1521-009X/44/3/398–408$25.00 http://dx.doi.org/10.1124/dmd.115.066795DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:398–408, March 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Solitary Inhibition of the Breast Cancer Resistance Protein EffluxTransporter Results in a Clinically Significant Drug-Drug Interaction

with Rosuvastatin by Causing up to a 2-Fold Increase inStatin Exposure

Robert Elsby,1 Paul Martin, Dominic Surry,2 Pradeep Sharma, and Katherine Fenner

DMPK, Drug Safety and Metabolism (R.E., D.S.), and Quantitative Clinical Pharmacology, (P.M.), AstraZeneca R&D Alderley Park,Macclesfield, Cheshire, United Kingdom; and DMPK, Drug Safety and Metabolism, AstraZeneca R&D Darwin, Cambridge,

Cambridgeshire, United Kingdom (P.S., K.F.)

Received August 13, 2015; accepted December 18, 2015

ABSTRACT

The intestinal efflux transporter breast cancer resistance protein(BCRP) restricts the absorption of rosuvastatin. Of the transportersimportant to rosuvastatin disposition, fostamatinib inhibited BCRP(IC50 = 50 nM) and organic anion-transporting polypeptide 1B1(OATP1B1; IC50 > 10 mM), but not organic anion transporter 3, invitro, predicting a drug-drug interaction (DDI) in vivo throughinhibition of BCRP only. Consequently, a clinical interaction studybetween fostamatinib and rosuvastatin was performed (and report-ed elsewhere). This confirmed the critical role BCRP plays in statinabsorption, as inhibition by fostamatinib resulted in a significant1.96-fold and 1.88-fold increase in rosuvastatin area under theplasma concentration–time curve (AUC) and Cmax, respectively. Anin vitro BCRP inhibition assay, using polarized Caco-2 cells androsuvastatin as probe substrate, was subsequently validated withliterature inhibitors and used to determine BCRP inhibitorypotencies (IC50) of the perpetrator drugs eltrombopag, darunavir,

lopinavir, clopidogrel, ezetimibe, fenofibrate, and fluconazole.OATP1B1 inhibition was also determined using human embryonickidney 293–OATP1B1 cells versus estradiol 17b-glucuronide. Cal-culated parameters of maximum enterocyte concentration [Igut max],maximum unbound hepatic inlet concentration, transporter fractionexcreted value, and determined IC50 value were incorporated intomechanistic static equations to compute theoretical increases inrosuvastatin AUC due to inhibition of BCRP and/or OATP1B1.Calculated theoretical increases in exposure correctly predictedthe clinically observed changes in rosuvastatin exposure andsuggested intestinal BCRP inhibition (not OATP1B1) to be themechanism underlying the DDIs with these drugs. In conclusion,solitary inhibition of the intestinal BCRP transporter can result inclinically significant DDIs with rosuvastatin, causing up to a maxi-mum 2-fold increase in exposure, which may warrant statin doseadjustment in clinical practice.

Introduction

Breast cancer resistance protein (BCRP; encoded by ABCG2) is oneof the two major clinically relevant human ATP-binding cassette(ABC)–transporter proteins (the other being P-glycoprotein; ABCB1)that use ATP hydrolysis to efflux drugs and xenobiotics out of cells(Giacomini et al., 2010). BCRP is ubiquitously expressed in theintestine (apical brush border membrane of enterocytes), liver (canalicularmembrane of hepatocytes), and kidney (apical brush border membrane of

renal proximal tubule cells) and can affect the absorption and eliminationof drugs that are substrates of this transporter, ultimately defining theirexposure (Mao and Unadkat, 2015).The important role that BCRP plays in drug disposition has been

demonstrated clinically in pharmacogenetic studies investigating theABCG2 single nucleotide polymorphism c.421C.A (Q141K, rs2231142),in which the impaired transport function phenotype (421CA or AA)resulted in increased plasma exposures of several BCRP substratedrugs, including rosuvastatin, atorvastatin, fluvastatin, and diflomotecan(Sparreboom et al., 2004; Keskitalo et al., 2009; Giacomini et al., 2013).Such observations also suggest that interindividual differences in BCRPfunction due to pharmacogenetics likely contribute to variability inbioavailability (absorption), exposure [area under the plasma concentration–time curve (AUC) and maximum plasma concentration (Cmax)], andefficacy of drugs that are BCRP substrates (Giacomini et al., 2010, 2013).

This research was funded by AstraZeneca. P.M., P.S., and K.F. are employeesof AstraZeneca, and R.E. and D.S. are former employees of AstraZeneca.

dx.doi.org/10.1124/dmd.115.066795.1Current affiliation: Drug Transporter Services, Cyprotex Discovery Ltd., Biohub

at Alderley Park, Macclesfield, Cheshire, United Kingdom.2Current affiliation (DS): Skill Supply, Sandbach, Cheshire, United Kingdom.

ABBREVIATIONS: ABC, ATP-binding cassette; AUC, area under the plasma concentration–time curve; B-A, basolateral to apical; BCRP, breastcancer resistance protein; DDI, drug-drug interaction; DMSO, dimethylsulfoxide; HBSS, Hanks’ balanced salt solution; HEK293, human embryonickidney 293; Ko143, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-Octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino-[19,29:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester; LC-MS/MS, liquid chromatography–tandem mass spectrometry; NTCP, sodium/taurocholate cotransport-ing peptide; OAT3, organic anion transporter 3; OATP1B1, organic anion-transporting polypeptide 1B1; R406, N4-(2,2-dimethyl-3-oxo-4-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine.

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Indeed, it has recently been reported that the observed ethnic differenceand variability in exposure of the BCRP substrates rosuvastatin andatorvastatin between Caucasian and Asian populations can be partlyexplained by the higher frequency of the ABCG2 c.421C.A poly-morphism, alongside a hypothesized lower intrinsic BCRP activity, inAsian populations. The resulting overall increase in statin absorptiongives rise to the higher plasma exposures observed clinically in Asiancompared with Caucasian subjects (Birmingham et al., 2015a).Aside from genetic polymorphisms, concomitant administration of a

drug that is an inhibitor of BCRP can alsomodulate the pharmacokineticsof a transported substrate, resulting in a drug-drug interaction (DDI),which may lead to toxicity due to increased exposure, or altered efficacy.DDIs attributed to BCRP have been observed clinically between topotecanand elacridar, resulting in increased topotecan absorption (Kruijtzer et al.,2002); between methotrexate and benzimidazoles, resulting in delayedrenal elimination of methotrexate (Breedveld et al., 2004); and, in part,between rosuvastatin and cyclosporine, resulting in increased rosuvastatinabsorption (Simonson et al., 2004; Tweedie et al., 2013). As highlighted

earlier, there is a persuasive body of evidence that supports the clinicalimportance of the BCRP efflux transporter in drug development.Rosuvastatin calcium is an 3-Hydroxy-3-methylglutaryl–coenzyme-A

reductase inhibitor (statin) that has been developed to treat dyslipidemiaby reducing low-density lipoprotein cholesterol (Crestor; AstraZeneca,Cambridge, UK). The critical disposition pathways of rosuvastatin andtheir individual contributions to overall clearance (fraction excretedvalues; ƒe), determined by Elsby et al. (2012) and derived from clinicalhuman mass balance, pharmacogenetic, and DDI evidence, are shown inFig. 1. These critical pathways include the intestinal BCRP effluxtransporter as the rate-determining barrier to rosuvastatin absorption(fraction absorbed = 0.5; therefore, BCRP ƒe = 0.5), the hepatic uptaketransporter organic anion-transporting polypeptide 1B1 (OATP1B1)responsible for hepatic elimination, and the renal uptake transporterorganic anion transporter 3 (OAT3) responsible for the active renalsecretion of rosuvastatin (Elsby et al., 2012). Metabolism of rosuvastatinby CYP2C9 constitutes only a minor pathway to overall disposition(#10%).

Fig. 1. Critical disposition pathways of rosuvastatin as described by Elsby et al. (2012).

