research article interaction of natural dietary and herbal...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 612527 7 pageshttpdxdoiorg1011552013612527

Research ArticleInteraction of Natural Dietary and Herbal Anionic Compoundsand Flavonoids with Human Organic Anion Transporters 1(SLC22A6) 3 (SLC22A8) and 4 (SLC22A11)

Li Wang and Douglas H Sweet

Department of Pharmaceutics Virginia Commonwealth University 410 N 12th Street Richmond VA 23298 USA

Correspondence should be addressed to Li Wang wangl4vcuedu

Received 19 January 2013 Accepted 22 February 2013

Academic Editor Khalid Rahman

Copyright copy 2013 L Wang and D H Sweet This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Active components of complementaryalternativemedicines and natural supplements are often anionic compounds and flavonoidsAs such organic anion transporters (OATs) may play a key role in their pharmacokinetic and pharmacological profiles andrepresent sites for adverse drug-drug interactions Therefore we assessed the inhibitory effects of nine natural products includingflavonoids (catechin and epicatechin) chlorogenic acids (13- and 15-dicaffeoylquinic acid) phenolic acids (ginkgolic acids (13 0)(15 1) and (17 1)) and the organic acids ursolic acid and 18120573-glycyrrhetinic acid on the transport activity of the human OATshOAT1 (SLC22A6) hOAT3 (SLC22A8) and hOAT4 (SLC22A11) Four compounds 13- and 15-dicaffeoylquinic acid ginkgolic acid(17 1) and 18120573-glycyrrhetinic acid significantly inhibited hOAT1-mediated transport (50 120583M inhibitor versus 1 120583Msubstrate) Fivecompounds 13- and 15-dicaffeoylquinic acid ginkgolic acids (15 1) and (17 1) and epicatechin significantly inhibited hOAT3transport under similar conditions Only catechin inhibited hOAT4 Dose-dependency studies were conducted for 13-dicaffeoyl-quinic acid and 18120573-glycyrrhetinic acid on hOAT1 and IC

50values were estimated as 12 plusmn 04 120583M and 27 plusmn 02 120583M respectively

These data suggest that 13-dicaffeoylquinic acid and 18120573-glycyrrhetinic acid may cause significant hOAT1-mediated DDIs in vivopotential should be considered for safety issues during use and in future drug development

1 Introduction

In concert with our growing knowledge of drug trans-porter expression and function transporter-mediated drug-drug interactions (DDIs) are being increasingly identifiedby numerous in vitro and in vivo studies [1 2] Recentlygovernment agencies in the United States (Food and DrugAdministration) and Europe (European Medicines Agency)as well as the pharmaceutical industry have acknowledgedthat transporters play a key role in the absorption distri-bution and excretion of many clinical therapeutics Organicanion transporter 1 (OAT1 SLC22A6) OAT3 (SLC22A8)and OAT4 (SLC22A11) are among the transporters identifiedthus far to impact the pharmacokinetics and hence dosingefficacy and toxicity of some drugs OAT1 and OAT3expressed in the basolateral membrane of renal proximaltubule cells in both humans and preclinical species function

as key mediators for organic solute flux from blood tothe glomerular filtrate [1 2] Additionally OAT4 whichis exclusively expressed in the apical membrane of humanproximal tubules reabsorbs anionic compounds from theurine [1] Further many endogenous substances includinghormones neurotransmitters and toxins have been iden-tified as substrates andor inhibitors of OATs [1 2] Thusthe potential clinical significance of OAT-mediated DDIs isfirmly recognized

Although many studies have exhibited OAT-mediatedDDIs for synthesized drugs relatively little is known aboutthe potential interaction between OATs and natural prod-ucts including various organic anions phenolic acids andflavonoids found in herbal supplements and food Severaldietary flavonoids and their metabolic conjugates (eg sul-fates and glucuronides) were identified as potent inhibitorsandor substrates of human (h) OAT1 hOAT3 and hOAT4

2 Evidence-Based Complementary and Alternative Medicine

[3 4] Phenolic acids for example contained in the widelyused Chinese herbal medicine Danshen (Salvia miltiorrhiza)or common berries such as strawberries or blueberries weresimilarly demonstrated to interact with these three trans-porters [5 6] These studies highlight the strong potentialfor hOAT-mediated natural product-drug interaction and theneed to investigate further anionic compounds and flavo-noids that are found in popular complementaryalternativemedicines and natural supplements

Catechin and epicatechin are major components of teaproducts possessing antioxidative and purported anticancerproperties [7 8] 13- and 15-dicaffeoylquinic acids are twoenantiomers of dicaffeoylquinic acid with various pharma-cological effects which are widely distributed in plants (egcoffee beans sweet potato leaves and fennels) [9] Indeed13-dicaffeoylquinic acid (also known as cynarin) is beingactively investigated for its anti-HIV and immunosuppressiveproperties [10] Extracts of Ginkgo biloba have gained pop-ularity as a herbal supplement because they are believed toimprove mental sharpness and memory while slowing brainaging and were hoped to be effective in relieving symptomsassociated with Alzheimerrsquos disease Regardless it is wellrecognized that several ginkgolic acids particularly (13 0)(15 1) and (17 1) (designating the number of carbon atomsin the alkyl side chain) can produce severe allergic cytotoxicmutagenic carcinogenic and genotoxic effects [11] Licoriceroot (Radix Glycyrrhizae) is employed to relieve a numberof maladies including stomach ulcers colic chronic gas-tritis sore throat bronchitis osteoarthritis liver disordersmalaria and tuberculosis One major component of RadixGlycyrrhizae preparations glycyrrhizin gives rise to twobioactive metabolites 18120572- and 18120573-glycyrrhetinic acidthought to play a role in these beneficial effects [12] Ursolicacid is a pentacyclic triterpene acid occurring naturally inherbs and fruits It exhibits anti-inflammatory and anticar-cinogenic activities [13] and is marketed as a herbal sup-plement to promote weight loss Since these compoundshave been identified as major components of first-linecom-plementaryalternative medicines foods and beverageshumans can be exposed to these compounds through clinicaltherapies and the daily diet Many of these compounds haveshown systemic exposure in humans (Table 1) Based ontheir chemical structures and previous studies these com-pounds have the potential to interact with OATsThus OAT-mediated DDIs may occur in vivo when combined withknown OAT substrates and such information should provehelpful in guiding the safe use and development of productsthat contain these compounds

