Contribution of Abcc4-Mediated Gastric Transport to the ......Jul 26, 2013 · Cancer Therapy:...

Cancer Therapy: Preclinical

Contribution of Abcc4-Mediated Gastric Transport to theAbsorption and Efficacy of Dasatinib

Brian D. Furmanski1, Shuiying Hu1, Ken-ichi Fujita1, Lie Li1, Alice A. Gibson1, Laura J. Janke2,Richard T. Williams3, John D. Schuetz1, Alex Sparreboom1, and Sharyn D. Baker1

AbstractPurpose: Several oral multikinase inhibitors are known to interact in vitro with the human ATP-binding

cassette transporter ABCC4 (MRP4), but the in vivo relevance of this interaction remains poorly understood.

We hypothesized that host ABCC4 activity may influence the pharmacokinetic profile of dasatinib and

subsequently affect its antitumor properties.

Experimental Design: Transport of dasatinib was studied in cells transfected with human ABCC4 or the

ortholog mouse transporter, Abcc4. Pharmacokinetic studies were done in wild-type and Abcc4-null mice.

The influence of Abcc4 deficiency on dasatinib efficacywas evaluated in amodel of Phþ acute lymphoblastic

leukemia by injection of luciferase-positive, p185(BCR-ABL)-expressing Arf(�/�) pre-B cells.

Results: Dasatinib accumulation was significantly changed in cells overexpressing ABCC4 or Abcc4

comparedwith control cells (P < 0.001). Deficiency of Abcc4 in vivowas associatedwith a 1.75-fold decrease

in systemic exposure to oral dasatinib, but had no influence on the pharmacokinetics of intravenous

dasatinib. Abcc4was found to be highly expressed in the stomach, and dasatinib efflux from isolatedmouse

stomachs ex vivowas impaired by Abcc4 deficiency (P < 0.01), without any detectable changes in gastric pH.

Abcc4-null mice receiving dasatinib had an increase in leukemic burden, based on bioluminescence

imaging, and decreased overall survival compared with wild-type mice (P ¼ 0.048).

Conclusions: This study suggests that Abcc4 in the stomach facilitates the oral absorption of dasatinib,

and it possibly plays a similar role for other orally administered substrates, such as acetylsalicylic acid. This

phenomenon also provides a mechanistic explanation for the malabsorption of certain drugs following

gastric resection. Clin Cancer Res; 19(16); 1–12. �2013 AACR.

IntroductionDasatinib, an orally administered inhibitor of Bcr/Abl

and Src kinases, has been approved for the treatment ofimatinib-resistant or imatinib-intolerant forms of chronicmyeloid leukemia (CML) and Philadelphia chromosome-positive (Phþ) acute lymphoblastic leukemia (ALL; ref. 1).A mass balance study has indicated that, following oral

administration, dasatinib accounts for only less than 1%and 19% of the dose recovered in urine and feces, respec-tively, suggesting that dasatinib is extensively metabolizedbefore being eliminated from the body (2). The primarypathways of dasatinib metabolism in humans include N-dealkylation (M4) and N-oxidation (M5) by CYP3A4 andflavin-containing monooxygenase 3, respectively (3).

As with other tyrosine kinase inhibitors, dasatinib issubject to extensive interindividual pharmacokinetic vari-ability. For example, the coefficient of variation in the areaunder the curve (AUC) of dasatinib is more than 50% inadult patients (4), and as high as 104% in a pediatric patientpopulation (5). The high degree of pharmacokinetic vari-ability observed with dasatinib has potentially importanttoxicologic and therapeutic ramifications. In particular, itwas previously shown that the steady-state trough concen-tration of dasatinib in patients with CML is strongly asso-ciated with treatment-related toxicities as well as dosereductions and interruptions (6). Furthermore, pharmaco-kinetic studies conducted in mice bearing CML xenograftshave suggested that the time course and extent of inhibitionof tumoral biomarkers of efficacy are correlated with theplasma levels of dasatinib (7).

Themechanistic basis underlying the unpredictable phar-macokinetic profile of dasatinib remains largely unknown.

Authors' Affiliations: Departments of 1Pharmaceutical Sciences, 2Pathol-ogy, and 3Oncology, St. Jude Children's Research Hospital, Memphis,Tennessee

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://cancerres.aacrjournals.org/).

Current address for B.D. Furmanski: Siga Technologies, Corvallis, Oregon;current address for Ken-ichi Fujita: Institute of Molecular Oncology, ShowaUniversity, Tokyo, Japan; and current address for R.T. Williams: PumaBiotechnology, Los Angeles, California.

B.D. Furmanski and S. Hu contributed equally to this work.

Corresponding Author: Alex Sparreboom, St. Jude Children's ResearchHospital, 262 Danny Thomas Place, CCC, Mail Stop 313, Room I5308,Memphis, TN 38105. Phone: 901-595-5346; Fax: 901-595-3125; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-0980

�2013 American Association for Cancer Research.

