1521-0103/358/1/94–102$25.00 http://dx.doi.org/10.1124/jpet.116.232025THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 358:94–102, July 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Interaction of the Sodium/Glucose Cotransporter (SGLT) 2 InhibitorCanagliflozin with SGLT1 and SGLT2: Inhibition Kinetics, Sidednessof Action, and Transporter-Associated Incorporation Accountingfor its Pharmacodynamic and Pharmacokinetic Featuress

Ryuichi Ohgaki, Ling Wei, Kazunori Yamada, Taiki Hara, Chiaki Kuriyama, Suguru Okuda,Kiichiro Ueta, Masaharu Shiotani, Shushi Nagamori, and Yoshikatsu KanaiDepartment of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan (R.O., L.W., S.O., S.N.,Y.K.); and Research Division, Mitsubishi Tanabe Pharma Corporation, Saitama, Japan (K.Y., T.H., C.K., K.U., M.S.)

Received January 12, 2016; accepted April 26, 2016

ABSTRACTCanagliflozin, a selective sodium/glucose cotransporter (SGLT)2 inhibitor, suppresses the renal reabsorption of glucoseand decreases blood glucose level in patients with type2 diabetes. A characteristic of canagliflozin is its modestSGLT1 inhibitory action in the intestine at clinical dosage. Toreveal its mechanism of action, we investigated the interactionof canagliflozin with SGLT1 and SGLT2. Inhibition kineticsand transporter-mediated uptake were examined in humanSGLT1- or SGLT2-expressing cells. Whole-cell patch-clamprecording was conducted to examine the sidedness of drugaction. Canagliflozin competitively inhibited SGLT1 andSGLT2, with high potency and selectivity for SGLT2. Inhibitionconstant (Ki) values for SGLT1 and SGLT2 were 770.5 and4.0 nM, respectively. 14C-canagliflozin was suggested to betransported by SGLT2; however, the transport rate was less

than that of a-methyl-D-glucopyranoside. Canagliflozin inhibiteda-methyl-D-glucopyranoside–induced SGLT1- and SGLT2-mediated inward currents preferentially from the extracellu-lar side and not from the intracellular side. Based on theKi value, canagliflozin is estimated to sufficiently inhibitSGLT2 from the urinary side in renal proximal tubules. The Kivalue for SGLT1 suggests that canagliflozin suppressesSGLT1 in the small intestine from the luminal side, whereasit does not affect SGLT1 in the heart and skeletal muscle,considering the maximal concentration of plasma-unboundcanagliflozin. Similarly, SGLT1 in the kidney would not beinhibited, thereby aiding in the prevention of hypoglycemia.After binding to SGLT2, canagliflozin may be reabsorbed bySGLT2, which leads to the low urinary excretion and pro-longed drug action of canagliflozin.

IntroductionUnder euglycemic conditions, glucose in the glomerular

filtrate is completely reabsorbed in the proximal tubules of thekidney so that it is not excreted into urine. In the process ofglucose reabsorption, glucose in the luminal fluid is taken upby tubular epithelial cells via sodium/glucose cotransporters

(SGLTs) in the apical membrane and then leaves the epithe-lial cells through the facilitative glucose transporters in thebasolateral membrane to the bloodstream (Wright et al.,2011). Under physiological conditions, the bulk of filteredglucose (∼90%) is reabsorbed by SGLT2 (SLC5A2), a low-affinity high-capacity transporter in the proximal convolutedtubule (particularly in the S1 segment) (Kanai et al., 1994;You et al., 1995). The remaining filtered glucose (∼10%) isreabsorbed by SGLT1 (SLC5A1), a high-affinity low-capacitytransporter in the proximal straight tubule (particularly inthe S3 segment) (Hediger et al., 1987; Lee et al., 1994). In thesmall intestine, SGLT1 is present in the apical membrane ofepithelial cells and is responsible for the intestinal absorptionof glucose and galactose (Wright et al., 2011). To target SGLT2in renal proximal tubules, selective SGLT2 inhibitors havebeen developed as antidiabetic agents. These compounds in-hibit the renal reabsorption of glucose to increase its urinaryexcretion, thereby lowering plasma glucose levels in an insulin-independent manner (Liang et al., 2012; Boyle and Wilding,2013; Fujita and Inagaki, 2014; Peene and Benhalima, 2014).

This work was supported by Grants-in-Aid for Scientific Research from theJapan Society for the Promotion of Science; the Ministry of Education, Science,Culture, Sports and Technology of Japan (MEXT); and the Mitsubishi TanabePharma Corporation. L.W. was supported by a scholarship from MEXT forforeign students.

This work was previously presented in part as follows: L.W., R.O., S.N., andY.K. Interaction of a novel SGLT2 inhibitor Canagliflozin with human SGLT2.The 57th Annual Meeting of the Japan Diabetes Society, 2014 May 22–24;Osaka, Japan; and R.O., L.W., S.N., K.Y., T.H., C.K., K.U., M.S., and Y.K.Study on the transport of SGLT2 inhibitor canagliflozin by human SGLT2 andits inhibitory action from extracellular and intracellular sides. The 58thAnnual Meeting of the Japan Diabetes Society, 2015 May 21–24; Fukuoka,Japan.

