characteristic analysis of intestinal transport in enterocyte-like cells … · transport in...

1521-009X/44/10/1662–1667$25.00 http://dx.doi.org/10.1124/dmd.116.069336DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1662–1667, October 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Characteristic Analysis of Intestinal Transport in Enterocyte-LikeCells Differentiated from Human Induced Pluripotent Stem Cells

Nao Kodama, Takahiro Iwao, Takahiro Katano, Kinya Ohta, Hiroaki Yuasa,and Tamihide Matsunaga

Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan (N.K., T.I.,T.M.), Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan

(T.K., K.O., H.Y.)

Received January 6, 2016; accepted July 13, 2016

ABSTRACT

We previously demonstrated that differentiated enterocytes fromhuman induced pluripotent stem (iPS) cells exhibited drug-metabolizing activities and cytochrome P450 CYP3A4 inducibility.The aimof this studywas to apply human iPS cell–derived enterocytesin pharmacokinetic studies by investigating the characteristics ofdrug transport into enterocyte-like cells. Human iPS cells cultured onfeeder cells were differentiated into endodermal cells using activin A.These endodermal-like cells were then differentiated into intestinalstem cells by fibroblast growth factor 2. Finally, epidermal growthfactor and small-molecule compounds induced the maturation of theintestinal stem cell-like cells. After differentiation, we performedtransepithelial electrical resistance (TEER) measurements, immuno-fluorescence staining, and transport studies. TEER values increasedin a time-dependentmanner and reachedapproximately 100V3cm2.

Efflux transport of Hoechst 33342, a substrate of breast cancerresistance protein (BCRP), was observed and inhibited by the BCRPinhibitor Ko143. The uptake of peptide transporter 1 substrateglycylsarcosine was also confirmed and suppressed when thetemperature was lowered to 4�C. Using immunofluorescence stain-ing, villin and Na+–K+ ATPase were expressed. These results suggestthat human iPS cell–derived enterocytes had loose tight junctions,polarity, as well as uptake and efflux transport functions. In addition,the rank order of apparent membrane permeability coefficient (Papp)values of these test compounds across the enterocyte-like cellmembrane corresponded to the fraction absorbance (Fa) values.Therefore, differentiated enterocytes from human iPS cells mayprovide a useful comprehensive evaluation model of drug transportand metabolism in the small intestine.

Introduction

The small intestine is an important organ in the pharmacokinetics oforally administered drugs owing to the presence of drug transporters anddrug-metabolizing enzymes (Choi et al., 2013; Li et al., 2013; Yoshidaet al., 2013; Kostewicz et al., 2014). Thus, Caco-2 cells, a human coloncarcinoma cell line, are widely used to evaluate the intestinal transport oforally administered drugs. Although immortalized cells offer manyadvantages, the extrapolation of data generated with these cell lines toin vivo conditions is often difficult. This is because these cells originatedfrom tumors and are therefore not representative of the natural physi-ologic environment (Le Ferrec et al., 2001). However, Caco-2 cells havedifferent characteristics compared with human enterocytes owing todifferences in the expression patterns of drug transporters (Sun et al.,2002; Harwood et al., 2016). Additionally, the level of cytochrome P450

CYP3A4, a major drug-metabolizing enzyme in the small intestine, isalso very low (Nakamura et al., 2002). Thus, it is difficult to estimatedrug transport and metabolism in the small intestine appropriately andcomprehensively using Caco-2 cells. To precisely predict intestinalpharmacokinetics, it is desirable to use human primary small intestinalepithelial cells. However, it is difficult to obtain such cells, and there is noappropriate model that exists for intestinal pharmacokinetic prediction.Human induced pluripotent stem (iPS) cells (Takahashi et al., 2007)

have the potential to form almost any type of cell and are expected to be auseful tool in regenerative medicine and drug discovery research. Thehepatic differentiation of human iPS cells has been frequently reported(Kondo et al., 2014; Takayama et al., 2014; Faulkner-Jones et al., 2015;Ishikawa et al., 2015): however, intestinal differentiation remainsrelatively unexplored in the literature. Spence et al. (2011) reportedthe generation of three-dimensional gut-like organoids from humaniPS cells and demonstrated that the organoids had the morphologiccharacteristics of intestinal-tract tissues. Ogaki et al. (2013, 2015)showed that all four intestinal differentiated cell types (absorptiveenterocytes, goblet cells, enteroendocrine cells, and Paneth cells) couldbe efficiently differentiated from human pluripotent stem cells. How-ever, the pharmacokinetic functions of organoids and intestinal cells