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Fostamatinib is an oral spleen tyrosine kinase inhibitor that has beeninvestigated as a treatment of rheumatoid arthritis (Weinblatt et al.,2014) and, due to patient comorbidities in this disease population, waslikely to be coadministered with statins, such as rosuvastatin andatorvastatin. Fostamatinib is a prodrug (R788; chemical name/structureprovided in Sweeny et al., 2010) that is rapidly and essentiallycompletely metabolized by dephosphorylation in the enterocytes of the

intestine to the active systemic metabolite R406 (N4-(2,2-dimethyl-3-oxo-4-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethyoxyphenyl)-2,4-pyrimidinediamine) (Sweeny et al., 2010; Baluom et al., 2013).Therefore, the first aim of this study was to investigate fostamatiniband R406 as inhibitors of the principal rosuvastatin transporters BCRP,OATP1B1, and OAT3 in vitro and, on the basis of inhibitionpredictions, to use fostamatinib as a mechanistic in vivo tool in a

Fig. 2. Inhibition of BCRP-, OATP1B1-, and OAT3-mediated probe substrate transport activity by fostamatinib, R406, and positive control inhibitors. Data are expressed asthe mean (n = 3 wells or n $ 5 oocytes).

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rosuvastatin clinical interaction study to confirm the impact that solitaryinhibition of BCRP has on rosuvastatin exposure. The second aim wasto validate a Caco-2 rosuvastatin BCRP inhibition assay, and then,using determined IC50 data and mechanistic static equations, to evaluatethe role BCRP inhibition plays in the clinical DDIs observed betweenrosuvastatin and a range of perpetrator drugs by comparing predictedtheoretical fold increases in exposure to the clinically observedrosuvastatin AUC increases.

Materials and Methods

Materials. Estrone 3-sulfate, sulfasalazine, estradiol 17b-glucuronide,rifamycin SV, Triton X, novobiocin, Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-Octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino-[19,29:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], cyclosporin A, panto-prazole, diclofenac, nifedipine, clopidogrel, fluconazole, and fenofibrate werepurchased from Sigma-Aldrich (St. Louis, MO; Poole, Dorset, UK). Eltrombopag,darunavir, and lopinavir were purchased from Selleck Chemicals (Houston, TX;Shanghai, China). Fluvastatin was obtained from Sequoia Research Products(Pangbourne, Reading, UK). [3H]estrone 3-sulfate, [3H]estradiol 17b-glucuronide,and Ultima Gold and Optiphase Supermix scintillation cocktails were purchasedfrom PerkinElmer Life and Analytical Sciences (Boston,MA; Buckinghamshire,UK). [3H]methotrexate and methotrexate were purchased from AmericanRadiolabeled Chemicals (St. Louis, MO). Fostamatinib and its active metaboliteR406 were supplied by AstraZeneca R&D (Alderley Park, Macclesfield, UK).Metoprolol, rosuvastatin, atorvastatin, and ezetimibe were supplied by AstraZenecaCompound Management Group. [3H]rosuvastatin was custom synthesized byQuotient Bioresearch (Cardiff, UK). Hanks’ balanced salt solution (HBSS;containing CaCl2 and MgCl2) and HEPES were purchased from Life Technol-ogies (Paisley, UK; Grand Island, NY). All other chemicals, solvents, andreagents were purchased from Sigma-Aldrich.

Sodium uptake buffer, ND96 buffer, BD Falcon HTS 96-well flat-bottomtissue culture treated plates, BD Biocoat Poly-D-lysine 24-well multiwell plates,humanBCRP (ABCG2)membrane vesicles, humanOAT3 (SLC22A8)–expressingXenopus laevis oocytes, and control (water-injected) oocytes were supplied by BDBiosciences Discovery Labware (Woburn/Bedford,MA;Oxford, UK). UniFilter-96GF/B filter plates and TopSeal A adhesive sealing film were supplied byPerkinElmer Life and Analytical Sciences. Millicell-96 multiwell cell cultureinsert plates (with polycarbonate membranes; 0.4mmpore size, 0.12 cm2 surfacearea) and Millicell 96-well transport companion plates were purchased fromMillipore (Watford, Hertfordshire, UK). Ninety-six–well deep well (2 ml)polypropylene or shallow round-bottomed polypropylene plates were suppliedby VWR International Ltd. (Lutterworth, Leicestershire, UK). HTS Transwell-96permeable supports and associated 96-well plasticware were obtained fromCorning Incorporated Life Sciences (Tewksbury, MA). A recombinant cell lineexpressing OATP1B1 [human embryonic kidney 293 (HEK293)–OATP1B1]was provided by AstraZeneca R&D. Caco-2 cells (HTB37, supplied at passagenumber 17) were purchased from American Type Culture Collection (Manassas,VA).

In Vitro BCRP Inhibition Assessment of Fostamatinib and R406. Uptakeof the BCRP probe substrate [3H]estrone 3-sulfate (1 mM) was determined (intriplicate wells per condition) according to a previously validated method (Elsbyet al., 2011a), using an incubation time of 2minutes at 32�C andBCRP-expressingmembrane vesicles plus/minus ATP (5 mM), in the absence and presence offostamatinib (0.0009–2 mM), R406 (0.0009–2 mM), and the positive controlinhibitor sulfasalazine (0.1–30 mM). Uptake solutions contained #2% (v/v)dimethylsulfoxide (DMSO).

In Vitro OATP1B1 Inhibition Assessment of R406. Uptake of theOATP1B1 probe substrate [3H]estradiol glucuronide (0.02 mM) was determinedusing 24-well plates according to a previously validated method (Sharma et al.,2010) in HEK293-OATP1B1 cells in the absence and presence of R406 (0.3–10 mM) and the positive control inhibitor rifamycin SV (0.001–100 mM).Incubations (triplicate wells per condition) were conducted in HBSS buffer(containing 10 mMHEPES, pH 7.4) at 37�C for 2 minutes, following a 15-minutepreincubation with test inhibitor. Uptake solutions contained #1% (v/v) DMSO.

In Vitro OAT3 Inhibition Assessment of R406. Uptake of the OAT3 probesubstrate [3H]methotrexate (10 mM) was determined according to a previouslydescribed method (Elsby et al., 2011b) using control and OAT3-expressingoocytes in the absence and presence of R406 (0.11–32 mM) and the positivecontrol inhibitor sulfasalazine (0.3–100 mM). Incubations (pools of 10 oocytesper condition) were conducted at room temperature for 60 minutes. Uptakesolutions contained #2% (v/v) DMSO.

Data Analysis. In brief, the amount of radioactivity (disintegrations perminute) taken up by vesicles, HEK293-OATP1B1 cells, or oocytes was determinedand used to calculate the amount (picomoles) of probe substrate, which wassubsequently converted to uptake rate (pmol/min/mg protein or pmol/h/oocyte)as described previously (Sharma et al., 2010; Elsby et al., 2011b). Backgroundcondition uptake rate (uptake in BCRP vesicles minus ATP, uptake in HEK293-OATP1B1 cells in the presence of 100 mM positive control inhibitor whereOATP1B1 is 100% inhibited, or uptake in control oocytes) was subtractedfrom that determined in transporter-expressing vesicles/cells/oocytes to givetransporter-specific uptake rate. This was then converted to percentage (vehicle)control transport activity, which was subsequently plotted against nominalinhibitor concentration. Curves were fitted using XLfit 5.1 (ID BusinessSolutions Ltd., Guildford, Surry, UK) [four-parameter logistic model, Eq. 201, aspublished for P-glycoprotein inhibition (Elsby et al., 2011c)] to determine theconcentration that produces half-maximal inhibition of probe substrate transport(IC50).

Clinical Interaction Study between Fostamatinib and Rosuvastatin.Recently, a clinical interaction study (NCT01725230, D4300C00039) betweenfostamatinib and rosuvastatin performed in healthy volunteers (62% Caucasian,38% black ethnicity; 95% male and 5% female between the ages of 18 and 55years, with body weight.50 kg and body mass index between 18 and 30 kg/m2)was fully described and reported elsewhere (Martin et al., 2016). In brief, thestudy was an open-label, fixed-sequence study that assigned eligible subjects toreceive 20 mg of rosuvastatin over two treatment periods: alone for period 1 andin combination with fostamatinib at steady state (100 mg twice daily for 5 days)for period 2. Plasma concentrations of rosuvastatin and R406 were quantifiedby validated bioanalytical liquid chromatography–tandem mass spectrometry

TABLE 1

Summary of fostamatinib (100 mg) dose-related concentration and inhibitory kinetic parameters for calculation of [I]/Ki ratios and R-value

R-value is equal to (1 + [Iinlet max u]/Ki).