R.O. and L.W. contributed equally to this work.dx.doi.org/10.1124/jpet.116.232025.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: AMG, a-methyl-D-glucopyranoside; 8-Br-cAMP, 8-bromo-cAMP; DMSO, dimethylsulfoxide; DOX, doxycycline; HBSS, Hank’sbalanced salt solution; IAMG, a-methyl-D-glucopyranoside–induced current; IAMG/Cm, current density; Ki, inhibition constant; PCR, polymerase chainreaction; SGLT, sodium/glucose cotransporter.

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Canagliflozin, a C-glucoside with a thiophene ring (Nomuraet al., 2010), is the first selective SGLT2 inhibitor approved bythe US Food and Drug Administration to improve glycemiccontrol in individuals with type 2 diabetes mellitus (Elkinsonand Scott, 2013). The European Medicines Agency alsoapproved canagliflozin for this use in 2013. The administra-tion of canagliflozin at a clinical dosage to patients with type 2diabetesmellitus inducesurinary glucose excretionat∼100g/day,which would not increase the risk of hypoglycemia (Devineniet al., 2012, 2013; Iijima et al., 2015). In addition to anti-hyperglycemic action, canagliflozin, as a selective SGLT2inhibitor, exhibits other favorable effects for the treatment oftype 2 diabetes mellitus, including loss in body weight andhypotensive effects (Sha et al., 2011; Lavalle-Gonzalez et al.,2013). Among the selective SGLT2 inhibitors currently avail-able for clinical use, a characteristic of canagliflozin is its mod-est SGLT1 inhibitory action in the intestine at clinical dosage(Grempler et al., 2012; Kuriyama et al., 2014). This is probablybecause of its relatively low SGLT2/SGLT1 selectivity as well asits relatively high clinical dosage, owing to its high plasmaprotein binding (Fujita and Inagaki, 2014;Devineni et al., 2015).A major question regarding the mechanisms of action of

SGLT2 inhibitors is the sidedness of action [i.e., whetherthese compounds inhibit SGLT2 from the extracellular side(luminal side) or from the cytoplasmic side]. A canonicalnonselective SGLT inhibitor, phlorizin, the parent compoundof selective SGLT2 inhibitors, exhibits a ∼600-fold lowerinhibition constant (Ki) value on SGLT1 from the extracellularside than it does from the intracellular side (Eskandari et al.,2005). Phlorizin is freely filtrated by the glomerulus andmainly excreted into urine, which is in line with the idea thatthe glomerular-filtrated phlorizin acts on SGLTs from theluminal side to increase urinary glucose excretion (Silverman,1974). In contrast, canagliflozin exhibits high plasma proteinbinding (99%), and its urinary excretion is 1% or less of a singleoral dosage (Inagaki et al., 2014; Devineni et al., 2015; KinoshitaandKondo, 2015). These observations raised the possibility thatcanagliflozin enters epithelial cells of the renal proximal tubulethrough the basolateral membrane to exert its inhibitory effecton SGLT2 in the apical membrane from the cytoplasmic side.There is currently no experimental evidence for the sidedness ofthe action of clinically used selective SGLT2 inhibitors. In thestudy using TA-3404, a compound with a canagliflozin-relatedstructure, it has been suggested that the SGLT2 inhibitionoccurs from the extracellular side (Ghezzi et al., 2014). In ad-dition, we speculated that canagliflozin is not only an inhibitorof SGLT2, but also a substrate for SGLT2, and that SGLT2-mediated active transport, at least in part, explains the low uri-nary excretion rate observed with the use of canagliflozin.In this study, to reveal the molecular mechanisms of action

of canagliflozin, we investigated its interaction with SGLTs invitro using cultured cell lines stably expressing humanSGLT1and SGLT2. We examined the concentration-dependent pro-files of inhibition to determine the Ki values of canagliflozinfor each SGLT, the uptake of canagliflozin by SGLTs usingradioactive 14C-canagliflozin, and the sidedness of the drugaction using whole-cell patch-clamp recording.

Materials and MethodsChemicals. Canagliflozin was synthesized by the Mitsubishi

TanabePharmaCorporation,MedicinalChemistryLaboratory (Saitama,

Japan). a-Methyl-D-glucopyranoside (AMG) was purchased fromSigma-Aldrich (St. Louis, MO). Phlorizin was obtained from TCI(Tokyo, Japan). 14C-Canagliflozin ((1S)-1,5-anhydro-1-C-(3-{[5-(4-fluorophenyl)thiophen-2-yl]-[14C]methyl}-4-methylphenyl)-D-glucitolhemihydrate) (2.15 GBq/mmol) was synthesized by Janssen Research& Development, LLC (Spring House, PA). 14C-AMG {methyl-a-D-[glucose-14C(U)]glucopyranoside} (10.69 GBq/mmol) was fromPerkinElmer (Boston, MA). Blasticidin S was purchased fromInvivoGen (San Diego, CA). 8-Bromo-cAMP (8-Br-cAMP) wasobtained from Sigma-Aldrich. Penicillin-streptomycin solution,hygromycin B, G418 sulfate, and doxycycline (Dox) were from WakoPure Chemical Industries (Osaka, Japan).