This work was supported, in part, by Grants-in-Aid from the Japan Society forthe Promotion of Science [Grant 23390036, Grant 25860120, Grant 26293036];Adaptable & Seamless Technology Transfer Program through Target-Driven R&D(A-STEP) of Japan Science and Technology Agency [AS262Z01122Q]; a grant ofthe Nakatomi Foundation [NF2014-32].

dx.doi.org/10.1124/dmd.116.069336.

ABBREVIATIONS: A-83-01, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide; BCRP, breast cancer resistanceprotein; ER, efflux ratio; Fa, fraction absorbance; FGF, fibroblast growth factor; GFR-Matrigel, Matrigel Matrix Growth Factor Reduced; HIEC, humansmall intestinal epithelial cell; iPS, induced pluripotent stem cells; 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-dimethylethylester; P-gp, P-glycoprotein; Papp, apparentmembrane permeability coefficient; PBS, phosphate-buffered saline; PD98059, 2-(2-amino-3-methoxyphenyl)4H-1-benzopyran-4-one; PEPT1,peptide transporter 1; TEER, transepithelial electrical resistance.

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were only briefly explored in these studies. Kauffman et al. (2013) andOzawa et al. (2015) have reported the pharmacokinetic characteristics ofhuman iPS–derived enterocyte-like cells, but the characteristics ofintestinal transport have been insufficiently investigated.We have previously established a method for differentiating human

iPS cells into enterocytes (Iwao et al., 2014, 2015) and found severalsmall-molecule compounds that were effective in promoting thedifferentiation of human iPS cells. In addition, these enterocytes wereassociated with a gain of pharmacokinetic function. Moreover, we alsodemonstrated that the enterocyte-like cells had various pharmacokineticfunctions such as drug-metabolizing activities by cytochromes P450,UDP-glucuronosyltransferase, and sulfotransferase, as well as CYP3A4induction by 1a,25-dihydroxyvitamin D3 and peptide uptake throughpeptide transporters.In the present study, we investigated the characteristics of drug

transport in enterocyte-like cells. The enterocyte-like cells had tightjunctions and exhibited activities of efflux transporter breast cancerresistance protein (BCRP). Moreover, our findings indicated that theenterocyte-like cell membrane may be able to predict fraction absor-bance (Fa) in humans. These results suggest that the human iPS cell–derived enterocyte-like cells would be useful for a human intestinalpharmacokinetic prediction that included transport and metabolism.

Materials and Methods

Materials. Fibroblast growth factor (FGF) 2, activin A, and epidermal growthfactor were purchased from PeproTech Inc. (Rocky Hill, NJ). BDMatrigelMatrixGrowth Factor Reduced (GFR-Matrigel) was purchased from BD Biosciences(Bedford, MA). KnockOut Serum Replacement was purchased from InvitrogenLife Technologies/Thermo Fisher Scientific (Carlsbad, CA). (+)-(R)-trans-4-(1-Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632),2-(2-amino-3-methoxyphenyl)4H-1-benzopyran-4-one (PD98059), 5-aza-29-deoxycytidine, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A-83-01), ibuprofen, paraformaldehyde, and Hoechst33342were purchased from Wako Pure Chemical Industries (Osaka, Japan). Ko143was purchased from Sigma-Aldrich (St. Louis, MO). Anti-villin and anti-BCRP/ABCG2 antibodies were purchased from Abcam (Cambridge, UK).Anti-Na+–K+ ATPase antibody purchased from GeneTex, Inc. (Irvine, CA).Anti–peptide transporter 1 (PEPT1) antibody was purchased from Santa CruzBiotechnology, Inc. (Dallas, TX). [N-methyl-14C]Antipyrine was purchased fromAmerican Radiolabeled Chemicals, Inc. (St. Louis, MO). [Ring-3H]Atenolol,[3H]metoprolol, and [3H]glycylsarcosine were purchased from Moravek Bio-chemicals, Inc. (Brea, CA). (2)-[Methoxy-3H]sulpiride and [14C]polyethyleneglycol 4000 were purchased from PerkinElmer, Inc. (Boston, MA). All otherreagents were of the highest quality available. Triton X-100 was purchased fromAMRESCO (Cleveland, OH).