Transporter I1 I2 IC50 [I1]/Ki [I2]/Ki Iinlet max u R-Value Potential for DDI ([I1]/Ki $ 0.1, [I2]/Ki $ 10, R-value $ 1.25)

mM mM mM mM

BCRP1.6a 691 0.050b 32 13,820 NA NA Yes (intestinal)

0.031c 52 NAOATP1B1 1.6a NA . 10 0.16 NA 0.284 1.03 No

I1, mean steady-state maximum plasma concentration (Cmax) value for total systemic drug (R406); I2, maximal theoretical gastrointestinal concentration of R788prodrug [calculated from dose (mol)/250 ml]; Iinlet max u, maximum unbound liver inlet concentration, calculated as fu � [(I1) + (Fa � ka � dose/Qh)], where fu is thefraction unbound (R406 = 0.018), Fa is the fraction absorbed (as default taken to be 1.0), ka is the absorption rate constant (as default taken to be 0.1 min

21), and Qh ishepatic blood flow (1500 ml/min); Ki, absolute inhibition constant (equates to IC50 in these assays as probe substrate concentration used is , ,,, Km); NA, notapplicable.

aDerived from plasma concentration data determined from multiple clinical studies.bFostamatinib (R788).cR406.

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(LC-MS/MS) methodologies and used to determine pharmacokinetic parameters(AUC and Cmax) by noncompartmental modeling, as reported in Martin et al.(2016).

Validation of an In Vitro Caco-2 BCRP Inhibition Assay UtilizingRosuvastatin as BCRP Probe Substrate. The following experimentalproperties were investigated in the validation of the Caco-2 BCRP inhibitionassay as a method to identify inhibitors of BCRP: 1) the suitability of novobiocinas a positive control inhibitor, 2) interassay variability, and 3) acceptance criteriato quality control future use of the assay. The basolateral-to-apical (B-A)transport of the probe substrate [3H]rosuvastatin was measured across polarizedCaco-2 cell monolayers (triplicate wells per condition) in the absence andpresence (in both donor and receiver compartments; added at the same time asrosuvastatin) of a range of concentrations (0.03–10, 0.1–30, or 0.3–100 mM) ofthe known BCRP inhibitors novobiocin, Ko143, cyclosporin A, pantoprazole,sulfasalazine, atorvastatin, diclofenac, fluvastatin, and nifedipine, and of thenoninhibitor metoprolol, to determine an apparent IC50 value for the inhibition ofBCRP-mediated rosuvastatin transport. Experiments determining the inhibitionof BCRP transport by the positive control inhibitor novobiocin were conductedon nine separate occasions spanning five cell passage numbers (24, 25, 26, 27,and 29) to ensure that the data generated were consistent over the passagesroutinely used for inhibition experiments (20–30). The remaining inhibitors wereeach assessed on three separate experimental occasions. The methodology usedwas a slight modification of the previously validated Caco-2 P-glycoproteininhibition assay (Elsby et al., 2008), which is in agreement with therecommendations of the International P-glycoprotein IC50 Working Group(Bentz et al., 2013). The main modifications were seeding of Caco-2 cells on96-well multiwell insert plates at a density of 270,000 cells/ml (27,000 cells perwell; 245,455 cells/cm2), use of monolayers in transport studies 14–21 days afterseeding, and use of a single incubation time point (90 minutes) in the absence ofshaking.

In brief, polarized Caco-2 cell monolayers were washed three times and thenpreincubated with warm transport buffer (HBSS containing 10 mM HEPES, pH7.4) for 10–15 minutes at 37�C prior to the addition of donor and receiversolutions. Donor solutions of transport buffer containing [3H]rosuvastatin(1mM; approximately 10-fold lower than Km for BCRP = 10.8mM;Huang et al.,2006) and the appropriate concentration of test inhibitor or DMSO (vehiclecontrol; at the equivalent volume of solvent vehicle used for test inhibitor) wereadded to the basolateral compartment of the monolayer (total volume = 300 ml).Receiver solutions of transport buffer containing either the correspondingconcentration of test inhibitor or DMSO (vehicle control) and the cell monolayerintegrity marker Lucifer yellow (100 mM) were added to the apical compart-ments (total volume = 100 ml). Donor and receiver solutions contained 0.5% (v/v)DMSO. Following a 90-minute incubation at 37�C, the amount of [3H]rosuvastatinappearing in the receiver compartment was determined by sampling (50 ml),quantified by liquid scintillation counting, and used to calculate apparentpermeability (Papp), as described previously (Elsby et al., 2008). The passivepermeability of rosuvastatin observed when BCRP is completely inhibited[derived from incubations containing the highest concentration (30 mM) of thepositive control inhibitor novobiocin] was subtracted from the determined B-APapp value in the absence or presence of test inhibitor, to give a correctedBCRP-mediated B-A Papp, which was subsequently converted to percentage(vehicle) control transport activity. The resulting inhibition curves were plottedand fitted as described earlier to determine IC50 values, which are equivalent toKi values (assuming competitive inhibition). Mass balance (percent recovery) ofrosuvastatin was calculated as described previously (Elsby et al., 2011c). Thepercentage mass (picomoles) transfer of coincubated Lucifer yellow across thecell monolayer was determined, and cell monolayer integrity was deemedacceptable if the transfer was #1.5%.

Mechanistic Static Predictions of AUC Changes for Known ClinicalDDIs with Rosuvastatin Based upon Determined In Vitro Caco-2 BCRPInhibitory Data. Eltrombopag, darunavir, lopinavir, clopidogrel, ezetimibe,fenofibrate, and fluconazole were assessed (over a range of six concentrations) inthe validated Caco-2 BCRP inhibition assay (using both radiolabeled and across-validated LC-MS/MS endpoint) to determine inhibitory potencies towardBCRP-mediated rosuvastatin transport. The IC50 (equating to Ki) valuesobtained for the aforementioned compounds and fostamatinib were incorporatedinto the following adapted Rowland-Matin mechanistic static equation, asdescribed previously by Elsby et al. (2012), to predict the change in rosuvastatin

AUC based upon inhibition of a fraction excreted (ƒe) value of 0.5 for intestinalBCRP:

Fold  D AUC ¼ 1feð1þ½I�=KiÞ þ ð12 feÞ

where Ki = absolute inhibition constant (equating to IC50 if the probe [S],,,,Km in the inhibition assay and assuming competitive inhibition, based on theCheng-Prusoff equation; Cheng and Prusoff, 1973) and [I] = maximumenterocyte concentration (Igut max; as described by Agarwal et al., 2013).

Additionally, the predicted change in rosuvastatin AUC based upon inhibitionof OATP1B1 (ƒe = 0.38) was also determined using Ki values derived from eitherthe aforementioned in vitro OATP1B1 inhibition assay (at 0.3, 1, 3, 10, 30, and100mM; using LC-MS/MS as the analytical endpoint) or the literature, in contextwith the unbound maximum hepatic inlet concentration as [I], to understand thecontribution (if any) of OATP1B1 inhibition toward the overall magnitude ofexposure change observed clinically through DDI.