Cell Lines. For the construction of the tetracycline-inducibleexpression vector, a plasmid backbone of pJTI R4 DEST [except theregion flanked by l integrase attR1 and attR2 sites (nucleotideposition 3842–5545)], and a partial fragment of pcDNA4/TO (nucleo-tide position 232–1319: composed of cytomegalovirus promoter,tetracycline operator, multiple cloning sites, and bovine growthhormone polyadenylation site) were amplified by polymerase chainreaction (PCR) using Pwo Super Yield DNA Polymerase (RocheDiagnostics, Mannheim, Germany). The obtained two PCR productswere then ligated together by homologous recombination using In-Fusion HD Cloning Kit (Clontech, Palo Alto, CA). The nucleotidesequences of the primer pairs were as follows: forward 59-GGCTT-GGCTCCGGTGcccgt-39 and reverse 59-GGCGTAATCATGGTCatagctg-39for pJTI R4 DEST; and forward 59- CACCGGAGCCAAGCCacgcgttga-cattgattattgactag-39 and reverse 59-GACCATGATTACGCCtccccag-catgcctgctattgtc-39 for pcDNA4/TO, wherein the complementarysequences between the primer sets are indicated by capital letters.

The coding sequences of human SGLT1 (GenBank accession#AB463272) and SGLT2 (GenBank accession #NM_003041.3) wereamplified by PCR from Flexi ORF SGLT1 Clone pF1KB8725(Promega, Madison, WI) and from a full-length synthetic SGLT2 CDSclone (GenScriptUSA, Piscataway, NJ), respectively. ThePCR productswere integrated into the tetracycline-inducible expression vector(HindIII/XbaI linearized) by homologous recombination using In-Fusion HD Cloning Kit (Clontech). The nucleotide sequences of theprimer pairs were as follows: forward 59-ACTTAAGCTTggtaccaccatg-gacagtagcacctggagc-39 and reverse 59-TTAAACGGGCCCTCTAGActcaggcaaaatatgcatggc-39 for SGLT1; and forward 59-GTTTAAACTT-AAGCTTccaccatggaggagcacacagaggcaggct-39 and reverse 59-AAAC-GGGCCCTCTAGAttaggcatagaagccccagaggaac-39 for SGLT2, whereinthe primer sequences identical to that of the tetracycline-inducibleexpression vector were indicated by capital letters.

To introduce an R4 attP retargeting site and promoter-lessneomycin resistance gene into the genomic PhiC31 pseudo-attP site,the TetR-expressing T-Rex-CHO and T-Rex-293 cells (Invitrogen,Carlsbad, CA) were cotransfected with pJTI phiC31 Int and pJTI/Neousing X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostics).After the selection for hygromycin B resistance and cloning, theestablished Jump-In TI platform cells (termed as JTREx-CHO andJTREx-293 cells) were further cotransfected with pJTI R4 Int andthe tetracycline-inducible expression vectors for human SGLT1 orSGLT2 constructed as described above. Successful retargeting of theexpression vectors at the R4 attP site confers neomycin resistance tothe Jump-In TI platform cells by cointegration of the EF1a promoterupstream of the promoter-less neomycin resistance gene. Retargetedcells were selected for G418 sulfate resistance, cloned, and used forthe measurements.

Cells were grown at 37°C in a humidified incubator supplied with5% CO2. Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12andDMEM (WakoPureChemical Industries) supplementedwith 10%fetal bovine serum (ThermoFisher Scientific, Grand Island, NY),1% (v/v) penicillin-streptomycin solution, and 5 mg/ml blasticidin Swere used to maintain the T-Rex-CHO and T-Rex-293 cells, respec-tively. Where indicated, hygromycin B (100 mg/ml) and G418 sulfate(500mg/ml) were additionally supplied to themedium for selection andmaintenance of the constructed stable cell lines.

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14C-AMG Uptake Measurement and Inhibition Experi-ments. Uptake of AMG, an SGLT-selective substrate, was measuredin the cell lines. Cells were seeded on 24-well plates and cultured for24 hours in the presence of 10 ng/ml DOX to induce the expression ofhuman SGLTs. Uptake measurements were performed as previouslydescribed (Wiriyasermkul et al., 2012). Briefly, cells were incubated at37°C in glucose-free HBSS containing 14C-AMG at concentrations of400 mM (25.4 MBq/mmol) and 1500 mM (50.8 MBq/mmol) for SGLT1and SGLT2, respectively. NaCl in glucose-free HBSS was replacedwith choline chloride for the sodium-free condition. For inhibitionexperiments, the indicated concentrations of phlorizin or canagliflozindissolved in dimethylsulfoxide (DMSO) were added to the assaybuffer. The assay buffer contained up to 0.1% DMSO (final concen-tration), unless otherwise noted. Up to 0.2% of DMSO did notsignificantly affect the uptake of 14C-AMG and 14C-canagliflozin (datanot shown). After cell lysis, the radioactivity was measured by liquidscintillation counting and normalized according to the proteinamount. The IC50 of phlorizin and canagliflozin was determined byfitting the data to inhibition curves using nonlinear regression (four-parameter Hill function; SigmaPlot 12.5; SYSTAT). To determine thekinetic properties of inhibition, uptakemeasurementswere performedwith varied concentrations of 14C-AMG in the presence or absence ofthe indicated concentrations of inhibitors. Uptake rates were plottedagainst 14C-AMG concentration and fitted to the Michaelis-Mentencurve.Ki was calculated using the following equation when competitiveinhibition was observed: V5 Vmax/{11 [Km/(S)] [11 (I)/Ki]}, where V isthe 14C-AMG uptake rate, Vmax is the maximal 14C-AMG uptake rate,Km is the Michaelis constant; [S] is the 14C-AMG concentration, and [I]is the inhibitor concentration. All analyses were performed using theenzyme kinetics module of SigmaPlot 12.5 (SYSTAT, San Jose, CA).