Human iPS Cell Culture. The human iPS cell lineWindy, whichwas derivedfrom the human embryonic lung fibroblast cell line MRC-5, was provided by Dr.Akihiro Umezawa of the National Center for Child Health and Development(Tokyo, Japan). Human iPS cells were maintained in a 1:1 mixture of Dulbecco’smodified Eagle’s medium and Ham’s nutrient mixture F-12 (DMEM/F12)containing 20% KnockOut Serum Replacement, 2 mM L-glutamine, 1% minimal

essential medium nonessential amino acid solution (NEAA), 0.1 mM2-mercaptoethanol, and 5 ng/ml of FGF2 at 37�C in humidified air with 5% CO2.The human iPS cells were cultured on a feeder layer of mitomycin C-treatedmouse embryonic fibroblasts, and the medium was changed daily.

Differentiation into Enterocyte-Like Cells. The human iPS cells weredifferentiated into enterocytes on the basis of our previous report (Iwao et al.,2015). Briefly, human iPS cells were differentiated into endodermal cells byincubating the cells in the presence of 100 ng/ml activin A for 72 hours, and theseendodermal-like cells were then differentiated into intestinal stem cells via250 ng/ml FGF2 for 96 hours. Finally, the cells were passaged on GFR-Matrigel–coated 24-well plates or cell culture inserts and cultured in medium containing20 ng/ml epidermal growth factor. Subsequently, 20 mM PD98059, 5 mM 5-aza-29-deoxycytidine, and 0.5 mM A-83-01 were also added to the medium on day14 after differentiation. The medium was subsequently changed every 3 days.Transepithelial electrical resistance (TEER) values were obtained to check theintegrity of the membrane before the transport assay.

Uptake Assay. The culture medium was removed, and the differentiated cellswere preincubatedwith the transport buffer (Hank’s balanced salt solution containing10 mMMES, pH 6.0) at 37�C for 15 minutes. Uptake assays were initiated by thereplacement of a transport buffer containing 135 nM [3H]glycylsarcosine at 37�C inthe presence or absence of 3 mM ibuprofen, a known PEPT1 inhibitor (Omkvistet al., 2010) or at 4�C. Assays were stopped by the addition of ice-cold transportbuffer, and the cells were washed twice with the same buffer. The cells weresolubilized with 0.2 M NaOH solution (0.5 ml) containing 0.5% sodium dodecylsulfate. Radioactivity was measured by liquid scintillation counting using 3 ml ofClear-sol I (Nakarai Tesque, Kyoto, Japan) as a scintillation fluid.

To correct for the uptake of glycylsarcosine, the total protein of the differen-tiated cells was measured using a Pierce BCA Protein Assay Kit (Thermo FisherScientific Inc., Waltham, MA), according to the manufacturer’s instructions.

Immunofluorescence Staining. Differentiated cells were washed three timeswith phosphate-buffered saline (PBS) with 1 mM CaCl2 and 1 mM MgCl2,following which they were fixed and permeabilized in methanol (220�C) for5 minutes at 4�C for staining villin, Na+–K+ ATPase, and PEPT1. Differentiatedcells were washed three times with PBS, following which they were fixed in a 4%(w/v) paraformaldehyde solution for 30 minutes at room temperature, andpermeabilized in a Triton X-100 solution for 5 minutes at room temperature forstaining BCRP. After washing three times with PBS, the cells were blocked inPBS containing 2% skim milk for 20 minutes at room temperature. Following theblocking step, the cells were incubated for 60 minutes at room temperature, withanti-villin 1 and Na+–K+ ATPase antibody diluted at 1:100. The cells wereincubated overnight at 4�C,with the Na+–K+ATPase antibody diluted at 1:100, orBCRP and PEPT1 antibodies diluted at 1:50. The cells were washed three timeswith PBS and incubated with a 1:500 dilution ofAlexa Fluor 488- and 568-labeledsecondary antibody for 60 minutes at room temperature. After washing threetimes with PBS, the cells were incubated with 1 mg/ml of 49,6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature and washed with PBS.The cells were mounted on a glass slide using a 9:1 mixture of glycerol and PBS(without magnesium and calcium) and were viewed using an LSM 510 Metaconfocal microscope (Carl Zeiss Inc., Oberkochen, Germany).

