellamurin Cyclosporin Cyclosporin Concentration (ng/ml) Concentration (ng/ml)

AUC0-3: 56% AUC: 56% Cmax: 47% Cmax: 77%

Hsiu et al. (2002) Life Sciences 72:227-235 Chen et al. (2002) Planta Med 68:138-141 Flavonoid-Drug Interactions

Flavonoid-drug interactions could occur upon coadministration but appear to be substrate dependent (and will be flavonoid dependent)

However, other factors (for example, other transporters) may be important – there may be poor prediction if only based on their interaction with P-glycoprotein and CYP3A4 Multidrug Resistance-Associated Protein 1 (MRP1, ABCC1)

MRP1 is a 190-kDa protein encoded by the MRP1 gene. Expressed in most normal tissues in the human body and in several types of tumors such as lung carcinoma, myeloid leukemia, neuroblastoma, and breast cancer Substrates of MRP1:

• Endogenous substrates: leukotriene C4, glutathione disulfide, steroid glucuronides (17β-estradiol 17-β-D-glucuronide) • Exogenous substrates: daunomycin, vinca alkaloid (vinblastine), methotrexate, fluorouracil, chlorambucil, calcein, drug conjugates Involvement of GSH in MRP1-mediated transport

GSSG

MRP1

ATP

GSSG ADP + Pi (oxidized) oxidative stress GSH MRP1 GSH reductase GSH (reduced) endogenous or exogenous GSTase compound GSH GSH vincristine ATP conjugate ADP + Pi ADP + Pi ATP

MRP1 GSH + GS-X vincristine Qingcheng Mao Flavonoids increase the accumulation of 3H-VBL in Panc-1 Cells

Control Positive control 700 Flavonol Flavone ** Flavanol 600 Isoflavone

500

** 400 ** ** **

Accumulation ** 300 **

* ** 200 H-VBL % 3 100

0 Control Verapamil Kaempferol Morin Quercetin Phloretin Chalcone Siymarin Biochanin-A Genistein

* p < 0.05 ** p < 0.01

Nguyen et al, J Pharm Sci 2003 Flavonoids that have no effect on the accumulation of 3H-VBL in Panc-1 cells

500 Control Positive control Flavone Flavonol 400 Flavonones Flavanol

lation Isoflavone u 300 Accum 200 L % B H-V

3 100

0 trol mil nin ysin min olin n etin utin etin nin gin in in Con rapa pige Chr Dios Lute alangi yric R sper ringe Narin atech Daizde Ve A G M He Na alloc Epig Inhibitory constants for 3H-LTC4 transport in MRP1 membrane vesicles

FLAVONOID Ki (µM) myricetin 13.3 ± 2.7 (SD) quercetin 8.1 ± 1.7 naringenin 20.8 ± 6.4 (+ GSH) kaempferol 2.4 ± 1.6 apigenin (+GSH) 4.9 ± 0.7

Leslie et al., Mol Pharmacol, 2001 Mechanisms involved in MRP1 Inhibition ¾Decreased intracellular GSH appears to be important for some, but not all, flavonoids ¾No effects on glutathione S-transferase were observed ¾ No effects on the expression of MRP1 were seen with longer- term incubations ¾Significant effects on MRP1 ATPase activity ¾Likely not substrates ¾Binding at a substrate or ATP domain is likely also involved Breast Cancer Resistance Protein (BCRP)

A new member of ABC transporter superfamily; Also known as ABCP (ABC transporter in placenta), MXR (mitoxantrone-resistance protein) ABCG2 (the 2nd family of ABC subgroup G)

Broad substrate specificity; mitoxantrone, topoisomerase I inhibitors, methotrexate, topotecan zidovudine, lamivudine, flavopiridol, sulfate conjugates, omeprazole genistein Breast Cancer Resistance Protein (BCRP)

Expression in tumors: • leukemia: AML • solid tumors: colon cancer, lung cancer, myeloma, endometrial tumor, etc. Role in clinical MDR Expression in normal tissues: • placenta • intestine (expression level is higher than P-glycoprotein) • liver canalicular membrane • brain microvessels An important determinant for drug disposition

Steinbach et al. (2002) Leukemia 16: 1443-1447 Cooray et al (2002) Neuroreport 13:2059-2063 Diestra et al (2002) J. Pathol. 198: 213-219 Maliepaard et al (2001) Cancer Res 61:3458-3464 Jonker et al. (2000) J Natl Cancer Inst 92:1651-1656 BCRP Clinical Implications