ClinicalCancer

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Model-based simulations have indicated that a criticaldeterminant of dasatinib’s pharmacokinetic variability isassociated with absorption-related processes and to a lesserextent with clearance (8). This supposition is consistentwith studies indicating that deficiency of certain ATP-bind-ing cassette (ABC) transporters normally expressed on theapical membrane of intestinal enterocytes, such as ABCB1(P-glycoprotein), is associated with an increased AUC ofdasatinib after oral (9) but not intravenous administration(10). Because dasatinib is poorly permeable (11), yet veryrapidly absorbed in humans compared with other tyrosinekinase inhibitors (12), its transport across both of theenterocytic membranes after oral administration is likelytransporter mediated. One candidate transporter involvedin the basolateral transport process is the multidrug resis-tance-associated protein 4 (MRP4; ABCC4), which isknown to interact with a number of tyrosine kinase inhi-bitors (13) and, on the basis of functional in vivo dataobtained with cefadroxil (14) and in vitro expression datain Caco-2 cells (15), may be localized on the basolateralmembrane of enterocytes. In the current study,we tested thehypothesis that dasatinib is a transported substrate ofABCC4 in vitro and that deficiency of the ortholog mousetransporter Abcc4 is associated with an altered pharmaco-kinetic and efficacy profile of dasatinib in vivo.

Materials and MethodsIn vitro transport studies

Dasatinib was obtained from LC Laboratories andimatinib was kindly provided by Novartis Pharma-ceuticals. [3H]Dasatinib, [3H]imatinib, [3H]adefovirdipivoxil [bis(POM)-PMEA; hereafter referred to as

PMEA], and unlabeled PMEA were purchased fromMoravek Biochemicals. [3H]Estradiol-17b-D-glucuronide(E2G), [14C]tetraethylammonium bromide (TEA), and[acetyl-14C]salicylic acid (aspirin) were obtained fromAmerican Radiolabeled Chemicals, and [7-14C]salicylicacid from PerkinElmer. Inside-out vesicles of Sf9cells expressing ABCC3 or ABCC4 (Genomembrane)were used to determine uptake of either [3H]E2G,[3H]imatinib, or [3H]dasatinib using 5-minute incuba-tions, as described (16), in the presence or absence ofglutathione (GSH; Life Technologies). The efflux trans-port of [3H]PMEA, [3H]imatinib, or [3H]dasatinib wasalso evaluated in Saos-2 cells transfected with an emptypcDNA or Abcc4 using 4-hour incubations, as described(17). The uptake of [14C]TEA or [3H]dasatinib was alsoevaluated in HEK293 cells containing an empty PMIG IIvector or a flag-tagged cDNA of mOct1 or mOct2. Theresults from the in vitro transport studies were normal-ized to uptake values in cells transfected with an emptyvector, after normalization to total protein content asmeasured by a Pierce BCA Protein Assay Kit (ThermoScientific).

In vivo pharmacokinetic studiesMale mice knockout for Abcc4 [Abcc4(�/�)] were bred

in-house and age-matched wild-type mice, all on aC57BL/6 background, were obtained from The JacksonLaboratory. Mice were housed in a temperature-con-trolled environment with a 12-hour light cycle and givena standard diet and water ad libitum. All in vivo experi-ments were approved by the Institutional Animal Careand Use Committee at St. Jude Children’s Research Hos-pital (Memphis, TN).

Dasatinib was formulated in DMSO (25 mg/mL) anddiluted 25-fold in 50 mmol/L sodium acetate buffer (pH4.6) to make a 1 mg/mL dosing solution immediatelybefore drug administration by oral gavage, or in propyleneglycol:deionizedwater (1:1, v/v) before intravenous admin-istration (18). Imatinib was dissolved in sterile water tomake a 10 mg/mL dosing solution. Mice were fasted for 3hours before and during the study, with unrestricted accessto drinking water. Dasatinib was administered by oralgavage or by tail vein injection at a dose of 10 mg/kg(18) and imatinib was given at a dose of 50 mg/kg (19).At selected time points after drug administration, bloodsamples (30 mL each) were taken from individual mice at0.25, 0.5, or 0.67, and 1 hour from the submandibular veinusing a lancet, and at 2 and 4 hours from the retro-orbitalvenous plexus using a heparinized capillary (Oxford Lab-ware). A final blood draw was obtained at 6 hours by acardiac puncture after anesthetization with isoflurane usinga syringe and needle. Plasma samples were analyzed byliquid chromatography/tandem mass spectrometry (LC/MS-MS), using a Waters ACQUITY separation system cou-pled to a TQD detector (see below). Pharmacokinetic para-meters were calculated using WinNonlin 6.2 (Pharsight).

The extent of binding of dasatinib to serum proteins (20)and blood cell partitioning of dasatinib were evaluated

Translational RelevanceDasatinib is an inhibitor of the Bcr/Abl and Src kinases

and is used in the treatment of chronicmyeloid leukemiaand Philadelphia chromosome-positive (Phþ) acutelymphoblastic leukemia (ALL). The interindividualpharmacokinetic variability for dasatinib treatment isextensive and largely unexplained, and this phenome-non may have important ramifications for the agent’sclinical activity. We speculated that differential expres-sion of the ATP-binding cassette transporter ABCC4(MRP4) might play a role in explaining this pharmaco-logic variability. We investigated the contribution ofABCC4 to thepharmacokinetics and efficacy of dasatinibusing an array of in vitro and in vivomodel systems. Ourresults indicate that ABCC4 is abundantly expressed inthe stomach, where it facilitates the gastric absorption ofdasatinib. Moreover, Abcc4 deficiency in mice was asso-ciated with reduced activity of dasatinib against a modelof Phþ-ALL. These results identified ABCC4 as an impor-tant, previously unrecognized contributor to the oralabsorption of dasatinib and this process may be relevantfor other substrate drugs, such as acetylsalicylic acid.

Furmanski et al.