Results

Assessment of Fostamatinib and R406 as Inhibitors of BCRP InVitro. Inhibition of estrone 3-sulfate uptake by the positive controlinhibitor sulfasalazine (IC50 = 0.61 mM) passed the acceptance criteriaset during the assay validation (Elsby et al., 2011a) and thereforeindicated that the vesicle test system was capable of detecting inhibitorsof BCRP. Both fostamatinib and R406 inhibited BCRP-mediatedtransport of estrone 3-sulfate with IC50 values of 0.050 and 0.031 mM,respectively (Fig. 2).Assessment of R406 as an Inhibitor of OATP1B1 and OAT3 In

Vitro. Inhibition of probe substrate uptake by the positive controlinhibitors rifamycin SV (IC50 = 0.02mM) or sulfasalazine (IC50 = 4.3mM)demonstrated that the transfected cell or oocyte test systems were ableto detect inhibitors of OATP1B1 or OAT3, respectively. AlthoughR406 did not inhibit OAT3-mediated transport, it did inhibitOATP1B1-mediated transport of estradiol 17b-glucuronide with anIC50 . 10 mM (27% inhibition at 10 mM; Fig. 2).Calculation of [I]/Ki Ratios and R-Value to Predict the DDI

Potential of Fostamatinib. The calculated [I]/Ki ratios and R-value forthe expected therapeutic dose of fostamatinib (100 mg) are shown inTable 1. The mean total Cmax value of R406 (at steady state), usedtoward the calculation of hepatic inlet concentration, is derived from theplasma concentration data determined from multiple clinical studies.The [I2]/Ki ratio predicted that there was a potential for a DDI withBCRP substrate drugs in vivo through inhibition of intestinal BCRP. Incontrast, R406 was unlikely to cause a DDI through inhibition ofhepatic OATP1B1 in vivo (as R-value , 1.25), nor through renalOAT3, as R406 was not an inhibitor of OAT3.Effect of Fostamatinib on the Pharmacokinetic Parameters of

the BCRP Substrate Rosuvastatin in Healthy Volunteers. Themean plasma concentration–time profiles (0–96 hours) of a single oraldose of rosuvastatin (20 mg) when administered alone or concomitantlywith 100mg of fostamatinib at steady state (dosed twice daily for 5 days)are shown in Fig. 3 (reprinted from Martin et al., 2016). Fostamatinibincreased the geometric least-squares mean AUC and Cmax ofrosuvastatin by 96% (90% confidence interval, 78–115) and 88%(90% confidence interval, 69–110), respectively, indicating a clinicalpharmacokinetic interaction (Martin et al., 2016).Validation of an In Vitro Caco-2 BCRP Inhibition Assay

Utilizing Rosuvastatin as BCRP Probe Substrate. Initial compar-ison of bidirectional apparent permeabilities confirmed that rosuvasta-tin (1 mM) exhibited suitable BCRP-mediated efflux in the Caco-2 celltest system: mean6 S.D. (n = 3 wells) A-B Papp = 0.396 0.05 and B-APapp = 15.66 1.75 � 1026 cm/s, giving an efflux ratio of 40. BCRP is

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solely responsible for rosuvastatin efflux in Caco-2 cells, as the effluxratio returned to unity in the presence of the BCRP inhibitor novobiocin(30 mM; data not shown), which did not inhibit P-glycoprotein (up to300 mM) in MDCK-MDR1 cells (R. Elsby, unpublished observation).The rosuvastatin B-A Papp values observed at cell passage numbers 24,25, 26, 27, and 29 were 16.46 1.80, 14.26 1.50, 16.16 3.10, 20.661.9, and 12.5 6 0.90 � 1026 cm/s, respectively, indicating consistentfunctional expression of BCRP over the range of cell passages used forinhibition experiments.The determined interassay mean IC50 values for the inhibition of

BCRP-mediated rosuvastatin transport by the positive control inhibitornovobiocin from nine separate occasions over five different cell passagenumbers, and by Ko143, cyclosporin A, pantoprazole, sulfasalazine,atorvastatin, diclofenac, fluvastatin, and nifedipine are shown inTable 2, proving that the Caco-2 cell test system is able to correctlyidentify known literature inhibitors of BCRP. Mean IC50 curves areshown in Fig. 4, which also shows that metoprolol did not inhibitBCRP-mediated transport of rosuvastatin. The mean IC50 calculated forthe positive control inhibitor novobiocin was 1.46 0.5mM (precision =36%), giving an acceptance range of#2.9mM for future quality controlof the assay (derived from the mean6 3 S.D.). For all BCRP inhibitorstested, the IC50 values determined from individual experimentaloccasions (Table 2) gave good interassay reproducibility, demonstrat-ing that future assessment of compounds as inhibitors of BCRP in thistest system can be acceptably performed just once, and need not be

repeated multiple times. The validated inhibition assay was determinedto be robust, reproducible, and fit for the routine evaluation ofcompounds as inhibitors of BCRP.Predicted versus Observed AUC Changes for Known Clinical

DDIs with Rosuvastatin Based upon Determined In Vitro Caco-2BCRP Inhibitory Data. Eltrombopag, darunavir, lopinavir, clopidog-rel, ezetimibe, and fenofibrate inhibited BCRP-mediated rosuvastatintransport with IC50 values of 2.1, 75, 8.7, 63, 2.9, and 170 mM,respectively (Table 3). In HEK293-OATP1B1 cells, darunavir,lopinavir, clopidogrel, and ezetimibe inhibited OATP1B1-mediatedestradiol 17b-glucuronide uptake with IC50 values of 4.3, 0.43, 1.8, and2.2 mM, respectively (Table 3). In contrast, fluconazole did not inhibitBCRP or OATP1B1 over the concentration ranges tested (data notshown).Using the previously determined drug inhibitory affinities and their

pharmacokinetic parameters given in Table 3, calculations wereperformed with mechanistic static equations to predict the theoreticalfold increase in rosuvastatin AUC that would occur as a consequence ofthese drugs’ inhibition of intestinal BCRP and/or hepatic OATP1B1.The calculated theoretical fold increases in exposure due to inhibition ofeach transporter alone, and in combination, are shown in Table 4. Foreach individual drug highlighted, the theoretical fold increase in AUCdue to inhibition of OATP1B1 alone was lower (ranging from 1.01- to1.25-fold) than the corresponding increase due to sole inhibition ofBCRP, which ranged from 1.15- to 2-fold (Table 4).

TABLE 2

Determined IC50 values for inhibition of rosuvastatin basolateral-to-apical flux across Caco-2 cell monolayers by known inhibitors of BCRP

InhibitorIC50

Published IC50/Ki (Probe Substrate Used) Test System ReferenceDetermined Values from Different Occasions Mean S.D. CV (%)

mM mM mM % mM

Novobiocin 0.4, 1.5, 2.3, 1.6, 1.6, 1.5, 1.4, 1.1, 1.2 1.4 0.5 36 0.4 (methotrexate) Vesicles Saito et al. (2006)Ko143 0.6, 0.2, 0.3 0.4 0.2 50 0.013 (estrone 3-sulfate) Vesicles Xia et al. (2007)Cyclosporin A 5.5, 4.1, 1.6 3.7 2.0 54 6.7 (estrone 3-sulfate) Vesicles Xia et al. (2007)Pantoprazole 8.5, 14.4, 5.9 9.6 4.4 46 13 (methotrexate) Vesicles Breedveld et al. (2004)Sulfasalazine 2.7, 4.7, 1.4 2.9 1.7 59 0.74 (estrone 3-sulfate) Vesicles Elsby et al. (2011a)Atorvastatin 0.5, 0.8, 0.4 0.6 0.2 33 14.3 (estrone 3-sulfate) Vesicles Hirano et al. (2005)Diclofenac 16.5, 19.1, 23.7 19.8 3.6 18 78 (methotrexate) Vesicles Lagas et al. (2009)Fluvastatin 1.3, 1.1, 0.9 1.1 0.2 18 5.43 (estrone 3-sulfate) Vesicles Hirano et al. (2005)Nifedipine 8.0, 11.1, 13.0 10.7 2.5 23 60 (methotrexate) Vesicles Saito et al. (2006)

CV, coefficient of variation.

Fig. 3. Semilogarithmic plot of the geometric mean(6 S.D.) rosuvastatin plasma concentration versustime following administration of a single oral dose ofrosuvastatin (20 mg) in the presence and absence of100 mg of fostamatinib. The figure is reprinted withpermission from Martin et al. (2016).

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Fig. 4. Mean concentration-dependent inhibition of the BCRP-mediated transport of [3H]rosuvastatin (1 mM) by a range of known literature BCRP inhibitors, and thenoninhibitor metoprolol, using polarized Caco-2 cell monolayers. Data are expressed as the mean (6 S.D.) for n $ 3 experimental occasions per inhibitor.