• Apparent oral bioavailability : 40.0% ⇒ 97.1%

With inhibitor

Effects on both P-gp and BCRP

Kruijtzer, C. M. et al., J. Clin. Oncol. 20, 2943–2950, 2002 Breast Cancer Resistance Protein (BCRP)

GF120918 + topotecan in P-gp knockout mice, oral administration Plasma concentration Biliary excretion of topotecan of topotecan

control

GF120918 % of dose) oncentration

GF120918 ( c

(ng/ml) control Cumulative biliary topotecan Topotecan

Jonker et al. (2000) J Natl Cancer Inst 92:1651-1656 Investigated Flavonoids

A total of 20 naturally occurring flavonoids were studied.

Flavones: Isoflavones: Flavanones: apigenin biochanin A hesperetin chrysin daidzein naringenin luteolin genistein silybin silymarin epigallocatechin (EGC) Flavonols: Chalcones: epigallocatechin gallate (EGCG) naringin fisetin phloretin kaempferol phloridzin morin myricetin quercetin Mitoxantrone Accumulation In MCF-7 Cells

n 600

o ) *** i ***

l t 500 o *** a ***

t *** u *** *** n 400 *** ***

c u 300 *** *** f c *** * *

200

X ( 100 ∗∗∗ ∗∗∗ ∗∗∗ M 0 n n n l n n n n n n ol in A in i C G in i ti o lin in ti i ti zi ti in in tr en in ys e C et te e er o r e eni ng e d e b ar TC n g n r idz EG is is er f te o ic g i or ri rc ly F o pi h a EG f n p p u m yr in ar l lo e si ym c a cha c d ge es em l r n ph h u il o h a m na p q s Bi k MCF-7/sensitive (-BCRP) Mitoxantrone: 3 µM MCF-7 MX100 (+BCRP) Flavonoid: 50 µM N = 6 or 7 Correlation Between MX Accumulation In H460 MX20 and MCF-7 MX100 Cells

500

400 F-7 MX100

C 300 M n i 200 ion 100

0 0 100 200 300 400 500 Accumult Accumulation in H460 MX20 R2=0.74, p < 0.05 Mitoxantrone Cytotoxicity in MCF-7 Cells

MCF-7/sensitive MCF-7 MX100 compounds 10 µM 50 µM 2.5 µM5 µM 10 µM 50 µM control 5.30 ± 2.22 199 ± 19.3 apigenin 3.66 ± 0.52 3.44 ± 0.35 219 ± 10.0 10.5 ±7.13*** 1.73 ± 1.42***

BA 2.40 ±0.27*** 1.86 ±0.35*** 107 ± 17.6*** 30.9 ± 5.18*** 9.23 ± 2.07*** 2.19 ± 1.04***

chrysin 0.95 ± 0.46*** 18.8 ± 0.06*** 6.25 ± 2.13*** 3.35 ± 1.70*** 1.13 ± 1.11***

genistein 6.98 ± 0.81 148 ± 23.2 29.3 ± 6.76*** 2.29 ± 0.86***

kaempferol 6.10 ± 0.96 196 ± 20.9 228 ± 13.8 0.95 ± 0.19***

hesperetin 88.6 ± 20.8*** 11.6 ± 0.83*** 1.23 ± 0.16***

naringenin 189 ± 11.6 163 ± 29.5 1.23 ± 0.16***

silymarin 99.1 ± 50.2*** 104 ± 35.5*** 53.0 ± 7.27***

FTC 2.30 ± 0.29*** 1.79 ± 1.52***

N = 1 experiment performed in quadruplicate in MCF-7/sensitive cells N = 3 independent experiments performed in triplicate in MCF-7 MX100 cells Combined Effects of Flavonoids on BCRP-mediated Transport

Characterization of dose-response profiles of individual flavonoids and flavonoid combinations;

Calculation of EC30 , EC50 and EC70;

Analysis of potential interactions using isobologram and Berenbaum’s interaction index methods