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according to published methods (18). Intrinsic intestinalpermeability was evaluated by determining the recoveryof D-[2-3H]mannitol (American Radiolabeled Chemicals)in plasma, kidney, and liver at 2 hours after oral dosing(1 mg/kg), as described (21). In a separate experiment, theurinary bladder, gall bladder, and spleens, after perfusion,were removed from wild-type or Abcc4(�/�) mice 5 min-utes after oral administration of dasatinib and analyzed forthe presence of dasatinib by LC/MS-MS.

Quantitative determination of dasatinib andmetabolites by LC/MS-MSFrozen samples were thawed at room temperature and 10

mL aliquots of standard, quality control, or mouse samplewere transferred to polypropylene microcentrifuge tubescontaining 60 mL of a methanolic internal standards solu-tion. The tubes were vortex-mixed for 60 seconds, followedby centrifugation at 10,000 rpm for 8 minutes at 4�C. Next,the supernatantswere transferred to autosampler vials and3mL volumes were injected into a Waters ACQUITY separa-tion system coupled to a TQD triple-quadrupole MS/MSdetector. Separationwas achievedonaBEHC18 column(1.7mm; 50� 2.1mm;Waters) using a column heater operatingat 40�C with an in-line filter. The autosampler temperaturewas maintained at 15 � 5�C. The flow rate of a gradientmobile phase, composed of 0.1% formic acid in acetonitrileand 10 mmol/L ammonium acetate in 0.5% formic acidwater, was set at 0.7 mL per minute, and separation wascompleted within 5minutes. The instrument was equippedwith an electrospray interface and was controlled by Mas-slynx 4.1 software. The analysis was conducted in MRMmode, as follows: m/z 488.16 > 401.11 for dasatinib, m/z444.17 > 275.11 forN-deshydroxy dasatinib (M4), andm/z504.17 > 387.12 for dasatinib N-oxide (M5). Dasatinib-d8,N-deshyhydroxy-d8 dasatinib, and dasatinib N-oxide-d8(Toronto Research Chemicals) were used as internal stan-dards (final concentration, 15 ng/mL). The MS-MS condi-tions included: capillary voltage, 0.6 kV; source tempera-ture, 150�C; desolvation temperature, 450�C; cone gas flow,10 L per hour; and desolvation gas flow, 900 L per hour.Calibration curves of dasatinib, M4, andM5were created

by plotting the peak area ratios of analyte to the internalstandard against the analyte concentrations in the spikedmatrix. The within-run and between-run precisions fordasatinib and its metabolites were less than 6.9% and themeasured concentrations were all less than 5.3% of thenominal value.

Ex vivo microsomal incubationsMouse liver and intestinal microsomes were prepared

as described (22). Assays for cytochrome P450 enzymeswere conducted as described by the manufacturer (BDBiosciences). Briefly, dasatinib (2 mmol/L) was incubatedwith liver or intestinal microsomes (1 mg/mL) for 55minutes at 37�C (3) and the reaction was terminated by100 mL ice-cold acetonitrile. Dasatinib and its mainrodent metabolites M4 and M5 (23) were determined byLC/MS-MS.

Gastric transport studiesThe ex vivo mucosal-to-serosal transport of dasatinib in

isolated stomachs from wild-type or Abcc4(�/�) mousewas determined on the basis of a publishedmethod (24), aswas the in vivo gastric absorption of dasatinib (25). Briefly,after anesthetizingmicewith isoflurane, the abdominalwallwas opened longitudinally and a ligature was tightenedaround the cardia, to prevent reflux into the esophagus.Next, a tube was introduced into the duodenum and gentlyforced through the pylorus into the stomach cavity. Toensure tightness of the system and to avoid blood refluxinto the stomach, 2 ligatures were fastened upstream anddownstream of the pyloric sphincter. The tube was con-nected to a syringe to introduce dasatinib (1 mg/mL) in 50mmol/L sodium acetate buffer (pH 4.6) or vehicle aloneinto the gastric lumen. At 5minutes after the administrationof dasatinib, a sodium heparin solution (0.1 mL; 500 IU)was injected into the inferior cava vein and immediatelythereafter, livers were obtained after cervical dislocation toensure euthanasia. Body temperature, pulse, respirationrate, and anesthetic administration were monitoredthroughout the procedure. Gastric pH was determinedusing an IQ150 pH Meter with a PH37-SS piercing-tipmicroprobe (Hach).

Transporter gene and protein expressionGene expression in the small intestine and stomach of

wild-type or Abcc4(�/�) mice was conducted on RNAextracted from whole organs using the RNEasy Mini Kit(Qiagen) and analyzed using the Mouse Transporter RT2

Profiles PCR array system (SABiosciences). Relative geneexpression was determined using the DDCt method andnormalized to the housekeeping gene, Gapdh.

Western blotting of Abcc4 (26) or Abcb1 and Abcg2 (27)was done according to published methods. The primary ratanti-humanABCC4antibody, which also reactswithmouseAbcc4, was obtained from Abcam, and used at 1:50. Thesecondary antibody, rabbit anti-rat (Vector), was used at1:600. Slides of 4 mm sections were cut from formalin-fixedparaffin-embedded tissues. All assay steps, including depar-affinization, rehydration, and epitope retrieval, were con-ducted on the Bond Max with Bond wash buffer (Leica)rinses between steps. Heat-induced epitope retrieval wasconducted by heating slides in ER2 (Leica) for 30 minutes.Background Punisher (BioCare Chemical) was incubatedon slides for 10 minutes. The Refine (Leica) detectionsystem was used. Briefly, slides were incubated sequentiallywith hydrogen peroxide (5minutes), primary antibody (15minutes), secondary antibody (10 minutes), anti-rabbithorseradish peroxidase–conjugated polymer (8 minutes),3,30 diaminobenzidine (10 minutes), and hematoxylin (8minutes). Normal human stomach samples were obtainedfrom ProSci.