Therefore the purpose of the present study was to in-vestigate the inhibitory potential of these nine compoundscatechin epicatechin 13- and 15-dicaffeoylquinic acidsginkgolic acids (13 0) (15 1) and (17 1) 18120573-glycyrrhetinicacid and ursolic acid on hOAT1- hOAT3- and hOAT4-mediated transport activities In addition to generating trans-porter specific inhibition profiles dose-response studies wereconducted for potent inhibitors in order to derive inhibitoryconstants (IC

50and 119870

119894values) to aid evaluation of their

potential for clinical OAT-mediated DDIs

2 Material and Methods

21 PurifiedChemicals Catechin 13- and 15-dicaffeoylquin-ic acids epicatechin ginkgolic acids (13 0) (15 1) and (17 1)and ursolic acid (allge97 purity) were purchased fromTautoBiotech (Shanghai China) 18120573-glycyrrhetinic acid (ge97purity) was obtained from Sigma-Aldrich (St Louis MO)The chemical structures of these compounds are shownin Figure 1 Tritiated p-aminohippuric acid ([3H]PAH) andestrone sulfate ([3H]ES) were purchased from PerkinElmerLife and Analytical Sciences (Waltham MA) and unlabeledPAH ES and probenecid were purchased from Sigma-Aldrich (St Louis MO)

22 Cell Culture Derivation and culture of stably transfectedChinese hamster ovary (CHO) cells expressing hOAT1(CHO-hOAT1) and hOAT4 (CHO-hOAT4) stably trans-fected human embryonic kidney 293 (HEK) cells expressinghOAT3 (HEK-hOAT3) and their corresponding empty vec-tor transfected background control cell lines have been des-cribed previously [6 14ndash16]

23 Cell Accumulation Assays The cell accumulation assayprotocol was performed as described in our recent publica-tions with minor modifications [14 17] In brief 2times 105 cellswell were seeded in 24-well tissue culture plates and grown inthe absence of antibiotics for two days For transport experi-ments cells were preincubated in transport buffer withoutsubstrates or inhibitors for 10min after which time the bufferwas replaced with 400 120583L of fresh transport buffer containing1 120583M [3H]PAH (05 120583CimL) or [3H]ES (025120583CimL) withor without test compounds After incubation for times spec-ified in the figure legends cellular uptake was quenched byquickly rinsing each well three times with fresh ice-coldtransport buffer Cells were lysed with 1M NaOH and radio-activity was measured with a liquid scintillation counter andreported as picomoles of substrate per milligram total pro-tein All intracellular uptake data were corrected for back-ground accumulation in corresponding empty vector trans-fected control cells Kinetic calculations were performedusing GraphPad Prism Software version 50 (GraphPad Soft-ware Inc San Diego CA) The half maximal inhibitory con-centrations (IC

50) and inhibitory constants (119870

119894) were calcu-

lated using nonlinear regression with the appropriatemodelsResults were confirmed by repeating all experiments at leastthree times with triplicate wells for each data point Forkinetic analysis hOAT1 expressing cells showed linear PAHuptake for the initial 3min with 119870

119898= 154 120583M [15] while

hOAT3 expressing cells exhibited linear ES uptake for theinitial 1min with119870

119898= 145 120583M [6]

24 Statistics Values are expressed as meanplusmn SD or meanplusmnSEM as indicated Statistical differences were assessed usingone-way analysis of variance (ANOVA) followed by post hocanalysis with Dunnettrsquos t-test (120572 = 005)

Evidence-Based Complementary and Alternative Medicine 3

OHO

OH

OH

OH

Catechin

OH

OHO

OH

OH

OH

OH

Epicatechin HOH

H COOH

Ursolic acid

OH OH

O

OH

O

O

OHOH

13-dicaffeoylquinic acid

OHO

OH

O

O

O

OH

OH

O

OHOH

OH


OH

OH

O OH

OH

O OH

OH

O

Ginkgolic acid C130 Ginkgolic acid C151 Ginkgolic acid C171

O

OH

OH

O

C15H14O6 MW 290 C15H14O6 MW 290 C30H48O3 MW 457

C25H24O12 MW 516 C25H24O12 MW 516


CH2(CH2)11CH3 (CH2)7CHCH(CH2)5CH3 (CH2)9CHCH(CH2)5CH3

Figure 1 Chemical structures of compounds investigated in this study MW molecular weight

Table 1 Maximum plasma concentration (119862max) reported in human subjects

Compound 119862max (120583M) Route of administration Dose (mg) ReferenceEpicatechin 020 Oral 37 [7]15-Dicaffeoylquinic acid 014 Oral 600 [23]18120573-Glycyrrhetinic acid 011ndash29a Oral 130ndash144 (glycyrrhizin) [12 24 25]Ursolic acid 74 iv 186 [13]aAssuming plasma concentration of 18120572- and 18120573-glycyrrhetinic acid is about 1 3 as reported in [12]