Equal molar concentration of each constituent is present in the combinations. Dose-Response Profiles for Single Flavonoids

apigenin biochanin A chrysin

e 1.0 1.0 1.0

eas 0.8 0.8 r 0.8 0.6 0.6 0.6 l inc tion of 0.4 c 0.4 0.4 a 0.2

Fr 0.2 0.2 0.0

maxima 0.0 0.0 0246810 0246810 012345 Concentration (µM) Concentration (µM) Concentration (µM)

hesperetin kaempferol genistein

e 1.0 1.0 1.0

eas 0.8 0.8 0.8 r 0.6 0.6 0.6 l inc tion of 0.4 c 0.4 0.4 a 0.2 Fr 0.2 0.2

maxima 0.0 0.0 0.0 0 1020304050 0 5 10 15 20 25 30 0 1020304050 Concentration (µM) Concentration (µM) Concentration (µM) naringenin silymarin

e 0.8 0.6 eas

r 0.6 0.4 0.4 l inc tion of c

a 0.2 0.2 Fr 0.0 0.0 maxima 0 20406080100 0 1020304050 Concentration (µM) Concentration (µM) Dose-Response Profiles for Flavonoid Combinations

AB BC

e 1.0 1.0

eas 0.8 AB: apigenin + biochanin A r 0.8 0.6 0.6 l inc tion of c 0.4 0.4 a BC: biochanin A + chrysin

Fr 0.2 0.2

maxima 0.0 0.0 0246810 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ABC: apigenin + biochanin A + chrysin Concentration (µM) Concentration (µM) ABC ABCGK ABCGK: apigenin + biochanin A + chrysin

e 1.2 1.0 1.0 + genistein + kaempferol eas

r 0.8 0.8 0.6

l inc 0.6 tion of c 0.4 ABCGKHNS: all the eight flavonoids

a 0.4

Fr 0.2 0.2 investigated

maxima 0.0 0.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 Concentration (µM) Concentration (µM)

ABCGKHNS

e 1.0 eas

r 0.8 0.6 l inc tion of

c 0.4 a 0.2 Fr 0.0 maxima 0.0 0.20.40.60.81.01.21.4 Concentration (µM) The concentration values indicate the concentrations for each individual flavonoid in the combination EC50, EC30, EC70 for Increasing MX Accumulation

Flavonoids EC30 (µM) EC50 (µM) EC70 (µM) Apigenin (A) 0.97 ± 0.38 1.66 ± 0.55 2.86 ± 0.80 Biochanin A (B) 0.70 ± 0.47 1.62 ± 1.02 3.72 ± 2.24 Chrysin (C) 0.24 ± 0.07 0.39 ± 0.13 0.61 ± 0.23 Genistein (G) 8.91 ± 2.35 14.9 ± 2.69 25.0 ± 2.58 Hesperetin (H) 7.12 ± 1.39 12.4 ± 2.21 21.8 ± 3.59 Kaempferol (K) 3.79 ± 0.33 6.04 ± 0.09 9.67 ± 0.65 Naringenin (N) 17.5 ± 2.36 32.0 ± 3.22 59.1 ± 10.5 Silymarin (S) 10.6 ± 1.01 33.7 ± 2.78 109 ± 28.0 AB 0.39 ± 0.04 0.81 ± 0.17 1.69 ± 0.55 BC 0.15 ± 0.08 0.32 ± 0.16 0.69 ± 0.32 ABC 0.16 ± 0.08 0.27 ± 0.01 0.48 ± 0.09 ABCGK 0.14 ± 0.07 0.23 ± 0.08 0.40 ± 0.10 ABCGKHNS 0.13 ± 0.06 0.20 ± 0.10 0.34 ± 0.19 • N = 3 independent experiments performed in triplicate • Concentrations for combinations indicate the concentration for individual flavonoid in the combination

Zhang et al. Pharm Res, 2004 Berenbaum’s Interaction Index Method

Berenbaum’s Interaction Index D 2 I = ∑ x,i

ECx,i x

nde

ECx,I: the concentration of the i

constituent “i” to produce x effect; n

o 1

Dx,I: the concentration of the a

constituent “i” in the combination e

that will produce x effect. n

I I = 1, additive; 0 B C C K S A B AB G N I < 1, synergistic BC 30 % A GKH BC I > 1, antagonistic. 50 % A 70 % Interactions were additive Zhang et al. (2004) Pharm Res, 21: 1263-73 SAR and QSAR Study

Objective:

To identify structural elements required for potent BCRP inhibition;

