Comparative Pharmacokinetics and Pharmacodynamics of a Novel Sodium-Glucose Cotransporter 2 Inhibitor, DWP16001, with Dapagliﬂozin and Ipragliﬂozin

pharmaceutics

Article Comparative Pharmacokinetics and Pharmacodynamics of a Novel Sodium-Glucose Cotransporter 2 Inhibitor, DWP16001, with Dapagliﬂozin and Ipragliﬂozin

Min-Koo Choi 1, So Jeong Nam 2, Hye-Young Ji 3, Mi Jie Park 3, Ji-Soo Choi 3 and Im-Sook Song 2,* 1 College of Pharmacy, Dankook University, Cheon-an 31116, Korea; [email protected] 2 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Korea; [email protected] 3 Life Science Institute, Daewoong Pharmaceutical, Yongin, Gyeonggido 17028, Korea; [email protected] (H.-Y.J.); [email protected] (M.J.P.); [email protected] (J.-S.C.) * Correspondence: [email protected]; Tel.: +82-53-950-8575

Received: 21 February 2020; Accepted: 12 March 2020; Published: 15 March 2020

Abstract: Since sodium-glucose cotransporter 2 (SGLT2) inhibitors reduced blood glucose level by inhibiting renal tubular glucose reabsorption mediated by SGLT2, we aimed to investigate the pharmacokinetics and kidney distribution of DWP16001, a novel SGLT2 inhibitor, and to compare these properties with those of dapagliflozin and ipragliflozin, representative SGLT2 inhibitors. The plasma exposure of DWP16001 was comparable with that of ipragliflozin but higher than that of dapagliflozin. DWP16001 showed the highest kidney distribution among three SGLT2 inhibitors when expressed as an area under curve (AUC) ratio of kidney to plasma (85.0 16.1 for DWP16001, ± 64.6 31.8 for dapagliflozin and 38.4 5.3 for ipragliflozin). The organic anion transporter-mediated ± ± kidney uptake of DWP16001 could be partly attributed to the highest kidney uptake. Additionally, DWP16001 had the lowest half-maximal inhibitory concentration (IC50) to SGLT2, a target transporter (0.8 0.3 nM for DWP16001, 1.6 0.3 nM for dapagliflozin, and 8.9 1.7 nM for ipragliflozin). ± ± ± The inhibition mode of DWP16001 on SGLT2 was reversible and competitive, but the recovery of the SGLT2 inhibition after the removal of SGLT2 inhibitors in CHO cells overexpressing SGLT2 was retained with DWP16001, which is not the case with dapagliflozin and ipragliflozin. In conclusion, selective and competitive SGLT2 inhibition of DWP16001 could potentiate the efficacy of DWP16001 in coordination with the higher kidney distribution and retained SGLT2 inhibition of DWP16001 relative to dapagliflozin and ipragliflozin.

Keywords: sodium-glucose cotransporter 2 (SGLT2) inhibitors; DWP16001; kidney distribution; inhibition mode

1. Introduction Achieving appropriate glycemic control for type 2 diabetes patients is a prerequisite for preventing cardiovascular and microvascular complications, and this can be guided by a combination of antidiabetic drugs with different modes of action [1]. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are the latest class of antidiabetic drugs that act through the inhibition of renal tubular glucose reabsorption and a reduction of blood glucose levels without stimulating insulin release [2]. SGLT2 is expressed in the S1 segment of proximal kidney tubules and is responsible for roughly 90% of the reabsorption of filtered glucose [3]. Additionally, SGLT2 inhibitors exhibit low hypoglycemia risk and have been associated with a significant reduction in major adverse cardiovascular events in clinical trials [4], garnering SGLT2 inhibitors increased attention.