3 Results

31 Inhibition of hOAT1 by Natural Anionic Compounds andFlavonoids Approximately threefold greater accumulationof PAH was observed in CHO-hOAT1 cells (630 plusmn097 pmolmg proteinminus1 10minminus1) than in empty vectortransfected background control cells (212 plusmn 019 pmolmgproteinminus1 10minminus1) The hOAT1-mediated PAH accumula-tion was almost completely inhibited by probenecid(Figure 2) The nine test compounds catechin 13- and15-dicaffeoylquinic acid epicatechin ginkgolic acids (13 0)(15 1) and (17 1) 18120573-glycyrrhetinic acid and ursolicacid were assessed for inhibitory effects on hOAT1-mediateduptake (Figure 2) 13- and 15-Dicaffeoylquinic acid (64and

22 inhibition resp) ginkgolic acid (17 1) (42 inhibition)and 18120573-glycyrrhetinic acid (56 inhibition) each signifi-cantly inhibited hOAT1-mediated PAH uptake at 50-foldexcess Ursolic acid produced a significant stimulation ofuptake and the other compounds were without effect Since13-dicaffeoylquinic acid and 18120573-glycyrrhetinic acid produc-ed inhibition greater than 50 they were further chara-cterized by dose-response studies (001ndash500 120583M shown inFigure 3) Estimated IC

50values were 12 plusmn 04 120583M for 13-

dicaffeoylquinic acid and 27 plusmn 02 120583M for 18120573-glycyrrhetinicacid Numerous studies investigating the mode of inhibitionproduced on hOAT1 and hOAT3 for a broad array of com-pounds have demonstrated the interaction to be competitive[5 18ndash22] Therefore assuming competitive inhibition of


18120573-Glycyrrhetinic acid

0 50 100 150PAH uptake ( of control)

lowastlowast

lowastlowastlowastlowastlowastlowast


lowastlowastlowast

Catechin13-Dicaffeoylquinic acid15-Dicaffeoylquinic acid

EpicatechinGinkgolic acid (130)Ginkgolic acid (151)Ginkgolic acid (171)

Ursolic acidProbenecid

CHO-hOAT1 (control)

Figure 2 Inhibitory effects of the nine test compounds on hOAT1-mediated PAH uptake Ten-minute uptake of [3H]PAH (1120583M) wasmeasured in CHO-hOAT1 cells in the absence and presence oftest compounds (50 120583M) or probenecid (1000120583M) Data representhOAT1-mediated transport specifically that is data have beencorrected for background PAH accumulation measured in emptyvector transfected cells Values are mean plusmn SD of triplicate valuesfrom a representative experiment lowastlowast denotes 119875 lt 001 andlowastlowastlowast denotes 119875 lt 0001 as determined by one-way ANOVA followedby Dunnettrsquos t-test

hOAT1 inhibition constants (119870119894) were estimated as 11 plusmn

02 120583M for 13-dicaffeoylquinic acid and 25 plusmn 02 120583M for18120573-glycyrrhetinic acid

32 Inhibition of hOAT3 by Natural Anionic Compounds andFlavonoids Human OAT3 expressing cells showed about4-fold greater accumulation of ES as compared to back-ground control cells (106 plusmn 05 versus 26 plusmn 03 pmol mgproteinminus1 10minminus1 resp) Similar to hOAT1 hOAT3-med-iated ES uptake was completely (gt96 inhibition) blocked byprobenecid (Figure 4) Five of the compounds 13- and 15-dicaffeoylquinic acid epicatechin and ginkgolic acids (15 1)and (17 1) significantly inhibited hOAT3-mediated trans-port at 50-fold excess (Figure 4) 13-Dicaffeoylquinic acidand ginkgolic acid (17 1) exhibited 41 inhibition while 30ndash35 reduction of hOAT3-mediated ES uptake was observedfor 15-dicaffeoylquinic acid epicatechin and ginkgolic acid(15 1) Catechin 18120573-glycyrrhetinic acid and ursolic acidfailed to produce significant inhibition Based on the level ofinhibition observed IC

50values for all of these compounds

would be greater than 50 120583M much higher than clinicallyrelevant concentrations (Table 1) Therefore further dose-response studies were not performed

33 Inhibition of hOAT4 by Natural Anionic Compoundsand Flavonoids Stably transfected hOAT4-expressing cellsshowed sim18 fold higher accumulation of ES than back-ground control cells (425 plusmn 51 versus 24 plusmn 02 pmolmgproteinminus1 10minminus1 resp)HumanOAT4-mediated ES uptakewas virtually completely blocked (gt96 inhibition) by self-inhibition (Figure 5) Catechin (50120583M) was the only com-pound that showed a significant inhibitory effect (32)on hOAT4 (Figure 5) 15-Dicaffeoylquinic acid and 18120573-glycyrrhetinic acid produced significant stimulation of ESuptake Accordingly the IC

50value for catechin on hOAT4

transport activity should be greater than 50 120583M and no fur-ther kinetic studies were performed