Activity of dasatinib against a model of Phþ ALLDetails of the mouse model have been reported previ-

ously (28). Briefly, 2 � 105 luciferase-positive, p185(BCR-ABL)-expressing Arf(�/�) leukemia-initiating pre-B cells

Abcc4 and Absorption of Dasatinib

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(LIC) were administered by tail vein injection into cohortsof healthy, nonconditioned, immunecompetent 10- to 12-week-oldmalewild-type orAbcc4(�/�)mice on aC57BL/6background. Dasatinib formulated in 80mmol/L citric acid(pH3.1)or vehicle alonewas administered byoral gavage torecipient mice at 10 mg/kg per dose once daily, 5 days perweek for 4 weeks, starting on day 10 after LIC injection.Whole-animal luminescent imaging was conducted in allmice to monitor tumor engraftment and data analysis wasconducted using a Xenogen IVIS-200 system and LivingImage Version 3.01 software (Caliper Life Sciences), asdescribed (29). Mice were observed daily and the experi-ment was terminated when animals showed signs of ter-minal illness, including hind-leg paralysis, inability to eat ordrink, and/or were moribund.

LIC viability was determined using a MTT, Cell Prolifer-ation Kit I (Roche), as previously described (30). The cellswere treated with dasatinib at increasing concentrations(0.001–30 mmol/L) for 48 hours (2 independent experi-ments were carried out with 8 replicates at each concentra-tion). The concentration inhibiting cell viability by 50%compared with vehicle-treated control cells (IC50) wasdetermined using the software program GraphPad Prismversion 5.0 (GraphPad Software). To evaluate growth inhib-

itory properties of dasatinib against LICs at average con-centrations observed in vivo in wild-type and Abcc4(�/�)mice, a correction factor was applied to account for antic-ipated differences in the fraction unbound dasatinib inplasma of wild-type mice, Abcc4(�/�) mice, and cell cul-ture medium, as described (31).

Statistical analysisAll data are presented as mean values with SE. Group

differences as a function of cell-type or mouse genotypewere evaluated using a Student t test. Two-tailed P values ofless than 0.05 were considered as statistically significant.Overall survival was assessed by the Kaplan–Meier method,followed by a Mantel–Cox test. Statistical calculations wereconducted using NCSS version 2004 (Number CruncherStatistical System).

ResultsIn vitro transport of dasatinib by ABCC4

Results from ABCC4-overexpressing inside-out vesiclesindicated that dasatinib was actively transported byABCC4 (Fig. 1A). When compared with control vesicles,dasatinib accumulation in the ABCC4-overexpressingvesicles increased by 3.0-fold (P ¼ 0.0014). Similarly, the

Figure 1. Influence of ABCC4 ondasatinib transport andpharmacokinetics. A, uptake of[3H]estradiol-17b-D-glucuronide(E2G; 10 mmol/L), a positive controlsubstrate, [3H]dasatinib (1 mmol/L),or [3H]imatinib (1 mmol/L) in controlinside-out vesicles of Sf9 cells orvesicles expressing humanABCC4. B, accumulation of[3H]PMEA (10 mmol/L), a positivecontrol substrate, [3H]dasatinib(100 mmol/L), or [3H]imatinib (100mmol/L) in Saos-2 cells transfectedwith an empty pcDNA (control) ormouse Abcc4. Data represent themean 2 to 4 independentexperiments and are expressed asthe average percent of uptakevalues in control cells. Error barsrepresent the SE. The asterisk (�)denotes a significant differencefrom control (P < 0.05). C, plasmaconcentration–time profile ofdasatinib (oral dose, 10 mg/kg) inwild-type mice and Abcc4(�/�)mice. D, plasma concentration–time profile of imatinib (oral dose,50 mg/kg) in wild-type mice andAbcc4(�/�) mice. Data representthe mean of 6 (imatinib) or 8(dasatinib) observations per timepoint, and error bars representthe SE.

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accumulation of the prototypical ABCC4 substrate E2Gwasincreased by 3.2-fold (P ¼ 0.0016), although this was notobserved for imatinib (1.3-fold;P¼0.098). The transport ofdasatinib into vesicles-expressing ABCC4 did not reach aplateau within a clinically relevant range of concentrations(Supplementary Fig. S1). This observation is consistentwiththe relatively high Michaelis–Menten constants (Km)reported for several other xenobiotic substrates of ABCC4,including 220 to 1,300mmol/L formethotrexate, 640mmol/L for leucovorin, and >1,000 mmol/L for adefovir andtenofovir (32). As several compounds undergo transportby ABCC4 only in the presence of physiologic concentra-tions of GSH (33), we also evaluated ATP-dependentcotransport of reduced GSH. While GSH (2 mmol/L)increased ABCC4-mediated transport of E2G by abouttwo-fold, this phenomenon was not observed for dasatinib(Supplementary Fig. S2A). Under the same experimentalconditions, dasatinib uptake was also facilitated by therelated transporter ABCC3 (MRP3), although the extent ofaccumulation (1.3-fold; P¼ 0.028) was less compared withthat observed for ABCC4 (Supplementary Fig. S2B).Dasatinib was also found to be transported by mouse

Abcc4, as exemplified by a significantly reduced accumula-tion in Saos-2 cells overexpressing Abcc4 compared withcontrol cells (Fig. 1B). In this model system, no noticeableefflux transport by Abcc4was notedwith imatinib (Fig. 1B).