TEER Value Measurements. The TEER values of the human iPS cell–derived enterocyte-like cell membrane on cell culture inserts were measured byMillicell ERS-2 (Millipore, Bedford, MA).

Membrane Transport Assay. The culture medium was removed, and thedifferentiated cells were preincubated with transport buffer (Hank’s balanced saltsolution containing 10 mM HEPES, pH 7.4) at 37�C for 15 minutes. Transport

Fig. 1. Immunofluorescence staining analysis of PEPT1in the differentiated enterocyte-like cells. After differen-tiation, the cells were stained with PEPT1 (red) andDAPI (blue). Scale bar, 50 mm.

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assays were performed by replacing with transport buffer containing substratessuch as 1.15mM [14C]antipyrine, 87.1 nM [3H]atenolol, 9.39 nM [3H]metoprolol,7.70 nM [3H]sulpiride, or 1.31 nM [14C]PEG4000 on the apical chambers. Thesolution was collected from the basal chambers at 30, 60, and 120 minutes.Radioactivity was measured by liquid scintillation counting using 3 ml Clear-sol I(Nakarai Tesque, Kyoto, Japan) as a scintillation fluid. In a bidirectional transportassay using Hoechst33342, after preincubation, the transport buffer containing20 mM Hoechst33342 was added to the apical or basal chambers, and the cellswere incubated at 37�C for 120 minutes in the presence or absence of 10 mMKo143. Samples were collected from the receiver chambers. The intensity of theHoeschst33342 fluorescence was measured using a fluorescence plate reader(ARVO MX 1420 Multilabel Counter; Perkin Elmer Inc., Waltham, MA) usingthe wavelengths 355 nm for excitation and 460 nm for emission.

Analysis of Apparent Membrane Permeability. The apparent membranepermeability coefficient (Papp) in transport assay was calculated as follows:

Papp ¼ dQdt ×1

A� C0 ð1Þ

where dQ/dt is the amount of the compound permeated per unit of time, A is thesurface area of Transwell membrane (0.3 cm2), and C0 is the initial compoundconcentration in the donor chamber. Efflux ratio (ER) of Hoechst33342 was

calculated by dividing Papp of the basal-to-apical transport by that of the apical-to-basal transport.

Statistical Analysis. The level of statistical significance was assessed usingStudent’s t test. The correlation between the Papp value and Fa of the fivecompounds was estimated by a P and R values. The best-fitting curves werecalculated by nonlinear regression using PASW Statistics 18 system software(IBM, Armonk, NY).

Results

Uptake of Glycylsarcosine in the Differentiated Enterocyte-LikeCells. The oligopeptide transporter SLC15A1/PEPT1 is expressed in thesmall intestine and plays an important role in peptide transport from thelumen (Liang et al., 1995; Giacomini et al., 2010). In previous studies,we confirmed mRNA expression, but not protein expression, of PEPT1in enterocyte-like cells. Therefore, we conducted immunofluorescencestaining of PEPT1, indicating that the protein was also expressed (Fig.1). Moreover, we performed uptake analyses of glycylsarcosine,which is a substrate of PEPT1 (Nakanishi et al., 1997). The uptake ofglycylsarcosine in the enterocyte-like cells was increased in a time-dependent manner at 37�C (Fig. 2). When the uptake temperature waslowered to 4�C, the uptake was significantly suppressed and reacheda plateau after 30 minutes. Moreover, at 37�C, in the presence ofibuprofen, the uptake was significantly suppressed to a similar extent asthat at 4�C. Therefore, these findings indicate that PEPT1-mediatedactive transport was quantitatively evaluated in the enterocyte-like cells.Characteristics of Enterocyte-Like Cells. To investigate whether