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Discussion

The BCRP transporter plays an important role in clinically observedDDIs, the majority of which are attributable to inhibition of intestinalBCRP, resulting in increased absorption of substrates such as topotecanand rosuvastatin (Kruijtzer et al., 2002; Elsby et al., 2012). Studying thepotential for a drug to perpetrate a BCRP-mediated DDI in the clinicinvolves initial in vitro evaluation of the drug as an inhibitor of BCRPand, where warranted, a follow-up interaction study with a clinicalBCRP probe substrate, such as rosuvastatin, to confirm interactionpotential in vivo (Giacomini et al., 2010; Food and Drug Administra-tion draft DDI guidance 2012, (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM292362.pdf). However, rosuvastatin would not be a reliable probe for such astudy, as BCRP is not the only pathway that is critical to the dispositionof rosuvastatin in vivo, since both OATP1B1 and OAT3 pathways arealso important (Elsby et al., 2012). Consequently, any clinical studyusing rosuvastatin would simply be a broad drug interaction studyunless the “interacting” drug is proven not to inhibit OATP1B1 andOAT3 in vitro or, if an in vitro inhibitor, not predicted to inhibit in vivo,and only then would it become a mechanistic study for assessing BCRPinhibition.On the basis of fulfilling these criteria, following assessment of IC50

values toward BCRP (R788; [I2]/Ki = 13,820; DDI likely), OATP1B1(R406; R-value = 1.03; no DDI potential), and OAT3 (R406; noinhibition), it was recognized that fostamatinib could be used as an invivo tool for a mechanistic clinical BCRP interaction study withrosuvastatin. Such a study would confirm the critical role BCRP playsin rosuvastatin absorption by evaluating, through DDI, the impactsolitary inhibition of BCRP has on statin exposure. Although rosuvas-tatin was not used as the in vitro probe to assess OATP1B1 and BCRPinhibition potential, estradiol 17b-glucuronide is a surrogate forOATP1B1-mediated rosuvastatin transport (Izumi et al., 2015), andfor BCRP, even if a fostamatinib IC50 value using rosuvastatin was100-fold higher than that obtained versus estrone 3-sulfate, it would notchange DDI potential or predictions. The data reproduced from the

clinical interaction study by Martin et al. (2016) demonstrated that soleinhibition of BCRP by fostamatinib resulted in a clinically significantapproximately 2-fold increase in rosuvastatin exposure (AUC andCmax). Examination of rosuvastatin concentration–time profiles (Fig. 3)with and without fostamatinib suggested that there was no change inrosuvastatin elimination rate (0.044 or 0.049 hour21, respectively), aselimination phases were parallel. Coupled with the finding that therewas no change in rosuvastatin half-life (15.7 vs. 14.2 hours, respectively),these data indicated that the DDI with fostamatinib was attributable toincreased rosuvastatin absorption due to inhibition of intestinal BCRP(Martin et al., 2016). Furthermore, the magnitude of the observedexposure change is consistent with a doubling in rosuvastatin absorption,which not only suggests complete inhibition of intestinal BCRP (ƒe = 0.5)by fostamatinib (conforming to Ki being .1000 times lower thananticipated intestinal concentration), but also confirms that BCRP effluxrestricts absorption of rosuvastatin to 50% in humans.Eltrombopag, darunavir, lopinavir, clopidogrel, ezetimibe, fenofi-

brate, and fluconazole are drugs that are listed on the Crestor FDA labelas perpetrating pharmacokinetic DDIs upon coadministration withrosuvastatin. Of the rosuvastatin disposition pathways, OAT3 inhibitionis unlikely to contribute to these DDIs because darunavir and fluconazoledo not inhibit OAT3 in vitro, and eltrombopag (Ki = 7.8 mM), lopinavir(Ki . 10 mM), and fenofibric acid (Ki = 2.2 mM) are not predicted toinhibit OAT3 in vivo based on Cmax u/Ki ratios being ,0.1 (Chu et al.,2007; Yoshida et al., 2012). Additionally, inhibition of OATP1B3 isunlikely to play a role since, although eltrombopag (Ki = 25.6 mM),darunavir (Ki = 4.51 mM), and ezetimibe (Ki . 4 mM) are inhibitors,R-value extrapolations indicate their potential for DDI as unlikely(,1.25), and lopinavir is not an inhibitor (De Bruyn et al., 2011, 2013;Takeuchi et al., 2011; Vildhede et al., 2014). Finally, inhibition ofsodium/taurocholate cotransporting peptide (NTCP) can be ruled out as acontributory factor since 1) despite being anNTCP inhibitor in vitro (Ki =25 mM; Dong et al., 2013), ezetimibe is not expected to cause a DDI viathis mechanism in vivo (R = 1.00); 2) lopinavir does not inhibit NTCP(Vildhede et al., 2014); and 3) although it is unknown whether the

TABLE 3

Inhibitory properties and pharmacokinetic parameters of drugs coadministered with rosuvastatin in a clinical interaction study

Perpetrator Drug Dose MW [I2] BCRP Ki [I2]/Ki Ratio [Igut max] Cmax total fu [Iinlet max u] OATP1B1 KiBiliary BCRP

Iinlet max u / Ki Ratio

mg mM mM mM mM mM mM

Fostamatinib 100 578.52 691 0.05 13,820R406 0.031 58 1.9 0.018 0.289 .10 9.3Eltrombopag 75 442.5 678 2.1 323 29a 17.7 0.01 0.236 2.7b 0.11Darunavir 600 547.73 4382 75 58 73c 10.6d 0.05 1.261 4.3 0.02Lopinavir 400 628.8 2545 8.7 293 42e 14.6 0.02 0.462 0.43 0.05Clopidogrel 75 321.9 932 63 15 78 0.0099 0.02f 0.311 1.8 0.005Clopidogrel 300 321.9 3728 63 59 311 0.130 0.02f 1.245 1.8 0.02Ezetimibe 10 409.4 98 2.9 34 2.4g 0.014 0.10 0.050 2.2 0.02Fenofibrate/fenofibric acid 67 360.83 743 170 4.4 62 25.8 0.01 0.398 20h NAFluconazole 200 306.3 2612 No inhibition NA 218 34.6f 0.89 69.536 No inhibition NA

Cmax total, mean steady-state maximum plasma concentration for total (bound plus unbound) drug measured in the clinical interaction study with rosuvastatin; fu, fraction unbound [taken from thedrug label accessed via the Pharmapendium database (http://www.pharmapendium.com)]; I2, maximal theoretical gastrointestinal concentration (calculated from dose [mol]/250 ml); Igut max, maximumenterocyte concentration, calculated as (Fa � ka � dose/Qent), where Fa is the fraction absorbed (as default taken to be 1.0), ka is the absorption rate constant (as default taken to be 0.1 min21), and Qentis the enterocyte blood flow (300 ml/min [ 18 l/h; Agarwal et al., 2013); Iinlet max u, maximum unbound liver inlet concentration, calculated as fu � [(Cmax total) + (Fa � ka � dose/Qh)], where Fa is thefraction absorbed (as default taken to be 1.0), ka is the absorption rate constant (as default taken to be 0.1 min

21), and Qh is hepatic blood flow (1500 ml/min); Ki, absolute inhibition constant (assumingcompetitive inhibition; equates to IC50 in these assays as probe substrate concentration used is , ,,, Km); MW, molecular weight [taken from the drug label accessed via Pharmapendium database(http://www.pharmapendium.com)]; NA, not applicable.

aValue for Fa taken to be 0.52 (drug label).bValue taken from Allred et al. (2011).cValue for ka taken to be 0.02 (Arab-Alameddine et al., 2014).dValue taken from Kakuda et al. (2011).eValue for ka taken to be 0.02 (Duangchaemkarn and Lohitnavy, 2013).fValue taken from drug pharmacokinetic profile on the University of Washington Drug Interaction Database (http://www.druginteractioninfo.org).gValue for ka taken to be 0.03 (Ezzet et al., 2001).hValue for fenofibric acid taken from Yamazaki et al. (2005).