Dasatinib pharmacokinetics in Abcc4-knockout miceWe next evaluated the possible importance of this trans-

porter for dasatinib inmicewith a genetic deletion of Abcc4.The AUC for dasatinib in these animals after oral admin-istration was significantly decreased by 1.75-fold comparedwith that observed in wild-type mice (Table 1). The respec-

tive concentration–time profiles of dasatinib suggest thatthe low systemic exposure in the Abcc4(�/�) mice ispossibly due to an absorption defect rather than an eventoccurring in the terminal phase (Fig. 1C). This is consistentwith the notion that the absorption rate constant is dra-matically reduced in Abcc4(�/�)mice, leading to a delayedtime-to-peak concentration, whereas the terminal half-livesof dasatinib were not significantly different betweenmousegenotypes (Table 1). As anticipated on the basis of in vitrotransport data, the pharmacokinetic profile of oral imatinibwas not substantially affected by Abcc4 deficiency (Fig. 1D).

To obtain further insights into the mechanism under-lying the pharmacokinetic changes observed with dasa-tinib in the Abcc4(�/�) mice, the drug was also admin-istered via intravenous bolus injection. The resultingconcentration–time profiles (Supplementary Fig. S3) andpharmacokinetic parameter estimates (Table 1) were notsignificantly affected by Abcc4 deficiency. Moreover, thebinding of dasatinib to serum proteins and its extent ofblood cell partitioning (18) were independent of mousegenotype (Table 1).

To rule out potentially altered, compensatory changes inthe expression of enzymes in the liver and intestine of Abcc4(�/�) mice at baseline, differential expression profiles ofCyp1a1, Cyp1b1, and Cyp3a11, the 3 isoforms with thehighest dasatinib turnover (18), were evaluated. Comparedwith levels in liver and intestine of wild-type mice, tran-scripts of these enzymes were not increased in the Abcc4(�/�) mice (Fig. 2A and B). Furthermore, there were nopotentially compensatory changes in enzyme activity,because Abcc4 knockout had no influence on the hepaticor intestinal microsomal metabolism of dasatinib to M4(Fig. 2C) or M5 (Fig. 2D). This finding is consistent with

Table 1. Dasatinib pharmacokinetic parameters in wild-type mice and Abcc4(�/�) mice after oral orintravenous administration of dasatinib (10 mg/kg)

Parameter Wild-type Abcc4(�/�) P

OralKa (1/h) 34.3 � 6.85 2.49 � 0.92 0.022Tmax (h) 0.306 � 0.223 2.19 � 1.06 <0.0001Cmax (ng/mL) 182 � 93.8 48.1 � 16.5 0.0014AUC (ng.h/mL) 442 � 152 253 � 86.8 0.0041T1/2 (h) 4.05 � 2.44 5.10 � 2.34 0.54F (%) 13.2 6.36

IntravenousCmax (ng/mL) 3,387 � 305 3,458 � 323 0.73AUC (ng.h/mL) 3,361 � 314 3,975 � 371 0.93T1/2 (h) 3.96 � 2.33 3.81 � 2.49 0.90CL (mL/min/kg) 49.6 41.9

Cblood/Cplasma 0.896 � 0.013 0.940 � 0.04 0.17Blood cell partitioning (%) 33.0 � 1.04 36.1 � 2.86 0.16Fraction unbound (%) 8.70 � 0.440 8.12 � 0.493 0.13

Abbreviations: Cblood/Cplasma, ratio of concentration in blood to concentration in plasma;Cmax, peakplasmaconcentration; F, apparentoral bioavailability; Ka, absorption rate constant; Tmax, time-to-peak plasma concentration; T1/2, half-life of the terminal phase.






recent data indicating that hepatic Cyp3a11 protein expres-sion is not altered in adult male Abcc4(�/�) mice (34).

As dasatinib is a substrate for Abcb1 and Abcg2 (35) andthese transporters have been implicated in restricting dasa-tinib absorption (9),we confirmed that their protein expres-sion levels in the small intestine were not altered in Abcc4(�/�)mice (Supplementary Fig. S4A and S4B). In addition,there were no differences in intrinsic intestinal permeabilitybetween wild-type and Abcc4(�/�) mice, as determinedfrom the recovery of mannitol in plasma, kidney, and liverafter oral mannitol dosing (Supplementary Fig. S4C). Like-wise, the altered systemic levels of dasatinib in Abcc4(�/�)mice cannot be explained by an intrinsically altered abilityto excrete dasatinib because drug levels remained undetect-able (<10 ng/mL) in the urinary bladder and gall bladder at5 minutes after oral administration.

Gastric transport as a mechanism for dasatinibabsorption

The notion that, in wild-type mice receiving oral dasati-nib, peak concentrations in plasma were already observedat the first collection time point, prompted us to considerthe possibility that dasatinib absorption occurs throughthe stomach. Consistent with this hypothesis, the Abcc4protein was readily detectable in stomach samples fromwild-type mice (Fig. 3A). In stomachs of wild-type mice,Abcc4 was detected in the mucosa of the fundus (Fig. 3B)with basal labeling of gastric pit mucosal cells andbasolateral labeling of the parietal and chief cells; in theparietal cells, the basal labeling was faint-to-weak andthe lateral labeling was strong. Labeling was absent in

Abcc4 (�/�) mice (Fig. 3C). In the human stomach,ABCC4 was detected in the gastric pit mucous cells, andbasolateral labeling was present in parietal and chief cells(Fig. 3D).