14C-Canagliflozin Uptake Measurements. For 14C-canagliflozinuptake measurement, cells were incubated with the indicated concen-trations of 14C-canagliflozin (2.15 GBq/mmol) in the presence or ab-sence of sodium. Phlorizin (50 mM)was added to the assay buffer whereindicated. To examine the temperature dependence of the uptake, themeasurementswere performed using 14C-canagliflozin (5 and 10nM) or14C-AMG (500 mM, 25.4 MBq/mmol) for 20 minutes on ice or at 37°C.

Whole-Cell Patch-Clamp Recordings. Cells were seeded onpoly-D-lysine–coated glass coverslips and cultured in the presenceof DOX (10 ng/ml) for 12–20 hours. To increase the activity of SGLT2,8-Br-cAMP (0.1 mM) was added to the medium 1 hour before themeasurements (Ghezzi and Wright, 2012). Whole-cell patch-clamprecording was performed at room temperature for SGLT1 (JTREx-293-SGLT1 cells) and at 32°C for SGLT2 (JTREx-293-SGLT2 cells) ina stage-top chamber on an Eclipse TE300 invertedmicroscope (Nikon,Tokyo, Japan) with gravitational perfusion of external solution[100 mM Mannitol, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and10 mM HEPES-Tris (pH 7.4)]. To elicit the transporter-mediatedcurrent, the indicated concentration of AMGwas added to the externalsolution, at which no significant inward currents were induced by theequivalent osmolarity of mannitol (Supplemental Fig. 4). Patchpipettes were prepared from borosilicate glass capillary GD-1.5(Narishige, Tokyo, Japan) pulled by a PD-7 pipette puller (Narishige),giving a resistance of 7–10 MVwhen filled with internal solution. Thecomposition of the standard internal solution was 145mMCsCl, 5mMNaCl, 11 mMEGTA, and 10mMHEPES-Tris (pH 7.4). A high-sodiuminternal solution [150 mM NaCl, 11 mM EGTA, and 10 mM HEPES-Tris (pH 7.4)] was used for the intracellular application of canagli-flozin. Canagliflozin was dissolved in DMSO, and was diluted intointernal and external solutions. External and internal solutions withandwithout canagliflozin contained 0.5%DMSO (final concentration),the concentration at which DMSO did not significantly affect theAMG-induced inward current (Supplemental Fig. 4). The whole-cellcurrents were recorded at a holding potential of 260 mV using anAXOPATCH200B amplifier (Axon Instruments, Foster City, CA)witha low-pass Bessel filter set at 5 kHz. Datawere digitized at 10 kHz by aDigidata 1320A AD/DA converter (Axon Instruments) with Clampex8.2 software (Molecular Devices, Sunnyvale, CA) and analyzed using

Clampfit 10.4 software (Molecular Devices). The whole-cell capacitance(Cm) was measured at the beginning of each measurement by thesquare-pulse method with the Membrane Test tool of the Clampex 8.2software. The current density [AMG-induced current (IAMG)/Cm] wascalculated to normalize the recorded IAMG.

To investigate the sidedness of inhibition by canagliflozin onSGLT1, the measurements were performed in the presence (n 5 10)or absence (n5 11) of intracellular canagliflozin (20mM). In the case ofexperiments on SGLT2, all the measurements were performed withintracellular application of canagliflozin (200 nM), and the IAMG/Cmvalues before and after the application of extracellular canagliflozin(200 nM) were compared (n 5 7). The indicated concentrations ofcanagliflozin itself did not induce a significant current when appliedextracellularly (Supplemental Fig. 4). Replacement of the intracellu-lar fluid with high-sodium internal solution containing canagliflozinwas confirmed under current-clamp mode (0 pA) after achievingwhole-cell configuration.

Statistical Analyses. Unpaired, two-tailed Student’s t tests wereperformedwithExcel 2013 software (Microsoft, Redmond,WA), wherep values ,0.05 were considered to be significant.

ResultsFunctional Expression of SGLT1 and SGLT2. To

confirm the functional expression of SGLT1 and SGLT2, 14C-AMG uptake was measured in doxycycline-treated JTREx-CHO-SGLT1 and JTREx-CHO-SGLT2 cells. Doxycyclineincreased 14C-AMG uptake in the JTREx-CHO-SGLT1 andJTREx-CHO-SGLT2 cells but not in the parental JTREx-CHOcell line (Fig. 1, A and B). The expressed 14C-AMG uptake wasNa1 dependent and was abolished in the absence of Na1.Phlorizin almost completely suppressed 14C-AMG uptake inboth cell lines (Fig. 1, A and B). The 14C-AMG uptakes bySGLT1 and SGLT2 were linearly dependent on the incubationtime for (at least) 30 and 90 minutes, respectively (Fig. 1, Cand D). These results indicate that SGLT1 and SGLT2 werefunctionally expressed in the cell lines constructed. Proteinexpression of SGLT1 and SGLT2 was also confirmed by Westernblotanalysisusingspecific antibodies (SupplementalFig. 1).Basedon the linear range observed over the time course, the uptakeswere measured for 5 and 30 minutes on SGLT1 and SGLT2cells, respectively, in the subsequent kinetic analyses.Kinetic Properties of the Inhibition of 14C-AMG