4 Discussion

Literally hundreds of therapeutic and endogenous com-pounds have been demonstrated to interact with OATs [1 2]Precisely because of their broad polyspecific nature OATsare likely sites for DDIs in vivo As a consequence they havebeen identified worldwide as a family of ldquoclinically relevantrdquotransporters in recent government guidances to the pharma-ceutical industry [1 2] Unlike Western drugs the pharma-cokinetic and pharmacological properties of botanical drugproducts and natural supplements have not been well inves-tigated Being naturally derived people often view these com-pounds as ldquosaferdquo and without potential for causing adverseevents However there is increasing evidence demonstratingpotent interactions between components of natural productsand OATs resulting in possible toxicity and DDIs [1 2] Forexample aristolochic acid a potent nephrotoxic contam-inant sometimes found in certain Chinese herbal thera-pies was identified as a high affinity substrate of murineOat1 Oat2 and Oat3 as well as a potent inhibitor ofhOAT1 hOAT3 and hOAT4 [26 27] Additional studies havedemonstrated the OAT-mediated DDI potential for commonflavonoid and phenolic acid components of foods and herbalmedicines [3ndash6] These investigations showed that manydietaryphytomedicine-derived organic acids and flavonoidshave a high inhibitory potency for hOAT1 hOAT3 andorhOAT4 and according to proposed guidelines established bythe FDA and EMA high likelihood to result in significantDDIs in vivo [1ndash6] Therefore further studies on OAT-med-iated natural product-drug interactions are needed in orderto establish informed safety and efficacy profiles for botanicalproducts

In the present study we characterized the interactions ofnine chemically diverse compounds including flavonoidschlorogenic acids phenolic acids and other organic acidsfound as major dietary or phytomedicine components withthree human OATs hOAT1 hOAT3 and hOAT4 As illus-trated most of the compounds produced significant inhibi-tion of hOAT1 and hOAT3 at a 50-fold excess concentration(Figures 1 and 3) In marked contrast only catechin signif-icantly inhibited hOAT4 under this condition (Figure 5)Interestingly ursolic acid caused a significant stimulation ofhOAT1-mediated PAH uptake while it was without effect oneither hOAT3 or hOAT4 transport activity Similarly 15-di-caffeoylquinic acid and 18120573-glycyrrhetinic acid appeared tosignificantly stimulate hOAT4 transport while decreasinghOAT1- and hOAT3-mediated uptake activities Such spo-radic transporter stimulationinhibition has been previouslyreported in the literature for other compounds For examplefor fluoroquinolone antimicrobials ciprofloxacin inhibitedhOAT3 but stimulated hOAT1 [14] and sparfloxacin wasreported to stimulatemultidrug resistance-associated protein2 activity [28] Such stimulation of transport activity in vitroalso has been reported for steroids chemotherapeutic agentsand nonsteroidal anti-inflammatory drugs [14 28ndash30] One


Control minus9 minus8 minus7 minus6 minus5 minus4 minus3

0

25

50

75

100

125

PAH

upt

ake (

o

f con

trol)

log[13-Dicaffeoylquinic acid] (M)

(a)

Control minus8 minus7 minus6 minus5 minus4 minus30

25

50

75

100

PAH

upt

ake (

o

f con

trol)

log[18120573-Glycyrrhetinic acid] (M)

(b)

Figure 3 Dose-response effects for 13-dicaffeoylquinic acid and 18120573-glycyrrhetinic acid on hOAT1-mediated PAH transport One-minuteuptake of [3H]PAH (1 120583M) in CHO-hOAT1 cells was measured in the presence of increasing concentrations (10minus7 to 5 times 10minus4 M) of testcompounds Data were corrected by subtracting background PAH accumulation measured in empty vector transfected cells IC

50values

were determined with nonlinear regression and the ldquolog(inhibitor) versus responserdquo model using GraphPad Prism software Experimentswere repeated three times with triplicate samples and graphs shown are from representative experiments with values plotted as mean plusmn SD(119899 = 3) The data were used to generate mean IC

50plusmn SEM estimates




CHO-hOAT3 (control)

0 50 100ES uptake ( of control)

lowastlowastlowast

lowastlowastlowastlowast



Figure 4 Inhibitory effects of the nine test compounds on hOAT3-mediated ES uptake Ten-minute uptake of [3H]ES (1120583M) wasmeasured in HEK-hOAT3 cells in the absence and presence oftest compounds (50 120583M) or probenecid (1000120583M) Data representhOAT3-mediated transport specifically that is data have been cor-rected for background ES accumulation measured in empty vectortransfected cells Values are mean plusmn SD of triplicate values from arepresentative experiment lowast denotes 119875 lt 005 lowastlowast denotes 119875 lt 001and lowastlowastlowast denotes 119901 lt 0001 as determined by one-way ANOVAfollowed by Dunnettrsquos t-test

potential explanation is allosteric binding of the compoundsto the transporters with subsequent alteration of substrateaffinity [28 29] However these transporters share a highdegree of sequence identity and no readily discernible struc-tural differences corresponding to such a binding site areapparent and these effects do not exhibit a consistent patternamong the hOATs Whether these effects will be observed invivo is yet to be determined however in such an instanceincreased (versus decreased) elimination and shortened (ver-sus lengthened) terminal half-life of victim drugs could occurcausing loss of efficacy

0 50 100 150 200ES uptake ( of control)

lowast

lowast

lowastlowastlowast

lowastlowastlowast



Ursolic acidES (self-inhibition)

CHO-hOAT4 (control)


Figure 5 Inhibitory effects of the nine test compounds on hOAT4-mediated ES uptake Ten minute uptake of [3H]ES (1 120583M) wasmeasured in CHO-hOAT4 cells in the absence and presence oftest compounds (50 120583M) or ES (1000120583M for self-inhibition) Datarepresent hOAT4-mediated transport specifically that is data havebeen corrected for background ES accumulation measured inempty vector transfected cells Values are mean plusmn SD of triplicatevalues from a representative experiment lowast denotes 119875 lt 005 andlowastlowastlowast denotes 119875 lt 0001 as determined by one-way ANOVA followedby Dunnettrsquos t-test