the enterocyte-like cell membrane was available for drug permeabilitystudies, we characterized the enterocyte-like cells. After seeding on cellculture inserts, the TEER values were increased in a time-dependentmanner and finally reached a plateau at approximately 100 V � cm2(Fig. 3). Therefore, these results suggest that the enterocyte-like cellsformed a membrane with a loose tight junction. Using immunofluores-cence staining, we found that almost all the cells expressed the intestinalepithelial marker villin, whereas Na+–K+ ATPase was located only onthe basal side (Fig. 4).Bidirectional Transport across the Enterocyte-Like Cell

Membrane. It has been found that BCRP is highly expressed in theapical membrane of the small intestinal epithelium and plays an importantrole in intestinal absorption of drug substrates (Giacomini et al., 2010).Thus, we used a bidirectional transport assay to examine the enterocyte-like cell membrane on the cell culture inserts. Apical-to-basal Papp valueswere 3.32 6 1.09 and 11.53 6 0.82 (� 1026 cm/s) in the absence orpresence of Ko143, respectively (n = 4). The basal-to-apical values were49.966 7.98 and 33.726 9.55 (� 1026 cm/s) in the absence or presence

Fig. 2. Uptake of glycylsarcosine in the differentiated enterocyte-like cells.Following differentiation, the enterocyte-like cells were incubated with a transportbuffer (pH 6.0) containing glycylsarcosine at 37�C, with or without 3 mM ibuprofen,or at 4�C. Data are represented as the mean 6 S.D. (A, n = 3; B, n = 4). Open andclosed symbols bars show the uptake at 37�C and 4�C, respectively, and the gray barshows the uptake with 3 mM ibuprofen at 37�C. Levels of statistical significancecompared with the uptake at 37�C: A, **P , 0.01; B, *P , 0.05.

Fig. 3. Time-dependent changes of TEER values in the enterocyte-like cell mem-brane. The enterocyte-like cells were seeded on GFR-Matrigel-coated cell cultureinserts. TEER values were measured every 3 days from day 4 after seeding. Datawere represented as the mean 6 S.D. (n = 22).

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of Ko143, respectively (n = 4). As shown in Fig. 5, Papp values ofHoechst 33342, a substrate of BCRP (Doyle and Ross, 2003), in thebasal-to-apical direction was significantly higher than for the apical-to-basal direction. The ER, calculated from the ratio of Papp in the basal-to-apical direction to that in the apical-to-basal direction, was 15. From theaddition of Ko143, a BCRP inhibitor (Allen et al., 2002), the basal-to-apical Papp values were decreased and the apical-to-basal Papp valueswere increased. Additionally, the ER value was reduced to 3. Moreover,in immunofluorescence staining, it was indicated that BCRPwas locatedon the apical side (Fig. 6). Therefore, these results indicate that theenterocyte-like cell membrane had also efflux transporter BCRPactivity.Permeability of Test Compounds across an Enterocyte-Like Cell

Membrane. We performed a membrane transport study using five testcompounds with various Fa (1–97%) values in humans. The Papp valuesof the five test compounds ranged from 2.04 to 9.99 (� 1026 cm/s;Table 1). The rank order of Papp values of these test compoundscorresponded to those of the Fa values. Moreover, the sigmoidalrelationship between the Papp values and the Fa values of the fivecompounds were observed in differentiated cells (Fig. 7). These resultssuggest that drug permeability in the enterocyte-like cell membrane maybe able to predict Fa in humans.