Uptake by Canagliflozin. The concentration dependenceof the effects of canagliflozin on 14C-AMG uptake wasexamined and compared with that of the effects of phlorizin.Phlorizin inhibited 14C-AMG uptake mediated by SGLT1 andSGLT2 in a concentration-dependent manner (Fig. 2, A andB). The IC50 values of phlorizin on SGLT1 and SGLT2 were439 and 56 nM, respectively. Similarly, the inhibition ofSGLT1 and SGLT2 by canagliflozin also exhibited clearconcentration dependence with an IC50 of 1550 nM for SGLT1and 6.8 nM for SGLT2 (Fig. 2, C and D).To further characterize the kinetic properties of the in-

hibitory effects of canagliflozin, the Ki values and mode ofinhibition were determined. Nonlinear regression analysis ofthe inhibitory effects of the compounds (Supplemental Fig. 2,A, C, E, and G) revealed that canagliflozin and phlorizincompetitively inhibit SGLT1- and SGLT2-mediated 14C-AMGuptake. The competitive inhibition was confirmed by Eadie-Hofstee plots (Supplemental Fig. 2, B, D, F, and H). The Ki

values were determined from three or five independentlyperformed trials, and are listed in Table 1.

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SGLT2-Mediated Uptake of 14C-Canagliflozin. To ex-amine whether canagliflozin is transported by SGLT1 andSGLT2, we measured the uptake of 14C-canagliflozin inthe JTREx-CHO-SGLT1 and JTREx-CHO-SGLT2 cells.No statistically significant uptake of 14C-canagliflozinwas detected for SGLT1 at 10 and 20 nM, although theJTREx-CHO-SGLT1 cells tended to exhibit a slightly higheruptake compared with mock cells in the presence of Na1

(Supplemental Fig. 3). In contrast, the JTREx-CHO-SGLT2cells, showed a significantly higher 14C-canagliflozin up-take compared with mock cells at 5–20 nM in the presence,but not in the absence, of Na1 (Fig. 3A). The considerable14C-canagliflozin uptake observed in the parental JTREx-CHO cells most likely represents the incorporation viapassive diffusion through the plasma membrane. SGLT2-mediated 14C-canagliflozin uptake increased in a concentration-dependent manner (Fig. 3A) and was completely inhibitedby phlorizin (Fig. 3B), consistent with the properties of

SGLT2-mediated transport. The uptake of 14C-canagliflozinvia SGLT2 increased during the first 20 minutes of theincubation time (Fig. 3C).To confirm that the SGLT2-associated increase of

14C-canagliflozin radioactivity is because of the SGLT2-mediated transport and not SGLT2 binding, its tempera-ture dependency was examined. When similar measurementswere performed on ice, uptake of the 14C-AMG in JTREx-CHO-SGLT2 cells decreased to the background level (i.e., to that ofthe parental JTREx-CHO cells), confirming the transporter-mediated uptake to be highly sensitive to temperature (Fig.3D). Similarly, the JTREx-CHO-SGLT2 cells showed ahigher 14C-canagliflozin uptake compared with the parentalJTREx-CHO cells at 37°C, whereas this difference disap-peared on ice (Fig. 3D). Such temperature dependencesuggests that the observed 14C-canagliflozin uptake repre-sents SGLT2-mediated transport but not the binding of14C-canagliflozin to SGLT2.

Fig. 1. Properties of 14C-AMG uptake in the JTREx-CHO-SGLT1 and JTREx-CHO-SGLT2 cells. (A and B) Na+ dependence and phlorizin sensitivity ofdoxycycline-induced 14C-AMG uptake. Doxycycline-treated (Dox+) or untreated (Dox2) cells were used to determine 14C-AMG uptake in the presence orabsence of Na+ and phlorizin (100mM). For the JTREx-CHO-SGLT1 cells (SGLT1) and parental JTREx-CHO cells (Mock), the uptake of 14C-AMG (400mM)was measured for 5 minutes (A). For the JTREx-CHO-SGLT2 cells (SGLT2) and parental JTREx-CHO cells (Mock), the uptake of 14C-AMG (1500 mM) wasmeasured for 30 minutes (B). (C and D) Time course of 14C-AMG uptake examined in the presence of Na+ (glucose-free HBSS). 14C-AMG of 400 mM wasused for the Dox-treated JTREx-CHO-SGLT1 (SGLT1) and parental JTREx-CHO (Mock) cells (C). 14C-AMG of 1500 mM was used for the Dox-treatedJTREx-CHO-SGLT2 cells (SGLT2) and parental JTREx-CHO cells (Mock) (D). Data shown depict the mean 6 S.E.M. n = 3–4. ***p , 0.001.