We next evaluated the ex vivo transport of dasatinib insacs prepared from mouse stomachs and found thatAbcc4 deficiency was associated with up to 3.9-fold(P ¼ 0.0025) reduced drug transport to the serosal side(Fig. 4A). To test whether the stomach is a site of absorp-tion of dasatinib in vivo, a surgical procedure was imple-mented on anesthetized mice, allowing us to introduce adasatinib dosing solution directly into the stomach, ligat-ed at both the cardia and the pylorus. Analysis of liversamples, as a surrogate for portal and systemic plasma,obtained 5 minutes after introducing the drug confirmedthat dasatinib can permeate the gastric mucosa throughan Abcc4-mediated mechanism (Fig. 4B). During thisexperiment, the gastric pH remained on average 4.36 �0.32 and 4.31 � 0.23 (P ¼ 0.81) in wild-type and Abcc4(�/�) mice, respectively. This suggests that Abcc4 defi-ciency is unlikely to cause changes in gastric pH thatcould subsequently cause altered dasatinib solubility andlead to impaired absorption (36).

Analysis of stomachs from Abcc4(�/�) mice revealedthat the expression of 84 ATP-binding cassette transporterand solute carrier genes was not substantially changedcompared with stomachs obtained fromwild-type animals,with the exception of a downregulation of Slc22a1 (4.85-fold) and Slc22a2 (4.35-fold; Fig. 4C). These genes encodethe organic cation transporters Oct1 and Oct2 that havebeen implicated in xenobiotic transport (37). However, the

Figure 2. Influence of Abcc4knockout ondasatinibmetabolism.AandB, comparative expressionofCyp1a1, Cyp1b1, and Cyp3a11 atbaseline in liver (A) and smallintestine (B) of wild-type mice andAbcc4(�/�) mice. C and D,comparative metabolism ofdasatinib toM4 (C) or M5 (D) in liveror small intestinal microsomes ofwild-type mice and Abcc4(�/�)mice. Data represent the mean of 4to 8 independent observations pergroup and are expressed as themean (bars) and the SE (error bars).

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altered expression of Slc22a1 and Slc22a2 in the stomach ofAbcc4(�/�) mice is unlikely to affect the pharmacokineticprofile of dasatinib because overexpression of Oct1 or Oct2inmammalian cells did not alter intracellular accumulationof dasatinib (Fig. 4D), which suggests that the drug is not asubstrate of these transporters.As the stomach drains either directly or indirectly into the

portal vein through short gastric veins from the fundus tothe splenic vein, we alsomeasured dasatinib concentrationsin the spleen, an organ that expresses high levels of Abcc4,which in turn can restrict splenic accumulation of substratessuch as topotecan (26) and PMEA (38). At 5 minutes afteroral dasatinib administration, however, the drug levelswere still higher in the spleen of wild-type mice (46.5 �11.8 ng/g) than in Abcc4(�/�) mice (26.7 � 7.51 ng/g).This suggests that splenic trapping of dasatinib after oraladministration is not substantially contributing to the lowsystemic plasma levels in Abcc4(�/�) mice.

Influence of Abcc4 deficiency on dasatinib efficacyIn view of the altered systemic exposure caused by Abcc4

deficiency and the established pharmacokinetic–pharmaco-dynamic relationships for dasatinib (7), we hypothesizedthat the antileukemic properties of dasatinib are dependenton host Abcc4 function. This hypothesis was evaluated inpaired wild-type and Abcc4(�/�) mice receiving daily oraldasatinib starting on day 10 following the injection ofluciferase-positive, p185(BCR-ABL)-expressing Arf(�/�)leukemia initiating pre-B cells (28). We found that thesecells express low levels of Abcc4, as detected by real-timereverse transcriptase PCR, with an Abcc4 toGapdh expressionratio of 0.002653. However, despite detectable transcripts,the intracellular accumulation or in vitro cell growth inhib-itory properties of dasatinib in p185(BCR-ABL)-expressingArf(�/�) leukemia initiating pre-B cells were not influencedby coincubation with the ABC transporter inhibitor, MK571(Supplementary Fig. S5A and S5B). Additional in vitro studies

Abcc4(-/-) MouseWild-type mouse B

Gastric pit cells Gastric pit cells

Parietal cellsParietal cells

Chief cells Chief cells

Human

Gastric pit cells

Parietal cells

Chief cells

A Abcc4(-/-)Wild-type

DCFigure 3. Expression of Abcc4 inmouse and human stomach. A,protein expression of Abcc4(upper bands) normalized tob-actin (lower bands) in stomachsof wild-type mice and Abcc4(�/�)mice. B–D, localization of Abcc4by immunohistochemistry in thestomach of a wild-type mouse (B),its absence in the stomach of anAbcc4(�/�) mouse (C), and thepresence of ABCC4 in stomach ofa human (D). Magnification, �20.






indicated that exposure of these cells to dasatinib at aconcentration equivalent to the average in vivo levels ofdasatinib in plasma of wild-type mice was associated witha 5.5-fold increase (P < 0.001) in cell growth inhibitioncompared with levels of dasatinib in plasma of Abcc4(�/�)mice (Supplementary Fig. S5C).