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remaining drugs are NTCP inhibitors, even if they were to have similarpotencies to those described earlier for OATP1B3, then R-values wouldstill indicate that DDI potential by this mechanism is unlikely.Consequently, this reasoning indicates inhibition of BCRP and/orOATP1B1 as being the predominant transporter pathway(s) to focuson to decipher the underlying mechanisms behind observed DDIs. TheIC50 values of these drugs toward BCRP were determined in the Caco-2BCRP inhibition assay to obtain clinically relevant parameters that couldbe used, alongside OATP1B1 potencies, in calculations of maximumtheoretical increases in rosuvastatin exposure to aid understanding of themechanism(s) behind each DDI. Apart from fluconazole, which did notinhibit BCRP, all of the drugs were inhibitors of BCRP-mediatedrosuvastatin transport and, other than fenofibrate, were predicted to causea DDI through intestinal BCRP inhibition in vivo ([I2]/Ki ratios .10;Table 3). Interestingly, none of these inhibitors were anticipated to inhibitbiliary BCRP in vivo (ratios of [Iinlet max u]/Ki were all #0.1; Table 3),supporting the notion that inhibition of intestinal BCRP is clinicallyimportant for BCRP-mediated DDIs. However, although all thedrugs (except fluconazole) were OATP1B1 inhibitors, only darunavir,lopinavir, and clopidogrel (300 mg) gave R-values.1.25 (= 1.29, 2.07,and 1.69, respectively), indicating potential for an interaction in vivovia OATP1B1. Collectively, these data suggested that inhibition ofintestinal BCRP may be the principal cause of the clinically observedDDIs perpetrated by eltrombopag, darunavir, lopinavir, clopidogrel,ezetimibe, and fenofibrate (Table 4).Predictions of AUC change were performed using determined IC50

values and the adapted Rowland-Matin equation, as described by Elsbyet al. (2012). Previously, a maximum 2-fold increase in exposure due tocomplete inhibition of intestinal BCRP efflux was predicted when [I2]/Ki. 10. However, such an approach likely overpredicts the impact dueto BCRP inhibition (particularly for those DDIs where the increase inobserved AUC is ,2-fold) because of high [I2] values, which may notreflect true concentrations once intestinal solubility is accounted for. Analternative parameter, maximum inhibitor concentration in the enter-ocyte [Igut max], may be a more accurate representation of intestinalconcentration as it is independent of solubility (Agarwal et al., 2013)(see Table 3 for equation). Consequently, in this study, we explored theuse of [Igut max] in the adapted Rowland-Matin equation, alongside aBCRP fe = 0.5, to establish whether taking into account varying degreesof intestinal BCRP inhibition (rather than assuming 100% inhibition)resulted in more accurate predictions of clinical exposure.

Comparison of predicted increases in rosuvastatin AUC due toinhibition of composite transporter pathways BCRP and OATP1B1confirmed intestinal BCRP inhibition to be the primary mechanismunderlying each DDI; as for each individual drug, the AUC increase dueto BCRP was higher than that attributed to OATP1B1 inhibition alone(Table 4). Additionally, all of the predicted AUC increases due to onlyOATP1B1 inhibition were#1.25. Overall, calculated fold increases inAUC (the product of the major AUC change from BCRPwith the minorfrom OATP1B1) accurately predicted the clinically observed dose-dependent fold increases in rosuvastatin exposure due to clopidogreland those increases due to eltrombopag, darunavir, lopinavir, ezetimibe,and fenofibrate, as all predictions were within 10% of the clinical value(Table 4). Furthermore, the accuracy of the predictions not only supportour earlier assumption that BCRP and (to a lesser extent) OATP1B1 arethe dominant pathways involved in the majority of rosuvastatin clinicalDDIs, but also that [Igut max] appears to be the correct concentrationparameter for use in predictions with rosuvastatin.The clinical role of intestinal BCRP inhibition in causing rosuvas-

tatin DDIs implies that the magnitude of such DDIs may be reduced inindividuals who are polymorphic for ABCG2 c.421C.A, due to theseindividuals having less functional BCRP activity to inhibit. This maytranslate to an ethnic difference in DDI susceptibility if the higherfrequency of ABCG2 polymorphism in a particular group results (inpart) in lower functional BCRP activity at the population level, as hasbeen demonstrated in Asian compared with Caucasian populations(Sakiyama et al., 2014; Birmingham et al., 2015a,b). Interestingly, suchan ethnic difference has been observed clinically with eltrombopag,where there was a larger 1.88-fold increase in rosuvastatin AUC in non-Asian subjects compared with only a 1.3-fold increase in Asian subjectsfollowing coadministration of eltrombopag (Allred et al., 2011). Thus,the much smaller magnitude of AUC change for the Asian group isconsistent with this group having less functional BCRP activity to beinhibited by eltrombopag. It was for this reason that the non-Asiangroup AUC change was used as the clinically observed AUC change forcomparison with predictions in this study, as it more accurately reflectsthe impact of inhibiting fully functioning BCRP.Finally, taking into account the analyses from both this study and that

of Elsby et al. (2012) toward understanding the likely mechanismsbehind clinically observed DDIs with rosuvastatin, it is interesting tonote that out of a total of 12 reported clinical DDIs (the first 10 listed onthe Crestor label, plus clopidogrel and fostamatinib) for which increases

TABLE 4

Predicted versus observed AUC changes of rosuvastatin with various coadministered drugs based upon inhibition of intestinal BCRP ( fe = 0.5) or hepatic OATP1B1 ( fe =0.38) transport

Perpetrator Drug Dose

Predicted Fold Increase in AUC Due toInhibition of Composite Pathways

Overall Predicted FoldIncrease in AUC

Clinically ObservedFold Increase in AUC

ReferencePrimary Mechanism

of DDIIntestinal BCRP

(Theoretical Max = 2.0)OATP1B1

(Theoretical Max = 1.6)

mg

Fostamatinib/R406 100 2.00 1.01 2.02 1.96 Martin et al. (2016) BCRP inhibitionEltrombopaga 75 1.87 1.03 1.93 1.88 Allred et al. (2011) BCRP inhibitionDarunavir 600 1.33 1.09 1.45 1.48 Samineni et al. (2012) BCRP inhibitionLopinavir 400 1.71 1.25 2.14 2.10 Kiser et al. (2008) BCRP inhibitionClopidogrel 75 1.38 1.06 1.46 1.40 Pinheiro et al. (2012) BCRP inhibitionClopidogrel 300 1.71 1.18 2.02 1.96 Pinheiro et al. (2012) BCRP inhibitionEzetimibe 10 1.29 1.01 1.30 1.21 Kosoglou et al. (2004) BCRP inhibitionFenofibrate/fenofibric acid 67 1.15 1.01 1.16 1.07 Martin et al. (2003) NAFluconazole 200 None None 1.10b 1.14 Cooper et al. (2002) CYP2C9 inhibition

NA, not applicable.aThe clinically observed fold-increase in exposure used for comparison with the prediction is the mean AUC change derived from non-Asian subjects within the clinical interaction study.bBased upon inhibition of CYP2C9 ( fe = 0.10, fluconazole CYP2C9 Ki = 7 mM; Kunze et al., 1996).

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in AUC were$1.2-fold, six (50%; Table 4) can be attributed to BCRPinhibition as the primary mechanism, two (17%; cyclosporine andatazanavir) can be attributed to complete inhibition of BCRP combinedwith inhibition of OATP1B1, and one (8%; gemfibrozil) can be attributedto the inhibition of both OATP1B and OAT3. The remaining three DDIs(tipranavir, dronedarone, and itraconazole) have not been studied, andtheir mechanisms wait to be determined. These findings confirm theimportance of intestinal BCRP inhibition toward manifesting clinicalDDIs with rosuvastatin.In conclusion, solitary inhibition of the intestinal BCRP efflux

transporter can result in a clinically significant DDI with rosuvastatincausing a maximum 2-fold increase in exposure, which may warrantstatin dose adjustment or dose capping in clinical practice.

Acknowledgments

The authors acknowledge the contract research services provided by BDGentest (now Corning Gentest; Woburn, MA) and Pharmaron Beijing Co.(Beijing, China) in performing some of the in vitro transporter experiments.

Authorship ContributionsParticipated in research design: Elsby, Surry, Martin.Conducted experiments: Elsby, Sharma, Surry.Performed data analysis: Elsby, Sharma, Surry, Martin, Fenner.Wrote or contributed to the writing of the manuscript: Elsby, Martin, Surry,

Sharma, Fenner.

References

Agarwal S, Arya V, and Zhang L (2013) Review of P-gp inhibition data in recently approved newdrug applications: utility of the proposed [I(1) ]/IC(50) and [I(2) ]/IC(50) criteria in the P-gpdecision tree. J Clin Pharmacol 53:228–233.

Allred AJ, Bowen CJ, Park JW, Peng B, Williams DD, Wire MB, and Lee E (2011) Eltrombopagincreases plasma rosuvastatin exposure in healthy volunteers. Br J Clin Pharmacol 72:321–329.

Antara® (fenofibrate) U.S. Food and Drug Administration drug label, revised October 2013;Lupin Pharma. Accessed via Pharmapendium database (www.pharmapendium.com) on September11, 2014.