To derive a QSAR equation for the prediction of flavonoid-BCRP interaction activity. Conclusions

Flavonoids can inhibit human BCRP with flavonoids such as chrysin, biochanin A, apigenin and benzoflavone having IC50 values in the sub- or low µM range Multiple flavonoids result in additive inhibition of BCRP The diversity of flavonoids allow the determination of SAR and QSAR for these compounds. SAR studies indicated the importance of lipophilicity, the placement of hydroxyl groups and the 2,3 double bond. Effects of flavonoids on topotecan pharmacokinetics in vivo Effect of GF120918 on Topotecan PK in SD Rats

Animal: SD female rats (180~220 g) 1000 control GF120918 (50mg/kg) Dosing regimen: iv topotecan (1mg/kg)

sma a

Control: vehicle: glycofurol, oral l 100 Treatment: 50 mg/kg GF120918, oral p

3 min later, topotecan 2mg/kg, oral 10 (in saline containing 5% glucose)

For iv dosing: topotecan (1mg/kg) Topotecan 1 concentration (ng/ml) 0 200 400 600 Topotecan analysis: validated HPLC method Time (min)

Parameters Control (n=7) GF120918 (50mg/kg) (n=4)

4 4 AUC0-360 (ng/ml•min) 1.74 ± 0.86 (×10 ) 7.65 ± 3.78 (×10 )** 4 4 AUC0-∞ (ng/ml·min) 1.80 ± 0.89 (×10 ) 7.91 ± 356 (×10 )** Tmax (min) 52 ± 85.3 75 ± 30 Cmax (ng/ml) 86.4 ± 42.9 257 ± 154*

terminal T1/2 (min) 127 ± 20.0 167 ± 65.1 F (%) 29.7± 14.8 130 ± 58.8** Effect of Chrysin on Topotecan PK in SD Rats

100 EC in MCF-7 MX100: 0.39± 0.13 µM control 50 chrysin (5mg/kg) substrate: mitoxantrone chrysin (50mg/kg) sma a Animal: SD female rats (180~220 g) l p 10 Dosing regimen: Control: vehicle: glycofurol, oral Treatment: 5 or 50 mg/kg chrysin, oral

3 min later, topotecan 2mg/kg, oral Topotecan

concentration (ng/ml) 1 (in saline containing 5% glucose) 0 200 400 600 Time (min)

Parameters Control (n=7) Chrysin (5mg/kg) (n=3) chrysin (50mg/kg) (n=6)

4 4 4 AUC0-360 (ng/ml•min) 1.74 ± 0.86 (×10 ) 1.29 ± 0.24 (×10 ) 0.88 ± 0.83 (×10 ) 4 4 4 AUC0-∞ (ng/ml·min) 1.80 ± 0.89 (×10 ) 1.34 ± 0.27 (×10 ) 0.93 ± 0.85 (×10 ) Tmax (min) 52 ± 85.3 35.0 ± 22.9 75.0 ± 82.2 Cmax (ng/ml) 86.4 ± 42.9 68.3 ±32.2 36.0 ± 37.9 terminal T1/2 (min) 127 ± 20.0 139 ± 40.4 173 ± 47.7 F (%) 29.7± 14.8 22.1 ± 4.47 15.3 ± 14.1 Effect of Chrysin on Topotecan PK in mdr1a/1b (-/-) mice

Animal: mdr1a/1b (-/-) mice (23~26.5 g) a 100 control

m 50 mg/kg chrysin Dosing regimen: Control: vehicle: olive oil, oral las

p 10 Treatment: 50 mg/kg chrysin, oral

3 min later, topotecan 2mg/kg, oral 1 (in saline containing 5% glucose)

Topotecan 0.1 concentration (ng/ml) 0 100 200 300 400 Time (min)

Chrysin (50mg/kg) Parameters Control (n=4) (n=4)

3 3 AUC0-360 (ng/ml·min) 4.56 ± 3.95 (×10 ) 4.17 ± 2.93 (×10 ) 3 3 AUC0-∞ (ng/ml·min) 5.01 ± 3.96 (×10 ) 4.65 ± 2.98 (×10 ) Tmax (min) 45.0 ± 46.4 33.7 ± 18.9 Cmax (ng/ml) 45.2 ± 47.3 50.8 ± 45.8 terminal T1/2 (min) 63.6 ± 42.7 90.6 ± 20.5 Possible Reasons for the In vitro and In vivo Discrepancy