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Sidedness of Canagliflozin Action. The sidedness ofcanagliflozin action on SGLTs was investigated using thewhole-cell configuration of the patch-clamp method byapplying canagliflozin to the outside or inside of the cells(Fig. 4A). Because AMG-induced electric currents in theJTREx-CHO-SGLT2 cells used for the uptake experimentswere too small for reliable electrophysiological recording, weprepared other stable cell lines (JTREx-293-SGLT1 andJTREx-293-SGLT2 cells) established from the JTREx-293cells and used them for whole-cell patch-clamp recording.When the AMG uptakes were measured in the two SGLT2-expressing cell lines, JTREx-293-SGLT2 cells showed a 2.32-fold higher uptake at 30 minutes than JTREx-CHO-SGLT2cells (data not shown). Furthermore, to enhance the activityof SGLT2, 8-Br-cAMPwas added to the medium, andmeasure-ments were conducted at 32°C (Hummel et al., 2011; Ghezziand Wright, 2012).For the intracellular application of the drug, the time course

for the replacement of intracellular fluid with a canagliflozin-containing pipette solution was estimated under current-clamp

mode (0 pA). After attaining the whole-cell configuration, theoriginal inside-negative membrane potential was promptlyshifted toward 0 mV, as expected from the ionic compositionof the internal solution and reached a plateau at ∼150 seconds(Supplemental Fig. 5), indicating complete replacement ofintracellular fluid with the drug-containing pipette solutionwithin this time range. Therefore, all the measurements wereperformed after 150 seconds.Neither the JTREx-293-SGLT1 nor the JTREx-293-SGLT2

cells, without induction by doxycycline treatment, showedany significant background inward current by the additionof AMG into the external solution (Fig. 4B). After the

Fig. 2. Concentration-dependent inhibition of 14C-AMG uptake by phlorizin and canagliflozin. 14C-AMG uptake mediated by SGLT1 or SGLT2 wasplotted against inhibitor concentration. SGLT1- or SGLT2-mediated uptake was obtained by subtracting 14C-AMG uptake in the parental JTREx-CHOcells from that in the JTREx-CHO-SGLT1 or JTREx-CHO-SGLT2 cells, respectively. To measure SGLT1-mediated uptake, cells were incubated with400 mM 14C-AMG for 5 minutes with varied concentrations of phlorizin or canagliflozin (A and C). To measure SGLT2-mediated uptake, cells wereincubated with 1500 mM 14C-AMG for 30minutes with varied concentrations of phlorizin or canagliflozin (B and D). The 14C-AMG uptake in the presenceof inhibitors is shown as the percentage of the uptake without inhibitors. Data shown depict the mean 6 S.E.M. n = 3–4.

TABLE 1Ki values of phlorizin and canagliflozin

Inhibitor Ki for SGLT1 (nM) Ki for SGLT2 (nM)

Phlorizin 545.7 6 56.89 (n = 3) 102.3 6 27.41 (n = 3)Canagliflozin 770.5 6 85.19 (n = 5) 4.0 6 0.77 (n = 3)

Values are presented as the mean 6 S.E.M.

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doxycycline treatment, a concentration of 10 mMAMG in theexternal solution induced an inward current in the JTREx-293-SGLT1 cells (Fig. 4C). This AMG-induced inwardcurrent was reverted to the basal level when either AMGor Na1 was washed out from the external solution duringthe measurement (Supplemental Fig. 4). The AMG-inducedcurrent was almost completely suppressed by extracellularapplication of 20 mM canagliflozin (Fig. 4C). In contrast,when the same concentration of canagliflozin was appliedintracellularly, no significant inhibition of the SGLT1-mediated AMG-induced current was observed (Fig. 4E).Similarly, an external solution of 20 mM AMG induced anappreciable inward current in the JTREx-293-SGLT2 cellsin the presence of intracellular 200 nM canagliflozin. Thisinward current was almost completely reverted to the basallevel when the same concentration of canagliflozin wasextracellularly applied (Fig. 4D). Figure 4F shows thecomparison of the current densities of the AMG-inducedcurrent before and after the addition of extracellularcanagliflozin, indicating that extracellular canagliflozinremarkably reduces AMG-induced currents. Taken to-gether, these results demonstrate that canagliflozinpreferentially inhibits SGLT1 and SGLT2 from the extra-cellular side.

DiscussionWe performed a kinetic analysis of the inhibitory effect of

canagliflozin and confirmed its high potency and selectivity forSGLT2 (Fig. 2 and Supplemental Fig. 2). This is the firstreport to describe the Ki values of canagliflozin, which areuseful to evaluate the in vivo action of canagliflozin on SGLT2and SGLT1 in various organs. Canagliflozin competitivelyinhibited human SGLT1 and SGLT2 with ∼200-fold higherselectivity for SGLT2 than for SGLT1, based on the ratio of Ki

values (Table 1). Based on the Ki values, canagliflozin hasan ∼25-fold higher affinity for SGLT2 than for phlorizin,whereas both inhibitors exhibited similar affinity for SGLT1(Table 1). These results are in line with those of previousreports, wherein the potency and selectivity of canagliflozinwere discussed based on IC50 values (Nomura et al., 2010;Grempler et al., 2012).Thewhole-cell patch-clampmethod allowed us to control the

composition of intracellular as well as extracellular fluid,enabling the delivery of canagliflozin into the cells forexamining the sidedness of the inhibition (Fig. 4). Wedemonstrated that canagliflozin preferentially inhibitsSGLT2 from the extracellular side and not from the intracel-lular side. This suggests that canagliflozin acts on SGLT2