The FDA guidance on drug interaction studies proposedusing the DDI index calculated as the ratio of unbound119862max over IC

50or 119870119894 as an indicator of the assessment of

a compoundrsquos DDI potential with a value greater than 01indicating the potential need to perform an in vivo DDIstudy for an investigational drug In the current study 18120573-glycyrrhetinic acid exhibited strong inhibition of hOAT1withan estimated IC

50of 27 plusmn 02 120583M or 119870

119894of 25 plusmn 02 120583M

(Figures 1 and 2) Human exposure studies reported 119862maxvalues ranging from 011 to 29 120583M (Table 1) perhaps due to


interindividual variability different doses andor dosing reg-imen (single dose versus repeated dose) As such the maxi-mum DDI for 18120573-glycyrrhetinic acid is sim11 although thisvalue does not account for plasma protein binding as this isunknown However if we assume it to be highly plasma pro-tein bound for example 90 the DDI index would be 012meeting the FDA guidance threshold of 01 Therefore 18120573-glycyrrhetinic acid may affect hOAT1-mediated renal elimi-nation of coadministered therapeutics that are hOAT1 sub-strates

13-Dicaffeoylquinic acid is a major component found inartichoke and Echinacea purpurea [31 32] Pharmacologicalstudies demonstrated that it exhibits antimicrobial and anti-oxidant activity [31 33] Moreover it has garnered increasedinterest because of its anti-HIV and immunosuppressive pro-perties 13-Dicaffeoylquinic acid blocked HIV-1 integraseactivity leading to interference of insertion of viral DNA intothe genome of the victim cell [10] Another study demon-strated that it inhibited the interaction between CD28 of T-cell receptor and CD80 of antigen presenting cells blockingldquosignal 2rdquo of T-cell activation [32] Due to its potential devel-opment as a therapeutic agent systemic exposure in clinicalapplications may reach high levels In the present study 13-dicaffeoylquinic acid showed high affinity with hOAT1(IC50=12 plusmn 04 120583M 119870

119894=11 plusmn 02 120583M) Therefore the dose-

systemic exposure relationship and plasma protein bindingshould be elucidated as part of the future drug developmentprocess for this compound

It has been reported that flavonoids can interact withOATs [3] Therefore catechin and epicatechin two flavonoidcomponents from green tea were investigated for potentialinteraction with hOAT1 hOAT3 and hOAT4 Both com-pounds exhibited significant inhibition of hOAT4 andhOAT3 when present at a 50-fold excess however no inhibi-tion was observed for hOAT1 Because of low reported clin-ical plasma concentrations (Table 1) these flavonoids weredetermined to be unlikely to cause DDIs after normal con-sumption of tea products However OATs might promoteentry of these antioxidants into renal proximal tubules pro-viding a nephroprotective effect

In summary we investigated the potential interaction ofnine flavonoids phenolic acids chlorogenic acids and otherorganic acids with hOAT1 hOAT3 and hOAT4 Among theexamined compounds 13-dicaffeoylquinic acid and 18120573-gly-cyrrhetinic acid showed marked affinity with hOAT1 Inhumans systemic exposure of 18120573-glycyrrhetinic acid mayinduce significant hOAT1-associated DDIs Future in vivointeraction studies between 18120573-glycyrrhetinic acid or 13-dicaffeoylquinic acid and clinical therapeutics known to behOAT1 substrates may be necessary to establish safety guide-lines for use of pharmaceutical products containing thesecompounds to avoid potential DDIs

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors especially thank Dr Tomas Cihlar for providingthe CHO-hOAT1 cells and Dr Guofeng You for providing theCHO-hOAT4 cells LWang acknowledges theVirginia Com-monwealth University Graduate School for financial supportin the form of a ThesisDissertation Assistantship award

References

[1] L Wang and D H Sweet ldquoRenal organic anion transporters(SLC22 Family) expression regulation roles in toxicity andimpact on injury and diseaserdquo The AAPS Journal vol 15 no69 pp 53ndash69 2013

[2] A L VanWert M R Gionfriddo and D H Sweet ldquoOrganicanion transporters discovery pharmacology regulation androles in pathophysiologyrdquo Biopharmaceutics and Drug Disposi-tion vol 31 no 1 pp 1ndash71 2010

[3] A C Whitley D H Sweet and T Walle ldquoThe dietary polyphe-nol ellagic acid is a potent inhibitor of hOAT1rdquoDrugMetabolismand Disposition vol 33 no 8 pp 1097ndash1100 2005

[4] C C Wong N P Botting C Orfila N Al-Maharik and GWilliamson ldquoFlavonoid conjugates interact with organic aniontransporters (OATs) and attenuate cytotoxicity of adefovirmediated by organic anion transporter 1 (OAT1SLC22A6)rdquoBiochemical Pharmacology vol 81 no 7 pp 942ndash949 2011

[5] LWang andDH Sweet ldquoActive hydrophilic components of themedicinal herb Salvia miltiorrhiza (Danshen) potently inhibitorganic anion transporters 1 (Slc22a6) and 3 (Slc22a8)rdquo Evi-dence-Based Complementary and Alternative Medicine vol2012 Article ID 872458 8 pages 2012

[6] L Wang and D H Sweet ldquoPotential for food-drug interactionsby dietary phenolic acids on human organic anion transporters1 (SLC22A6) 3 (SLC22A8) and 4 (SLC22A11)rdquo BiochemicalPharmacology vol 84 no 8 pp 1088ndash1095 2012