In subsequent in vivo experiments, whole-animal lumi-nescent imaging was conducted to monitor tumor engraft-ment (Fig. 5A). Consistent with the prediction from the invitro experiments, 2 weeks after start of treatment withdasatinib (day 24), the luminescent signal was increasedby 1.6-fold in wild-type mice and by more than 5-fold inAbcc4(�/�)mice (P¼ 0.016; Fig. 5B). This suggests that theinitial antileukemic activity of dasatinib given at the applieddose and schedule against this p185(BCR-ABL)-expressingArf(�/�) leukemia is dependent on host Abcc4 genotype.While all mice receiving vehicle only died within 2 weeksirrespective of genotype, there was a statistically significantsurvival advantage for wild-type mice receiving dasatinibcompared with Abcc4(�/�) mice (median survival, 35 vs.

31 days; P ¼ 0.048; Fig. 5C). Importantly, the observedsystemic concentrations of dasatinib in these tumor-bearingmice receiving multiple daily doses of the drug were sim-ilarly dependent on Abcc4 genotype compared with resultsobtained in non tumor-bearingmice receiving a single dose(Fig. 5D).

DiscussionIn this study, we found that dasatinib is a substrate for the

ATP-binding cassette transporter ABCC4, that the oralabsorption of dasatinib is at least partially dependent onABCC4-mediated gastric transport, and that this processinfluences the drugs’ antitumor efficacy. The current datacomplement previous knowledge on the interaction oftyrosine kinase inhibitors with ATP-binding cassette trans-porters and may have important practical implications fortheir optimal use.

The oral absorption of small molecules is believed to beprimarily occurring in the small intestine of the gastro-intestinal tract, with the stomach playing a supporting

Figure 4. Influence of Abcc4 on gastric transport of dasatinib. A, time course of the ex vivo mucosal-to-serosal transport of dasatinib in isolated sacs fromwild-type or Abcc4(�/�) mouse stomachs. Data represent the mean of 4 independent observations per group and are expressed as the mean (bars)and the SE (error bars). B, the in vivo gastric absorption of dasatinib in wild-type or Abcc4(�/�) mice with ligated stomachs. See Materials and Methods fordetails. Data represent the mean of 4 observations per group and are expressed as the mean (bars) and the SE (error bars). The asterisk (�) denotes asignificant difference from wild-type (P < 0.05). C, pairwise comparison of gene expression in stomachs of wild-type mice and Abcc4(�/�) mice of 84genes encoding ATP-binding cassette transporters or solute carriers. Each symbol represents a single gene, the solid line is the line of identity, and thedotted lines are the 95% confidence intervals. Expression of the highlighted genes Slc22a1 and Slc22a2 is decreased in the Abcc4(�/�) mice. D, transport ofTEA (1 mmol/L), a positive control substrate, or [3H]dasatinib (1 mmol/L) in HEK293 cells containing an empty PMIG II vector (VC), or a flag-tagged cDNA ofmOct1 or mOct2 for the organic cation transporters mOct1 and mOct2. Data represent the mean of 6 to 12 observations and are expressed as the averagepercent of uptake values (bars) with SE (error bars) in VC cells. The asterisk (�) denotes a significant difference from VC (P < 0.05).

Furmanski et al.





role in the solubilization of weakly basic compounds(39). Indeed, only a handful of molecules, includingethanol, acetylsalicylic acid (aspirin), and certain antho-cyanin-flavonoids in the Vitis vinifera grape (CabernetSauvignon), have been shown to be absorbed from thegastric compartment (25). Conventionally, for smallmolecules to cross cellular membranes by simple diffu-sion they are thought to possess certain physicochemicalproperties, most notably those defined by Lipinski’s ruleof five (40). However, mounting recent evidence points todrug transport proteins playing a more significant role inthe transfer of small molecules across cellular membranesthan held previously (41).In the case of aspirin, for example, the drug is rapidly

absorbed from the stomach by crossing the luminalmembrane in a nonionized form, which is favored bythe highly acidic environment (42). The resulting changein pH caused by crossing the gastric mucosa would switchaspirin to a predominately ionized form that requires theassistance of a transporter to facilitate its movementacross the basal side of the lumen before entering thebloodstream. Interestingly, aspirin was recently identifiedas a transported substrate of human ABCC4 (43) and wefound that both aspirin and its metabolite salicylic acid

are also substrates of mouse Abcc4 (Supplementary Fig.S6). This finding supports the possibility that aspirin, likedasatinib, may be entering the circulation through aprocess that is dependent, at least in part, on a gastricATP-binding cassette transporter.