Arab-Alameddine M, Lubomirov R, Fayet-Mello A, Aouri M, Rotger M, Buclin T, Widmer N,Gatri M, Ledergerber B, and Rentsch K, et al.; Swiss HIV Cohort Study (2014) Populationpharmacokinetic modelling and evaluation of different dosage regimens for darunavir andritonavir in HIV-infected individuals. J Antimicrob Chemother 69:2489–2498.

Baluom M, Grossbard EB, Mant T, and Lau DT (2013) Pharmacokinetics of fostamatinib, aspleen tyrosine kinase (SYK) inhibitor, in healthy human subjects following single and mul-tiple oral dosing in three phase I studies. Br J Clin Pharmacol 76:78–88.

Bentz J, O’Connor MP, Bednarczyk D, Coleman J, Lee C, Palm J, Pak YA, Perloff ES, Reyner E,and Balimane P, et al. (2013) Variability in P-glycoprotein inhibitory potency (IC₅₀) usingvarious in vitro experimental systems: implications for universal digoxin drug-drug interactionrisk assessment decision criteria. Drug Metab Dispos 41:1347–1366.

Birmingham BK, Bujac SR, Elsby R, Azumaya CT, Wei C, Chen Y, Mosqueda-Garcia R,and Ambrose HJ (2015a) Impact of ABCG2 and SLCO1B1 polymorphisms on pharmacoki-netics of rosuvastatin, atorvastatin and simvastatin acid in Caucasian and Asian subjects: a classeffect? Eur J Clin Pharmacol 71:341–355.

Birmingham BK, Bujac SR, Elsby R, Azumaya CT, Zalikowski J, Chen Y, Kim K, and AmbroseHJ (2015b) Rosuvastatin pharmacokinetics and pharmacogenetics in Caucasian and Asiansubjects residing in the United States. Eur J Clin Pharmacol 71:329–340.

Breedveld P, Zelcer N, Pluim D, Sönmezer O, Tibben MM, Beijnen JH, Schinkel AH, vanTellingen O, Borst P, and Schellens JHM (2004) Mechanism of the pharmacokinetic interactionbetween methotrexate and benzimidazoles: potential role for breast cancer resistance protein inclinical drug-drug interactions. Cancer Res 64:5804–5811.

Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1) and theconcentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction.Biochem Pharmacol 22:3099–3108.

Chu XY, Bleasby K, Yabut J, Cai X, Chan GH, Hafey MJ, Xu S, Bergman AJ, Braun MP,and Dean DC, et al. (2007) Transport of the dipeptidyl peptidase-4 inhibitor sitagliptin byhuman organic anion transporter 3, organic anion transporting polypeptide 4C1, and multidrugresistance P-glycoprotein. J Pharmacol Exp Ther 321:673–683.

Cooper KJ, Martin PD, Dane AL, Warwick MJ, Schneck DW, and Cantarini MV (2002) Theeffect of fluconazole on the pharmacokinetics of rosuvastatin. Eur J Clin Pharmacol 58:527–531.

Crestor® (rosuvastatin) U.S. Food and Drug Administration drug label, revised August 2013;AstraZeneca PLC. Accessed via Pharmapendium database (www.pharmapendium.com) onSeptember 10, 2014.

De Bruyn T, Fattah S, Stieger B, Augustijns P, and Annaert P (2011) Sodium fluorescein is aprobe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3. J Pharm Sci100:5018–5030.

De Bruyn T, van Westen GJ, Ijzerman AP, Stieger B, de Witte P, Augustijns PF, and Annaert PP(2013) Structure-based identification of OATP1B1/3 inhibitors. Mol Pharmacol 83:1257–1267.

Diflucan® (fluconazole) U.S. Food and Drug Administration drug label, revised March 2014;Pfizer Inc. Accessed via Pharmapendium database (www.pharmapendium.com) on September11, 2014.

Dong Z, Ekins S, and Polli JE (2013) Structure-activity relationship for FDA approved drugs asinhibitors of the human sodium taurocholate cotransporting polypeptide (NTCP). Mol Pharm10:1008–1019.

Duangchaemkarn K and Lohitnavy M (2013) Compartmental pharmacokinetic modeling oflopinavir in humans. Conf Proc IEEE Eng Med Biol Soc 2013:3523–3526.

Elsby R, Fox L, Stresser D, Layton M, Butters C, Sharma P, Smith V, and Surry D (2011b) Invitro risk assessment of AZD9056 perpetrating a transporter-mediated drug-drug interactionwith methotrexate. Eur J Pharm Sci 43:41–49.

Elsby R, Gillen M, Butters C, Imisson G, Sharma P, Smith V, and Surry DD (2011c) The utilityof in vitro data in making accurate predictions of human P-glycoprotein-mediated drug-druginteractions: a case study for AZD5672. Drug Metab Dispos 39:275–282.

Elsby R, Hilgendorf C, and Fenner K (2012) Understanding the critical disposition pathways ofstatins to assess drug-drug interaction risk during drug development: it’s not just aboutOATP1B1. Clin Pharmacol Ther 92:584–598.

Elsby R, Smith V, Fox L, Stresser D, Butters C, Sharma P, and Surry DD (2011a) Validation ofmembrane vesicle-based breast cancer resistance protein and multidrug resistance protein 2assays to assess drug transport and the potential for drug-drug interaction to support regulatorysubmissions. Xenobiotica 41:764–783.

Elsby R, Surry DD, Smith VN, and Gray AJ (2008) Validation and application of Caco-2 assaysfor the in vitro evaluation of development candidate drugs as substrates or inhibitors ofP-glycoprotein to support regulatory submissions. Xenobiotica 38:1140–1164.

Ezzet F, Krishna G, Wexler DB, Statkevich P, Kosoglou T, and Batra VK (2001) A populationpharmacokinetic model that describes multiple peaks due to enterohepatic recirculation ofezetimibe. Clin Ther 23:871–885.

Giacomini KM, Balimane PV, Cho SK, Eadon M, Edeki T, Hillgren KM, Huang S-M, SugiyamaY, Weitz D, and Wen Y, et al.; International Transporter Consortium (2013) InternationalTransporter Consortium commentary on clinically important transporter polymorphisms. ClinPharmacol Ther 94:23–26.

Giacomini KM, Huang S-M, Tweedie DJ, Benet LZ, Brouwer KLR, Chu X, Dahlin A, Evers R,Fischer V, and Hillgren KM, et al.; International Transporter Consortium (2010) Membranetransporters in drug development. Nat Rev Drug Discov 9:215–236.

Hirano M, Maeda K, Matsushima S, Nozaki Y, Kusuhara H, and Sugiyama Y (2005) In-volvement of BCRP (ABCG2) in the biliary excretion of pitavastatin. Mol Pharmacol 68:800–807.

Huang L, Wang Y, and Grimm S (2006) ATP-dependent transport of rosuvastatin in membranevesicles expressing breast cancer resistance protein. Drug Metab Dispos 34:738–742.

Izumi S, Nozaki Y, Maeda K, Komori T, Takenaka O, Kusuhara H, and Sugiyama Y (2015)Investigation of the impact of substrate selection on in vitro organic anion transporting poly-peptide 1B1 inhibition profiles for the prediction of drug-drug interactions. Drug Metab Dispos43:235–247.

Kakuda TN, Abel S, Davis J, Hamlin J, Schöller-Gyüre M, Mack R, Ndongo N, Petit W,Ridgway C, and Sekar V, et al. (2011) Pharmacokinetic interactions of maraviroc withdarunavir-ritonavir, etravirine, and etravirine-darunavir-ritonavir in healthy volunteers: resultsof two drug interaction trials. Antimicrob Agents Chemother 55:2290–2296.

Kaletra® (lopinavir/ritonavir) U.S. Food and Drug Administration drug label, revised November2013; AbbVie Inc. Accessed via Pharmapendium database (www.pharmapendium.com) onSeptember 11, 2014.

Keskitalo JE, Zolk O, Fromm MF, Kurkinen KJ, Neuvonen PJ, and Niemi M (2009) ABCG2polymorphism markedly affects the pharmacokinetics of atorvastatin and rosuvastatin. ClinPharmacol Ther 86:197–203.