Fig. 3. 14C-canagliflozin uptakemediated by SGLT2. (A and B) Themeasurement of 14C-canagliflozin uptake by SGLT2. The uptake of 14C-canagliflozin(5, 10, and 20 nM) was measured for 20 minutes in the JTREx-CHO-SGLT2 (SGLT2) and parental JTREx-CHO (Mock) cells with or without Na+ (A) andphlorizin (50 mM) (B). (C) Time course of 14C-canagliflozin uptake by SGLT2. The uptake of 5 nM 14C-canagliflozin was measured in the JTREx-CHO-SGLT2 (SGLT2) and parental JTREx-CHO (Mock) cells in the presence of Na+. (D) Temperature dependence of 14C-canagliflozin uptake by SGLT2. Theuptakes of 14C-canagliflozin (5 and 10 nM) (left) and 14C-AMG (500 mM) (right) were measured either on ice or at 37°C for 20 minutes in the presenceof Na+ using the Dox-treated JTREx-CHO-SGLT2 (SGLT2) and parental JTREx-CHO (Mock) cells. Data shown depict the mean6 S.E.M. n = 3–6. *p,0.05; **p , 0.01; ***p , 0.001.

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Fig. 4. Sidedness of canagliflozin action on SGLTs. (A) Schematic representation of whole-cell recording of transporter-mediated AMG-induced electriccurrents. JTREx-293-SGLT1 and JTREx-293-SGLT2 cells were subjected to whole-cell patch-clamp recording at a holding potential of260 mV. (B) Thecurrent trace of whole-cell recording demonstrating the absence of the background AMG-induced current in JTREx-293-SGLT1 (top) and JTREx-293-SGLT2 (bottom) cells without the induction by doxycycline. AMG (10 mM for SGLT1 and 20 mM for SGLT2) was added to the external solution. Thetraces of AMG-induced currents showing the inhibitory effect after extracellular application of canagliflozin on SGLT1 (C) and SGLT2 (D). AMG (10 mMfor SGLT1 and 20 mM for SGLT2) and canagliflozin (20 mM for SGLT1 and 200 nM for SGLT2) were added to the external solution as indicated.

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from the luminal side because SGLT2 is localized on the apicalmembrane of proximal tubules (Wright et al., 2011). Becausethe plasma protein binding of canagliflozin is ∼99% (Devineniet al., 2015) and the maximal plasma concentration after oraladministration of clinical doses is 2.1–6.14 mM (Kinoshita andKondo, 2015; Sha et al., 2015), the maximal concentration ofunbound canagliflozin in plasma and in the glomerular filtrateis ∼20–60 nM. SGLT2 should be exposed to this approximatecanagliflozin concentration from the luminal side. This isabove the determined Ki value of canagliflozin for SGLT2(4.0 nM), suggesting that canagliflozin fully inhibits SGLT2from the luminal side. In the concentration range of 20–60 nM,canagliflozin is estimated to inhibit SGLT2 by 68–86%under euglycemic conditions [100 mg/dl (5.6 mM) D-glucose]and by 49–74% when the blood glucose level is elevated to300 mg/dl (16.7 mM); this is based on the following equation:V5 Vmax/{11 [Km/(S)] [11 (I)/Ki]}, where V is the uptake ratefor D-glucose, Vmax is the maximal uptake rate, Km 5 4.0 mMfor D-glucose (Lu et al., 2014), [S] is the D-glucose concen-tration, [I] is the unbound canagliflozin concentration, andKi 5 4.0 nM. This supports the idea that canagliflozininhibits the renal tubular SGLT2 by acting from the luminalside but not from the basolateral side.Previous studies demonstrated that the oral administration

of canagliflozin delays postprandial glucose absorption fromthe intestine and enhances glucose-induced glucagon-likepeptide-1 secretion (Polidori et al., 2013; Kinoshita andKondo, 2015; Sha et al., 2015). Because other SGLT2 inhib-itors with higher selectivity to SGLT2 have not been reportedto elicit such effects, transient inhibition of SGLT1 in theupper small intestine seems to be the reason underlying theeffect on intestinal glucose absorption (Polidori et al., 2013;Oguma et al., 2015a,b). Like SGLT2, we showed that SGLT1 ispreferentially inhibited by canagliflozin from the extracellularside, with a Ki value of 770.5 nM. Because of the high plasmaprotein binding ratio (Devineni et al., 2015), the clinicaldosage of canagliflozin is higher (100–300 mg) than otherSGLT2 inhibitors. Therefore, it is reasonable to suppose thatthe luminal concentration of canagliflozin in the upper smallintestine is transiently elevated after oral administration to alevel considerably higher than the Ki value for SGLT1,implying that canagliflozin could inhibit SGLT1 from theluminal side of the intestine. The intraluminal concentrationof canagliflozin in the small intestine is substantially elevatedafter oral administration (Oguma et al., 2015b).In a survey of the expression of SGLTs in human tissues by

quantitative PCR, SGLT1 was expressed at high levels in theheart and skeletal muscle, in addition to the small intestine(Chen et al., 2010). In the heart, SGLT1 mRNA was detectedin cardiomyocytes by in situ hybridization (Zhou et al., 2003),and SGLT1 protein was localized to the cardiac myocytesarcolemma (Banerjee et al., 2009). SGLT1 expression levelswere increased in diabetic or ischemic cardiomyopathy(Banerjee et al., 2009). Although the functional roles of SGLT1have not been determined in such nonepithelial tissues, it has