Metabolism Substrate dependence Species difference Inhibition of topotecan uptake transporter Effect of 7,8-benzoflavone (BF) on Topotecan PK in SD Rats

control

ion Benzoflavone (10 mg/kg) EC50 in MCF-7 MX100: 0.07± 0.02 µM 100 Benzoflavone (50 mg/kg)

substrate: mitoxantrone g/ml) (n Animal: SD female rats (180~220 g) 10 Dosing regimen: Control: vehicle: glycofurol, oral

Treatment: 10 or 50 mg/kg BNF, oral topotecan of Plasma concentrat 3 min later, topotecan 2mg/kg, oral 1 (in saline containing 5% glucose) 0 200 400 600 Time (min)

benzoflavone (10 mg/kg) benzoflavone (50mg/kg) Parameters Control (n=7) (n=9) (n=8)

4 4 4 AUC0-360 (ng/ml·min) 1.74 ± 0.86 (×10 ) 2.04 ± 0.48 (×10 ) 2.50 ± 1.14 (×10 ) 4 4 4 AUC0-∞ (ng/ml·min) 1.80 ± 0.89 (×10 ) 2.15 ± 0.43 (×10 ) 2.57 ± 1.15 (×10 ) Tmax (min) 52.0 ± 85.3 82.4 ± 91.4 107 ± 91.2 Cmax (ng/ml) 86.4 ± 42.9 98.0 ± 45.7 101 ± 50.9 terminal T1/2 (min) 127 ± 20.0 150 ± 63.6 127 ± 37.3 F (%) 29.7± 14.8 35.5 ± 7.33 42.5 ± 7.09 Possible Reasons for the In vitro and In vivo Discrepancy

Metabolism Substrate dependence Species difference Inhibition of topotecan uptake transporter Effect of Flavonoids on Topotecan Accumulation in MCF-7 cells

350 *** Accumulation time: 10 min; 300 *** *** Topotecan concentration: 5 µM; 250 *** Cells were harvested and 200 ccumulation

sonicated a 150 Topotecan in cell lysate was 100 (% of control) assayed by HPLC tecan o Accumulation were normalized 50 by protein content Top 0 l 33 C C in F tro 8 FT FT ys B n C hr M N = 4 co PS µM µM c µ M 0 0 M 5 µ 1 1 µ 10 + 50 33 C8 PS M µ 10

Chrysin and BF can inhibit BCRP-mediated MCF-7 MX100 efflux of topotecan MCF-7/sensitive Possible Reasons for the In vitro and In vivo Discrepancy

Metabolism Substrate dependence Species difference Inhibition of topotecan uptake transporter Effect of Flavonoids on Topotecan Accumulation in MDCK-bcrp1 Cells

500 Accumulation time: 10 min; *** ***

Topotecan concentration: 5 µM; 400 *** Cells were harvested and ntrol) 300 sonicated o ccumulation Topotecan in cell lysate was a 200 assayed by HPLC * Accumulation were normalized (% of c 100 by protein content Topotecan N = 4 0 ) ol ) ) ) M ) ) tr µM µM M µ µM µM n 0 0 µ 0 0 5 co 1 1 10 (1 5 ( ( ( ( C ( F 33 8 C T in B 8 91 T F ys C 0 F )+ r PS 12 M ch GF µ 10 ( 33 C8 Chrysin and BF may only have weak PS inhibition activity on mouse bcrp1 MDCK-mock MDCK-bcrp1 Summary

Chrysin and BF did not change topotecan PK in rats or mice

Tentative explanation for the discrepancy: species difference with respect to inhibition of topotecan (species difference not seen with mitoxantrone)

Other possibilities could not be excluded, such as involvement of other transporters Flavonoid Interactions with ABC Transporters

¾Flavonoids are widely-present in food and herbal products. ¾Inhibitory interactions occur with P-glycoprotein, MRP1 and BCRP. These interactions may be beneficial for the reversal of multidrug resistance in cancer. Flavonoids may also increase the bioavailability and decrease the clearance of drugs. ¾Concentrations achievable in vivo in the gastrointestinal tract are likely high enough to result in significant interactions with ABC transporters. This is particularly true with respect to flavonoid concentrations after herbal medicines.