[7] M Renouf K Redeuil K Longet et al ldquoPlasma pharmaco-kinetics of catechin metabolite 41015840-O-Me-EGC in healthyhumansrdquo European Journal of Nutrition vol 50 no 7 pp 575ndash580 2011

[8] H H Chow and I A Hakim ldquoPharmacokinetic and chemopre-vention studies on tea in humansrdquo Pharmacological Researchvol 64 pp 105ndash112 2011

[9] K Schram P Miketova J Slanina O Humpa and E TaborskaldquoMass spectrometry of 13- and 15-dicaffeoylquinic acidsrdquo Jour-nal of Mass Spectrometry vol 39 no 4 pp 384ndash395 2004

[10] P J King G Ma W Miao et al ldquoStructure-activity relation-ships analogues of the dicaffeoylquinic and dicaffeoyltartaricacids as potent inhibitors of human immunodeficiency virustype 1 integrase and replicationrdquo Journal ofMedicinal Chemistryvol 42 no 3 pp 497ndash509 1999

[11] SMahadevan and Y Park ldquoMultifaceted therapeutic benefits ofGinkgo biloba L chemistry efficacy safety and usesrdquo Journal ofFood Science vol 73 no 1 pp R14ndashR19 2008

[12] Q Zou P Wei J Li Z X Ge and P Ouyang ldquoSimultaneousdetermination of 18120572- and 18120573-glycyrrhetic acid in humanplasma by LC-ESI-MS and its application to pharmacokineticsrdquoBiomedical Chromatography vol 23 no 1 pp 54ndash62 2009

[13] Y Xia GWei D Si and C Liu ldquoQuantitation of ursolic acid inhuman plasma by ultra performance liquid chromatographytandem mass spectrometry and its pharmacokinetic studyrdquoJournal of Chromatography B vol 879 no 2 pp 219ndash224 2011


[14] A L VanWert C Srimaroeng and D H Sweet ldquoOrganic aniontransporter 3 (Oat3Slc22a8) interacts with carboxyfluoro-quinolones and deletion increases systemic exposure to cipro-floxacinrdquo Molecular Pharmacology vol 74 no 1 pp 122ndash1312008

[15] E S Ho D C Lin D B Mendel and T Cihlar ldquoCytotoxicityof antiviral nucleotides adefovir and cidofovir is induced by theexpression of human renal organic anion transporter 1rdquo Journalof the American Society of Nephrology vol 11 no 3 pp 383ndash3932000

[16] F Zhou W Xu M Hong et al ldquoThe role of N-linked glyco-sylation in protein folding membrane targeting and substratebinding of human organic anion transporter hOAT4rdquo Molecu-lar Pharmacology vol 67 no 3 pp 868ndash876 2005

[17] G W Schnabolk B Gupta A Mulgaonkar M Kulkarni andD H Sweet ldquoOrganic anion transporter 6 (Slc22a20) specificityand sertoli cell-specific expression provide new insight onpotential endogenous rolesrdquo Journal of Pharmacology and Ex-perimental Therapeutics vol 334 no 3 pp 927ndash935 2010

[18] Y Uwai Y Ozeki T Isaka H Honjo and K Iwamoto ldquoIn-hibitory effect of caffeic acid on human organic anion trans-porters hOAT1 and hOAT3 a novel candidate for food-druginteractionrdquo Drug Metabolism and Pharmacokinetics vol 26pp 486ndash493 2011

[19] P Duan and G You ldquoNovobiocin is a potent inhibitor forhuman organic anion transportersrdquo Drug Metabolism and Dis-position vol 37 no 6 pp 1203ndash1210 2009

[20] N Bakhiya BMonien H Frank A Seidel andH Glatt ldquoRenalorganic anion transporters OAT1 and OAT3 mediate the cell-ular accumulation of 5-sulfooxymethylfurfural a reactivenephrotoxic metabolite of the Maillard product 5-hydroxy-methylfurfuralrdquo Biochemical Pharmacology vol 78 no 4 pp414ndash419 2009

[21] K Y Jung M Takeda D K Kim et al ldquoCharacterization ofochratoxin A transport by human organic anion transportersrdquoLife Sciences vol 69 no 18 pp 2123ndash2135 2001

[22] L Wang and D H Sweet ldquoCompetitive inhibition of humanorganic anion transporters 1 (SLC22A6) 3 (SLC22A8) and 4(SLC22A11) by major components of the medicinal herb Salviamiltiorrhiza (Danshen)rdquo Drug Metabolism and Pharmacokinet-ics 2012

[23] R Gu G Dou J Wang J Dong and Z Meng ldquoSimultaneousdetermination of 15-dicaffeoylquinic acid and its active meta-bolites in human plasma by liquid chromatography-tandemmass spectrometry for pharmacokinetic studiesrdquo Journal ofChromatography B vol 852 no 1-2 pp 85ndash91 2007

[24] B Ploeger T Mensinga A Sips W Seinen J Meulenbelt andJ DeJongh ldquoThe pharmacokinetics of glycyrrhizic acid evalu-ated by physiologically based pharmacokineticmodelingrdquoDrugMetabolism Reviews vol 33 no 2 pp 125ndash147 2001

[25] B Ploeger T Mensinga A Sips C Deerenberg J Meulenbeltand J DeJongh ldquoA population physiologically based pharma-cokineticpharmacodynamic model for the inhibition of 11-120573-hydroxysteroid dehydrogenase activity by glycyrrhetic acidrdquoToxicology and Applied Pharmacology vol 170 no 1 pp 46ndash552001