been reported that SGLT1 is involved in the pathophysiology ofa murine model of PRKAG2 cardiomyopathy (Ramratnamet al., 2014). Because the maximal plasma level of unboundcanagliflozin in clinical studies (20–60 nM, see above) is ∼1/30to 1/10 of the Ki value for SGLT1 (770.5 nM), the oraladministration of clinical doses of canagliflozin would notinhibit SGLT1 in the heart or skeletal muscle. In theconcentration range of 20–60 nM, it is estimated that canagli-flozin inhibits SGLT1 by 0.2–0.7% under euglycemic conditions[100 mg/dl (5.6 mM) D-glucose] and by 0.1–0.2% when the bloodglucose level is elevated to 300mg/dl (16.7mM); this is based onthe assumption that the Km for D-glucose is 0.5 mM in SGLT1(Lu et al., 2014). Similarly, oral administration of canagliflozinat clinical dosage would not inhibit SGLT1 in the proximalstraight tubules of the kidney, aiding in the prevention ofhypoglycemia in conjunction with the paradoxical increase inendogenous glucose production caused by SGLT2 inhibition(Abdul-Ghani et al., 2013; Cefalu, 2014).Pharmacokinetic studies have revealed that the excretion

of canagliflozin in urine is ,1% of the administered dose(Inagaki et al., 2014; Devineni et al., 2015; Kinoshita andKondo, 2015). We showed that canagliflozin is incorporatedinto the SGLT2-expressing cells by SGLT2-mediated trans-port in addition to passive diffusion through the plasmamembrane (Fig. 3). Because the exogenous expression ofSGLT2 in conventional expression systems is, for an unknownreason, generally low in efficiency (Kanai et al., 1994), theuptake of 14C-canagliflozin by SGLT2 was low (Fig. 3).Therefore, based on the data obtained, it is difficult to estimatethe contribution of the possible SGLT2-mediated reabsorptionof canagliflozin to total tubular canagliflozin reabsorption,with the latter also involving the passive diffusion of thecompound through the renal tubule because of its highhydrophobicity. We propose that the tubular reabsorption ofcanagliflozin involving SGLT2-mediated transport and thelow filtration rate because of its high plasma protein bindingcontribute to the low urinary excretion of canagliflozin. More-over, a local circulation of canagliflozin through the apicalmembrane of renal proximal tubules may be facilitated by theSGLT2-mediated reabsorption of canagliflozin with the pos-sible involvement of its efflux transporters. This would retaincanagliflozin in the proximity of SGLT2, enabling the re-current inhibition of SGLT2 from the luminal side, whichexplains the prolonged drug action beyond the decrease inblood canagliflozin levels (Inagaki et al., 2014; Kuriyamaet al., 2014). The significance of SGLT2-mediated canagliflozinuptake in pharmacodynamics as well as pharmacokinetics invivo remains to be investigated.

14C-canagliflozin uptake was not detected for SGLT1 overthe concentration range tested (Supplemental Fig. 3). Higherconcentrations of 14C-canagliflozin could not to be examinedbecause of a high background cellular incorporation. However,considering its lower affinity to SGLT1, it is important to testthe uptake of canagliflozin by SGLT1 at a higher concentra-tion using high-expression systems.

Canagliflozin (200 nM) was also contained in the internal solution for (D). (E) The IAMG/Cm in JTREx-293-SGLT1 cells in the presence or absence ofintracellularly applied 20 mM canagliflozin [shown as (+) and (2), respectively]. Data shown depict the mean6 S.E.M. [n = 11 for (2) and n = 10 for (+)].(F) Comparison of the current densities for the currents induced by 20 mM AMG before (2) and after (+) the application of extracellular canagliflozin(200 nM) under the same condition as described in (D). Data shown are the mean 6 S.E.M. n = 7. *p , 0.05.

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In conclusion, canagliflozin inhibited SGLT2 and SGLT1 ina competitive manner with high potency and selectivity forSGLT2. Canagliflozin acted from the extracellular side, sug-gesting that the inhibition occurs from the urinary side in renalproximal tubules. We also suggested that canagliflozin is, atleast in part, transported by SGLT2 after binding, whichmaycontribute to its pharmacodynamic and pharmacokineticcharacteristics.

Acknowledgments

The authors thank Takanori Kobayashi and Miyuki Kurauchi fortheir technical assistance. Canagliflozin was developed by MitsubishiTanabe Pharma Corporation in collaboration with Janssen Research& Development, LLC.

Authorship Contributions

Participated in research design: Kanai, Ueta, Shiotani, andNagamori

Conducted experiments: Ohgaki, Wei, Yamada, Hara, Ueta, andKuriyama

Performed data analysis: Ohgaki and WeiWrote or contributed to the writing of the manuscript: Ohgaki, Wei,

Yamada, Hara, Kanai, Okuda, and Kuriyama

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Address correspondence to: Dr. Yoshikatsu Kanai, Department of Bio-system Pharmacology, Graduate School of Medicine, Osaka University, 2-2Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: ykanai@pharma1.med.osaka-u.ac.jp

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