Discussion

We previously reported that human iPS cell–derived enterocyte-likecells had metabolic functions, such as drug-metabolizing enzymeactivities and CYP3A4 inducibility (Iwao et al., 2015). Moreover, inthis study, we demonstrated the drug transport characteristics in theenterocyte-like cells.Human peptide transporter PEPT1 is primarily responsible for the

transport of dietary di- and tripeptides from the lumen of the small

intestine. PEPT1 has been exploited with prodrugs designed to introducepeptide and peptide bond-like moieties onto the parent molecule. Thismethod was demonstrated to significantly increase the absorption ofdrugs with poor oral bioavailability (Gomez-Orellana, 2005; Hammanet al., 2005; Leonard et al., 2006; Majumdar and Mitra, 2006). In the

Fig. 4. Immunofluorescence staining analysis of villin and Na+–K+ ATPase in the differentiated enterocyte-like cells. The enterocyte-like cells were seeded on GFR-Matrigel–coated cell culture inserts. After differentiation, the cells were stained with villin (green), Na+–K+ ATPase (red), and DAPI (blue). Scale bar, 50 mm. I and II arecross-sectional views along the red and green lines, respectively. A, apical side; B, basal side.

Fig. 5. Bidirectional permeability of Hoechst33342 across the enterocyte-like cellmembrane. The enterocyte-like cells were seeded on GFR-Matrigel–coated cell cultureinserts. After differentiation, the cells were incubated with the transport buffer (pH 7.4)containing Hoechst33342 (20 mM) for 120 minutes at 37�C in the presence or absenceof Ko143 (10 mM). Data are represented as the mean 6 S.D. (n = 4). White or blackbars show apical-to-basal or basal-to-apical Papp values, respectively. ER ofHoechst33342 was calculated by dividing Papp of the basal-to-apical transport bythat of the apical-to-basal transport. Levels of statistical significance compared with theeach Papp value in the absence of Ko143: *P , 0.05, **P , 0.01; and compared witheach Papp values for apical-to-basal transport:

†P , 0.01.


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enterocyte-like cells, we confirmed the expression of the PEPT1 protein(Fig. 1) and also quantitatively evaluated the PEPT1-mediated uptakeactivity of glycylsarcosine (Fig. 2). However, PEPT1-mediated uptakeusing fluorescence-labeled di- or tripeptides was also qualitativelyevaluated in previous reports (Iwao et al., 2014; Ozawa et al., 2015).Therefore, it was considered that the enterocyte-like cells may be used asa quantitative evaluation model of uptake transporters such as PEPT1.To our knowledge, there are few reports involving drug membrane

permeability using human iPS–derived enterocytes (Kauffman et al.,2013; Ozawa et al., 2015). However, this evaluation used only TEERmeasurements and the permeability of a nonabsorbable marker (fluo-rescein isothiocyanate-dextran, molecular weight 4 or 150 kDa) in thesereports. Thus, we performed a bidirectional transport assay and drugmembrane permeability assay to characterize the enterocyte-like cells.TEER values of the enterocyte-like cell membrane on the cell cultureinserts were gradually increased and finally reached a plateau atapproximately 100 V � cm2 (Fig. 3). It was reported that TEER valuesin the human small intestine were approximately 40 V � cm2 (Sjoberget al., 2013). In addition, we previously reported that the values in humansmall intestinal epithelial cell (HIEC) monolayer were 98.9 V � cm2,even lower in the Caco-2 cell monolayer (900V�cm2) (Takenaka et al.,2014). It was indicated that the TEER value of the enterocyte-like cellmembrane was comparable to that of the HIEC monolayer and thehuman small intestine. We were also able to use immunofluorescencestaining to visualize the presence of the intestinal epithelial marker villinand Na+–K+ ATPase located in the cytoplasm and on the basal side ofthe enterocyte-like cell membrane, respectively (Fig. 4). Taken together,these results suggest that the enterocyte-like cell membrane formed a

loose tight junction similar to the small intestine and also exhibited apolarity.In the intestine, transporters are localized on the brush border

membrane and the basal side of intestinal cells. Four major ATP-binding cassette efflux transporters have been shown to localize at theapical/luminal membrane of enterocytes. These are thought to form abarrier to intestinal absorption of the substrate drugs: P-glycoprotein(P-gp), BCRP, multidrug resistance–associated protein (MRP)2, andMRP4 (Englund et al., 2006; Takano et al., 2006;Maubon et al., 2007). Theexpression levels of these substrate drugs differed between the segmentsof the intestine. In general, BCRP,MRP2, and P-gp are expressed at highlevels in the small intestine and are considered to be a limiting barrier tooral drug absorption (Shirasaka et al., 2008; Giacomini et al., 2010).In the Food and Drug Administration (FDA) and the Ministry of HealthLabor andWelfare guidelines for drug interactions (http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf), it is indicated that drug candidates should be evaluatedto determine whether they are substrates of efflux transporters such asP-gp and BCRP. Thus, we examined the BCRP-mediated transportactivity in the enterocyte-like cells. The basal-to-apical Papp values of