[26] N Bakhiya V M Arlt A Bahn G Burckhardt D H PhillipsandHGlatt ldquoMolecular evidence for an involvement of organicanion transporters (OATs) in aristolochic acid nephropathyrdquoToxicology vol 264 no 1-2 pp 74ndash79 2009

[27] K G Dickman D H Sweet R Bonala T Ray and A WuldquoPhysiological and molecular characterization of aristolochic

acid transport by the kidneyrdquo Journal of Pharmacology andExperimental Therapeutics vol 338 no 2 pp 588ndash597 2011

[28] J M Pedersen P Matsson C A S Bergstrom U Norinder JHoogstraate and P Artursson ldquoPrediction and identificationof drug interactions with the human ATP-binding cassettetransporter multidrug-resistance associated protein 2 (MRP2ABCC2)rdquo Journal of Medicinal Chemistry vol 51 no 11 pp3275ndash3287 2008

[29] J Kindla FMullerMMiethM F Fromm and J Konig ldquoInflu-ence of non-steroidal anti-inflammatory drugs on OrganicAnion Transporting Polypeptide (OATP) 1B1- and OATP1B3-mediated drug transportrdquo Drug Metabolism and Dispositionvol 39 no 6 pp 1047ndash1053 2011

[30] A Koenen K Kock M Keiser W Siegmund H K KroemerandM Grube ldquoSteroid hormones specifically modify the activ-ity of organic anion transporting polypeptidesrdquo European Jour-nal of Pharmaceutical Sciences vol 47 pp 774ndash780 2012

[31] X Zhu H Zhang and R Lo ldquoPhenolic compounds from theleaf extract of artichoke (Cynara scolymus L) and their antimi-crobial activitiesrdquo Journal of Agricultural and Food Chemistryvol 52 no 24 pp 7272ndash7278 2004

[32] G C Dong P H Chuang K C Chang et al ldquoBlocking effect ofan immuno-suppressive agent cynarin on CD28 of T-cell re-ceptorrdquo Pharmaceutical Research vol 26 no 2 pp 375ndash3812009

[33] F Pellati S Benvenuti L Magro M Melegari and F SoragnildquoAnalysis of phenolic compounds and radical scavenging activ-ity of Echinacea spprdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 35 no 2 pp 289ndash301 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


[3 4] Phenolic acids for example contained in the widelyused Chinese herbal medicine Danshen (Salvia miltiorrhiza)or common berries such as strawberries or blueberries weresimilarly demonstrated to interact with these three trans-porters [5 6] These studies highlight the strong potentialfor hOAT-mediated natural product-drug interaction and theneed to investigate further anionic compounds and flavo-noids that are found in popular complementaryalternativemedicines and natural supplements

Catechin and epicatechin are major components of teaproducts possessing antioxidative and purported anticancerproperties [7 8] 13- and 15-dicaffeoylquinic acids are twoenantiomers of dicaffeoylquinic acid with various pharma-cological effects which are widely distributed in plants (egcoffee beans sweet potato leaves and fennels) [9] Indeed13-dicaffeoylquinic acid (also known as cynarin) is beingactively investigated for its anti-HIV and immunosuppressiveproperties [10] Extracts of Ginkgo biloba have gained pop-ularity as a herbal supplement because they are believed toimprove mental sharpness and memory while slowing brainaging and were hoped to be effective in relieving symptomsassociated with Alzheimerrsquos disease Regardless it is wellrecognized that several ginkgolic acids particularly (13 0)(15 1) and (17 1) (designating the number of carbon atomsin the alkyl side chain) can produce severe allergic cytotoxicmutagenic carcinogenic and genotoxic effects [11] Licoriceroot (Radix Glycyrrhizae) is employed to relieve a numberof maladies including stomach ulcers colic chronic gas-tritis sore throat bronchitis osteoarthritis liver disordersmalaria and tuberculosis One major component of RadixGlycyrrhizae preparations glycyrrhizin gives rise to twobioactive metabolites 18120572- and 18120573-glycyrrhetinic acidthought to play a role in these beneficial effects [12] Ursolicacid is a pentacyclic triterpene acid occurring naturally inherbs and fruits It exhibits anti-inflammatory and anticar-cinogenic activities [13] and is marketed as a herbal sup-plement to promote weight loss Since these compoundshave been identified as major components of first-linecom-plementaryalternative medicines foods and beverageshumans can be exposed to these compounds through clinicaltherapies and the daily diet Many of these compounds haveshown systemic exposure in humans (Table 1) Based ontheir chemical structures and previous studies these com-pounds have the potential to interact with OATsThus OAT-mediated DDIs may occur in vivo when combined withknown OAT substrates and such information should provehelpful in guiding the safe use and development of productsthat contain these compounds

Therefore the purpose of the present study was to in-vestigate the inhibitory potential of these nine compoundscatechin epicatechin 13- and 15-dicaffeoylquinic acidsginkgolic acids (13 0) (15 1) and (17 1) 18120573-glycyrrhetinicacid and ursolic acid on hOAT1- hOAT3- and hOAT4-mediated transport activities In addition to generating trans-porter specific inhibition profiles dose-response studies wereconducted for potent inhibitors in order to derive inhibitoryconstants (IC