The ability of dasatinib to permeate the gastric mucosathrough an ABCC4-mediated mechanism could be at thebasis of the fast absorption kinetics of dasatinib in rodentsas well as in humans (12). Although absorption is likely toalso occur in the upper intestine, the relative contribution ofthe stomach in the process of dasatinib absorption remainsto be determined. In this context, it is worthwhile pointingout that the fate of dasatinib in lower segments of thedigestive tract is severely complicated by its strong pH-dependent aqueous solubility, which ranges from 18.4mg/mL at pH 2.6 to only 0.008 mg/mL at pH 6.0 (36). Asthe intraluminal pH inhumans acutely changes fromhighlyacidic in the stomach to about pH 6 in the duodenum, andthen gradually to pH7.4 in the terminal ileum(44), it is verylikely that the stomach contributes substantially to theabsorption of oral dasatinib in patients. This possibility isconsistent with previous studies indicating that dasatinib isexquisitely sensitive to agents that modulate the luminalpH of the stomach by suppressing gastric acid secretion,

d10 d240

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Figure 5. Influence of Abcc4on antileukemic efficacy ofdasatinib. A and B, noninvasivebioluminescence imagingimmediately before drugadministration (day 10) and 2weeks after start of a daily oraldose (10 mg/kg) of dasatinib (day24) inmice injectedwith luciferase-positive, p185(BCR-ABL)-expressing Arf(�/�)LIC. Datarepresent the mean (bars) and SE(error bars) of 14 animals pergroup. C, overall survivalprobability for wild-type mice andAbcc4(�/�) mice receiving notreatment or dasatinib at a dailyoral dose (10 mg/kg) relative to thetime of LIC injection. D, observedsystemic concentrations ofdasatinib in plasma of wild-typemice or Abcc4(�/�) mice withoutleukemia (1 hour after a singledose) or with leukemia (1 hour afterthe eighth daily oral dose). Datarepresent the mean (bars) and SE(error bars) of 8 to 14 animals pergroup. The asterisk (�) denotes asignificant difference from wild-type (P < 0.05).






such as H2-receptor antagonists and proton pump inhibi-tors (45, 46).

It is possible that gastric transport also contributes to theoral absorption of other clinically important tyrosine kinaseinhibitors. Although direct evidence of this is lacking, somerecent studies have shown that major gastrectomy, but notsmall-bowel resection, is associated with significantlydecreased plasma levels of imatinib in patients with gas-trointestinal stromal tumors (47, 48). Similar findings havebeen reported for nilotinib (49), and in some patients,major gastrectomy was resulting in rapid disease progres-sion. Theobserveddecreases in exposure of these drugs havebeen attributed to impaired pH-dependent drugdissolutionand solubility and/or an altered gastric motility resultingfrom the surgery. However, these explanations seem inad-equate in view of the fact that the pharmacokinetics ofimatinib (50) and nilotinib (51) are not substantiallyaffected by omeprazole or its S-enantiomer esomeprazole,agents that both increase the pH of the gut as well as delaygastric emptying (52).

It is interesting to note that previous studies showed that,after oral administration, the pharmacokinetic profile of theABCC4 substrates methotrexate (24) and cefadroxil (14)was unchanged in Abcc4(�/�) mice. The reasons underly-ing the apparent differences in outcome of these previousstudies and our current results for dasatinib are not entirelyclear, but it is likely that differential mechanisms regulatingdrug entry on the apical side of the gut lumen contribute. Inaddition, unlike dasatinib, methotrexate and cefadroxil arerelatively high-affinity substrates of Abcc3 and this trans-porter may compensate for the loss of Abcc4 in the knock-outmice. Indeed, concentrations of cefadroxil in portal andperipheral blood, after injection of the drug directly into aligated jejunum, were significantly decreased in mice defi-cient for both Abcc3 and Abcc4, compared with wild-typemice or animals lacking only one of the transporters (14).Regardless of the exact mechanism, our current findingsprovide further evidence that ABCC4 can affect the phar-macokinetic properties of a remarkably broad range ofsubstrates.

Collectively, our results indicate that in humans andmiceABCC4 is abundantly expressed in the stomach, where itfacilitates the gastric absorption of dasatinib. Moreover,Abcc4 deficiency in mice was associated with reduced activ-ity of dasatinib against a model of Phþ-ALL. These resultsidentified ABCC4 as an important, previously unrecog-nized, contributor to the oral absorption of dasatinib andthis process may be relevant for other substrate drugs, suchas aspirin. In view of the established exposure–efficacyrelationships for dasatinib (53), we suggest that caution iswarranted when dasatinib has to be administered togetherwith agents that potently inhibit ABCC4.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Hu, R.T. Williams, J.D. Schuetz, A. Sparreboom,S.D. BakerDevelopment of methodology: S. Hu, A.A. GibsonAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): B.D. Furmanski, S. Hu, Ken-ichi Fujita, A.A.GibsonAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S. Hu, A.A. Gibson, L.J. Janke, R.T.Williams, A. Sparreboom, S.D. BakerWriting, review, and/or revision of the manuscript: B.D. Furmanski, S.Hu, Ken-ichi Fujita, R.T. Williams, J.D. Schuetz, A. Sparreboom, S.D. BakerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Li, A.A. GibsonStudy supervision: A. Sparreboom, S.D. Baker

AcknowledgmentsThe authors thank Sarah Colvin, Chaoxin Hu, Markos Leggas, Shelley

Orwick, Praveen Potukuchi, Laura Ramsey, and Samantha Wilcox for theirvarious contributions to this work.

Grant SupportThis work was supported in part by the American Lebanese Syrian

Associated Charities, USPHS CCSG grant no. 3P30CA021765 (to S.D.Baker), and NIH grant no. 2R01 GM060904 (to J.D. Schuetz).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 11, 2013; revised May 20, 2013; accepted June 17, 2013;published OnlineFirst June 21, 2013.

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Published OnlineFirst June 21, 2013.Clin Cancer Res Brian D. Furmanski, Shuiying Hu, Ken-ichi Fujita, et al. Absorption and Efficacy of DasatinibContribution of Abcc4-Mediated Gastric Transport to the

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