Fig. 6. Immunofluorescence staining analysis of BCRP in the differentiated enterocyte-like cells. The enterocyte-like cells were seeded on GFR-Matrigel–coated cell cultureinserts. After differentiation, the cells were stained with BCRP (green) and DAPI (blue). Scale bar, 50 mm. I and II are cross-sectional views along the red and green lines,respectively. A, apical side; B, basal side.

TABLE 1

Papp values of test compounds in the enterocyte-like cell membrane

Compounds Papp (Mean 6 S.D.) Faa

�1026 cm/sec %Antipyrine 9.99 6 2.59 97Metoprolol 7.54 6 1.51 85Atenolol 6.10 6 1.19 50Sulpiride 5.41 6 2.74 44PEG4000 2.04 6 0.43 .1

Papp values were represented as the mean 6 S.D. (n = 4).aFa values were obtained from published data (Rozehnal et al., 2012).

Fig. 7. Relationship between Fa values and Papp of test compounds across theenterocyte-like cell membrane. The enterocyte-like cells were seeded on GFR-Matrigel–coated cell culture inserts. Following differentiation, the cells wereincubated with the transport buffer (pH 7.4) containing antipyrine, atenolol,metoprolol, sulpiride, or PEG4000 for 120 minutes at 37�C. The correlation curvewas fitted by using the following formula: Fa = 1 / (0.01 + 5.28 * 0.36

Papp). Datawere represented as the mean 6 S.D. (n = 4). P , 0.01; R = 0.99.

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Hoechst33342, a substrate of BCRP, were higher than the apical-to-basal Papp values (Fig. 5). By addition of Ko143, a BCRP inhibitor,basal-to-apical Papp values were decreased and apical-to-basal Pappvalues were increased. ER values were also decreased by the presenceKo143. In addition, BCRP was located on the apical side (Fig. 6). Theseresults demonstrated that the enterocyte-like cells had BCRP-mediatedtransport activity and that the cell membrane was available for theevaluation of efflux transport.In our membrane permeability study, the rank order of Papp values of

these test compounds corresponded to those of the Fa values (Fig. 7).Takenaka et al. (2014) reported that the HIEC monolayer with loosetight junctions was able to accurately predict the oral absorption ofparacellularly absorbed compounds. The enterocyte-like cell membranecould also be useful for the prediction of the Fa values of drugs,including such compounds.In conclusion, we demonstrated that the enterocyte-like cells

exhibited: 1) functions of uptake and efflux transporters; 2) loose tightjunctions similar to the human small intestine; and 3) apical/basalpolarity. Moreover, it was indicated that the Fa of drugs in humans canbe estimated from permeability data of the enterocyte-like cellmembrane. In our previous study, we found that the differentiated cellsperformed drug-metabolizing enzyme activities and CYP3A4 induc-ibility (Iwao et al., 2015). Taken together, the enterocyte-like cells maybe useful as an appropriate model to comprehensively predict drugtransport and metabolism in the intestine.

Acknowledgments

The authors thank Drs. Hidenori Akutsu, Yoshitaka Miyagawa, Hajime Okita,Nobutaka Kiyokawa, Masashi Toyoda, and Akihiro Umezawa for providinghuman iPS cells. The authors also thank Enago (www.enago.jp) for the Englishlanguage review.

Authorship ContributionsParticipated in research design: Kodama, Iwao, Ohta, Yuasa, Matsunaga.Conducted experiments: Kodama, Iwao, Katano, Ohta.Performed data analysis: Kodama, Iwao, Katano, Ohta.Wrote or contributed to the writing of the manuscript: Kodama, Iwao, Ohta,

Yuasa, Matsunaga.

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Address correspondence to: Dr. Takahiro Iwao, Department of Clinical Pharmacy,Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan. E-mail: [email protected]


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