Kiser JJ, Gerber JG, Predhomme JA, Wolfe P, Flynn DM, and Hoody DW (2008) Drug/Druginteraction between lopinavir/ritonavir and rosuvastatin in healthy volunteers. J Acquir ImmuneDefic Syndr 47:570–578.

Kosoglou T, Statkevich P, Yang B, Suresh R, Zhu Y, Boutros T, Maxwell SE, Tiessen R,and Cutler DL (2004) Pharmacodynamic interaction between ezetimibe and rosuvastatin. CurrMed Res Opin 20:1185–1195.

Kruijtzer CMF, Beijnen JH, Rosing H, ten Bokkel Huinink WW, Schot M, Jewell RC, Paul EM,and Schellens JHM (2002) Increased oral bioavailability of topotecan in combination with thebreast cancer resistance protein and P-glycoprotein inhibitor GF120918. J Clin Oncol 20:2943–2950.

Kunze KL, Wienkers LC, Thummel KE, and Trager WF (1996) Warfarin-fluconazole. I. In-hibition of the human cytochrome P450-dependent metabolism of warfarin by fluconazole: invitro studies. Drug Metab Dispos 24:414–421.

Lagas JS, van der Kruijssen CM, van de Wetering K, Beijnen JH, and Schinkel AH (2009)Transport of diclofenac by breast cancer resistance protein (ABCG2) and stimulation ofmultidrug resistance protein 2 (ABCC2)-mediated drug transport by diclofenac and benz-bromarone. Drug Metab Dispos 37:129–136.

Mao Q and Unadkat JD (2015) Role of the breast cancer resistance protein (BCRP/ABCG2) indrug transport–an update. AAPS J 17:65–82.

Martin PD, Dane AL, Schneck DW, and Warwick MJ (2003) An open-label, randomized, three-way crossover trial of the effects of coadministration of rosuvastatin and fenofibrate on thepharmacokinetic properties of rosuvastatin and fenofibric acid in healthy male volunteers. ClinTher 25:459–471.

Martin P, Gillen M, Ritter J, Mathews D, Brealey C, Surry D, Oliver S and Elsby R (2016) Effectsof fostamatinib on the pharmacokinetics of oral contraceptive, warfarin, and the statins rosu-vastatin and simvastatin: results from phase I clinical studies. Drugs in R&D Epub ahead ofprint Jan 9 2016.

Pinheiro LF, França CN, Izar MC, Barbosa SP, Bianco HT, Kasmas SH, Mendes GD, Povoa RM,and Fonseca FA (2012) Pharmacokinetic interactions between clopidogrel and rosuvastatin:effects on vascular protection in subjects with coronary heart disease. Int J Cardiol 158:125–129.

Plavix® (clopidogrel) U.S. Food and Drug Administration drug label, revised December 2013;Sanofi-Aventis. Accessed via Pharmapendium database (www.pharmapendium.com) on Sep-tember 11, 2014.

Prezista® (darunavir) U.S. Food and Drug Administration drug label, revised April 2014; JanssenPharmaceuticals. Accessed via Pharmapendium database (www.pharmapendium.com) onSeptember 12, 2014.

Inhibition of Intestinal BCRP Increases Rosuvastatin Exposure 407

at ASPE

T Journals on A

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d.aspetjournals.orgD

ownloaded from

http://www.pharmapendium.comhttp://www.pharmapendium.comhttp://www.pharmapendium.comhttp://www.pharmapendium.comhttp://www.pharmapendium.comhttp://www.pharmapendium.comhttp://dmd.aspetjournals.org/

Promacta® (eltrombopag) U.S. Food and Drug Administration drug label, revised April 2014;GlaxoSmithKline. Accessed via Pharmapendium database (www.pharmapendium.com) onSeptember 11, 2014.

Saito H, Hirano H, Nakagawa H, Fukami T, Oosumi K, Murakami K, Kimura H, Kouchi T,Konomi M, and Tao E, et al. (2006) A new strategy of high-speed screening and quantitativestructure-activity relationship analysis to evaluate human ATP-binding cassette transporterABCG2-drug interactions. J Pharmacol Exp Ther 317:1114–1124.

Sakiyama M, Matsuo H, Takada Y, Nakamura T, Nakayama A, Takada T, Kitajiri S, Wakai K,Suzuki H, and Shinomiya N (2014) Ethnic differences in ATP-binding cassette transporter,sub-family G, member 2 (ABCG2/BCRP): genotype combinations and estimated functions.Drug Metab Pharmacokinet 29:490–492.

Samineni D, Desai PB, Sallans L, and Fichtenbaum CJ (2012) Steady-state pharmacokineticinteractions of darunavir/ritonavir with lipid-lowering agent rosuvastatin. J Clin Pharmacol 52:922–931.

Sharma P, Holmes VE, Elsby R, Lambert C, and Surry D (2010) Validation of cell-basedOATP1B1 assays to assess drug transport and the potential for drug-drug interaction to supportregulatory submissions. Xenobiotica 40:24–37.

Simonson SG, Raza A, Martin PD, Mitchell PD, Jarcho JA, Brown CD, Windass AS,and Schneck DW (2004) Rosuvastatin pharmacokinetics in heart transplant recipients admin-istered an antirejection regimen including cyclosporine. Clin Pharmacol Ther 76:167–177.

Sparreboom A, Gelderblom H, Marsh S, Ahluwalia R, Obach R, Principe P, Twelves C, VerweijJ, and McLeod HL (2004) Diflomotecan pharmacokinetics in relation to ABCG2 421C.Agenotype. Clin Pharmacol Ther 76:38–44.

Sweeny DJ, Li W, Clough J, Bhamidipati S, Singh R, Park G, Baluom M, Grossbard E, and LauDTW (2010) Metabolism of fostamatinib, the oral methylene phosphate prodrug of the spleentyrosine kinase inhibitor R406 in humans: contribution of hepatic and gut bacterial processes tothe overall biotransformation. Drug Metab Dispos 38:1166–1176.

Takeuchi K, Sugiura T, Umeda S, Matsubara K, Horikawa M, Nakamichi N, Silver DL, IshiwataN, and Kato Y (2011) Pharmacokinetics and hepatic uptake of eltrombopag, a novel platelet-increasing agent. Drug Metab Dispos 39:1088–1096.

Tweedie D, Polli JW, Berglund EG, Huang SM, Zhang L, Poirier A, Chu X, and Feng B;International Transporter Consortium (2013) Transporter studies in drug development: expe-rience to date and follow-up on decision trees from the International Transporter Consortium.Clin Pharmacol Ther 94:113–125.

Vildhede A, Karlgren M, Svedberg EK, Wisniewski JR, Lai Y, Norén A, and Artursson P (2014)Hepatic uptake of atorvastatin: influence of variability in transporter expression on uptakeclearance and drug-drug interactions. Drug Metab Dispos 42:1210–1218.

Weinblatt ME, Genovese MC, Ho M, Hollis S, Rosiak-Jedrychowicz K, Kavanaugh A, MillsonDS, Leon G, Wang M, and van der Heijde D (2014) Effects of fostamatinib, an oral spleentyrosine kinase inhibitor, in rheumatoid arthritis patients with an inadequate response tomethotrexate: results from a phase III, multicenter, randomized, double-blind, placebo-controlled, parallel-group study. Arthritis Rheumatol 66:3255–3264.

Xia CQ, Liu N, Miwa GT, and Gan LS (2007) Interactions of cyclosporin a with breast cancerresistance protein. Drug Metab Dispos 35:576–582.

Yamazaki M, Li B, Louie SW, Pudvah NT, Stocco R, Wong W, Abramovitz M, Demartis A,Laufer R, and Hochman JH, et al. (2005) Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediatedtransport. Xenobiotica 35:737–753.

Yoshida K, Maeda K, and Sugiyama Y (2012) Transporter-mediated drug–drug interactionsinvolving OATP substrates: predictions based on in vitro inhibition studies. Clin PharmacolTher 91:1053–1064.

Zetia® (ezetimibe) U.S. Food and Drug Administration drug label, revised January 2012; Merck& Co. Accessed via Pharmapendium database (www.pharmapendium.com) on September 11, 2014.

Address correspondence to: Katherine Fenner, DMPK, Drug Safety andMetabolism, AstraZeneca R&D Darwin, Cambridge, Cambridgeshire, CB4 0WG,United Kingdom. E-mail: Katherine.Fenner@astrazeneca.com

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