50and 119870

119894values) to aid evaluation of their

potential for clinical OAT-mediated DDIs

2 Material and Methods

21 PurifiedChemicals Catechin 13- and 15-dicaffeoylquin-ic acids epicatechin ginkgolic acids (13 0) (15 1) and (17 1)and ursolic acid (allge97 purity) were purchased fromTautoBiotech (Shanghai China) 18120573-glycyrrhetinic acid (ge97purity) was obtained from Sigma-Aldrich (St Louis MO)The chemical structures of these compounds are shownin Figure 1 Tritiated p-aminohippuric acid ([3H]PAH) andestrone sulfate ([3H]ES) were purchased from PerkinElmerLife and Analytical Sciences (Waltham MA) and unlabeledPAH ES and probenecid were purchased from Sigma-Aldrich (St Louis MO)

22 Cell Culture Derivation and culture of stably transfectedChinese hamster ovary (CHO) cells expressing hOAT1(CHO-hOAT1) and hOAT4 (CHO-hOAT4) stably trans-fected human embryonic kidney 293 (HEK) cells expressinghOAT3 (HEK-hOAT3) and their corresponding empty vec-tor transfected background control cell lines have been des-cribed previously [6 14ndash16]

23 Cell Accumulation Assays The cell accumulation assayprotocol was performed as described in our recent publica-tions with minor modifications [14 17] In brief 2times 105 cellswell were seeded in 24-well tissue culture plates and grown inthe absence of antibiotics for two days For transport experi-ments cells were preincubated in transport buffer withoutsubstrates or inhibitors for 10min after which time the bufferwas replaced with 400 120583L of fresh transport buffer containing1 120583M [3H]PAH (05 120583CimL) or [3H]ES (025120583CimL) withor without test compounds After incubation for times spec-ified in the figure legends cellular uptake was quenched byquickly rinsing each well three times with fresh ice-coldtransport buffer Cells were lysed with 1M NaOH and radio-activity was measured with a liquid scintillation counter andreported as picomoles of substrate per milligram total pro-tein All intracellular uptake data were corrected for back-ground accumulation in corresponding empty vector trans-fected control cells Kinetic calculations were performedusing GraphPad Prism Software version 50 (GraphPad Soft-ware Inc San Diego CA) The half maximal inhibitory con-centrations (IC

50) and inhibitory constants (119870

119894) were calcu-

lated using nonlinear regression with the appropriatemodelsResults were confirmed by repeating all experiments at leastthree times with triplicate wells for each data point Forkinetic analysis hOAT1 expressing cells showed linear PAHuptake for the initial 3min with 119870

119898= 154 120583M [15] while

hOAT3 expressing cells exhibited linear ES uptake for theinitial 1min with119870

119898= 145 120583M [6]

24 Statistics Values are expressed as meanplusmn SD or meanplusmnSEM as indicated Statistical differences were assessed usingone-way analysis of variance (ANOVA) followed by post hocanalysis with Dunnettrsquos t-test (120572 = 005)


OHO

OH

OH

OH

Catechin

OH

OHO

OH

OH

OH

OH

Epicatechin HOH

H COOH

Ursolic acid

OH OH

O

OH

O

O

OHOH


OHO

OH

O

O

O

OH

OH

O

OHOH

OH


OH

OH

O OH

OH

O OH

OH

O


O

OH

OH

O


C25H24O12 MW 516 C25H24O12 MW 516






3 Results








lowastlowast



lowastlowastlowast




CHO-hOAT1 (control)










4 Discussion





0

25

50

75

100

125

PAH

upt

ake (

o

f con

trol)


(a)


25

50

75

100

PAH

upt

ake (

o

f con

trol)


(b)


50values






CHO-hOAT3 (control)


lowastlowastlowast







lowast

lowast

lowastlowastlowast

lowastlowastlowast




CHO-hOAT4 (control)






50of 27 plusmn 02 120583M or 119870

119894of 25 plusmn 02 120583M











Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OHO

OH

OH

OH

Catechin

OH

OHO

OH

OH

OH

OH

Epicatechin HOH

H COOH

Ursolic acid

OH OH

O

OH

O

O

OHOH


OHO

OH

O

O

O

OH

OH

O

OHOH

OH


OH

OH

O OH

OH

O OH

OH

O


O

OH

OH

O


C25H24O12 MW 516 C25H24O12 MW 516






3 Results








lowastlowast



lowastlowastlowast




CHO-hOAT1 (control)










4 Discussion





0

25

50

75

100

125

PAH

upt

ake (

o

f con

trol)


(a)


25

50

75

100

PAH

upt

ake (

o

f con

trol)


(b)


50values






CHO-hOAT3 (control)


lowastlowastlowast







lowast

lowast

lowastlowastlowast

lowastlowastlowast




CHO-hOAT4 (control)






50of 27 plusmn 02 120583M or 119870

119894of 25 plusmn 02 120583M











Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















lowastlowast



lowastlowastlowast




CHO-hOAT1 (control)










4 Discussion





0

25

50

75

100

125

PAH

upt

ake (

o

f con

trol)


(a)


25

50

75

100

PAH

upt

ake (

o

f con

trol)


(b)


50values






CHO-hOAT3 (control)


lowastlowastlowast







lowast

lowast

lowastlowastlowast

lowastlowastlowast




CHO-hOAT4 (control)






50of 27 plusmn 02 120583M or 119870

119894of 25 plusmn 02 120583M











Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

25

50

75

100

125

PAH

upt

ake (

o

f con

trol)


(a)


25

50

75

100

PAH

upt

ake (

o

f con

trol)


(b)


50values






CHO-hOAT3 (control)


lowastlowastlowast







lowast

lowast

lowastlowastlowast

lowastlowastlowast




CHO-hOAT4 (control)






50of 27 plusmn 02 120583M or 119870

119894of 25 plusmn 02 120583M











Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article interaction of natural dietary and herbal...

Documents