Pharmaceutics 2020, 12, 268; doi:10.3390/pharmaceutics12030268 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 268 2 of 16

Several SGLT2 inhibitors have been approved for the treatment of type 2 diabetes, including canagliflozin (Invokana®), dapagliflozin (Farxiga®), empagliflozin (Jardiance®), ipragliflozin (Suglat®), and tofogliflozin (Apleway®)[3,5]. Additionally, there are several other similar compounds in the pipeline that may be approved in the near future. DWP16001 (Figure1), a selective SGLT2 inhibitor, is under development by Daewoong Pharmaceutical Co. Ltd. (Yongin, Korea) and is currently undergoing phase 2 clinical trials Pharmaceutics(Registration 2020 No., 12, NCT04014023).x 3 of 17

FigureFigure 1. 1. StructureStructure of of (A (A) )DWP16001, DWP16001, (B (B) D4-DWP16001) D4-DWP16001 (IS), (IS), (C ()C dapagliflozin,) dapagliﬂozin, and and (D ()D ipragliflozin.) ipragliﬂozin.

2.2. AnimalsRecently, and Tahara Ethical et Approval al. [5] compared the pharmacokinetics, pharmacodynamics, and pharmacological characteristics of six SGLT2 inhibitors, such as ipragliflozin, dapagliflozin, tofogliflozin, canagliflozin, Male Institute of Cancer Research (ICR) mice (7–8-weeks-year-old, 30−35 g) were purchased empagliflozin, and luseogliflozin. The study showed that all the SGLT2 inhibitors induced urinary from Samtako Co. (Osan, Korea). Animals were acclimatized for 1 week in an animal facility at glucose excretion in a dose-dependent manner but the duration of action differed among the six Kyungpook National University. Food and water were available ad libitum. All animal procedures drugs. Ipragliflozin and dapagliflozin showed persistent duration of action; these two drugs exhibited were approved by the Animal Care and Use Committee of Kyungpook National University increased urinary glucose excretion even 18 h post dose but the others showed about 12 h of duration. (Approval No. 2016-0138) and carried out in accordance with the National Institutes of Health In addition, ipragliflozin and dapagliflozind showed the lowest blood glucose and insulin level guidance for the care and use of laboratory animals. following the same daily dose (3 mg/kg) in diabetic mice. The long duration of action and glucose lowering efficacy of these two drugs closely correlated with the drug distribution and retention in the 2.3. Pharmacokinetic Study kidney and elimination half-life [5], suggesting the importance of kidney distribution and elimination profileICR is mice prerequisite were randomly in the edividedfficacy ofinto SGLT2 three inhibitors,groups and as were well fasted as the for potent at least SGLT2 12 h before inhibition. the oralTherefore, administration this study of aimedDWP16001, to compare dapagliflozin, the pharmacokinetic and ipragliflozin properties but had andfree access kidney to distribution water. On theof DWP16001day of pharmacokinetic with those of dapagliflozinstudy, the mice and received ipragliflozin, DWP16001, representative dapagliflozin, SGLT2 or inhibitors ipragliflozin that solutionshowed potentat a dose and of long 1 mg/kg duration (dissolved efficacy [5in] anda mixture to compare of 10% the DMSO selectivity and and 90% mode saline) of inhibition using oral of gavage.DWP16001 Blood on SGLT2samples with were dapagliflozin collected andat 0.5, ipragliflozin 1, 2, 4, 8, to evaluate24, 48, and the potency72 h following of DWP16001 the overoral administrationother SGLT2 inhibitors. (1 mg/kg each) of DWP16001, dapagliflozin, or ipragliflozin and centrifuged at 12,000 g for 1 min to separate the plasma. An aliquot (30 µL) of each plasma sample was stored at −2.80 Materials °C until the and analysis. Methods Kidney samples were also isolated at 8, 24, 48, and 72 h following the oral administration of DWP16001, dapagliflozin, or ipragliflozin, minced thoroughly, and homogenized 2.1. Materials with four volumes of saline using tissue grinder. An aliquot (50 µL) of each kidney homogenate sampleDWP16001 was stored and at − D4-DWP1600180 °C until the analysis. (for internal standard (IS)), were obtained from Daewoong PharmaceuticalFor the analysis Co. Ltd. of SGLT2 (Yongin, inhibitors, Korea). Dapagliflozin aliquots of plasma and ipragliflozin (30 µL) and were kidney obtained homogenate from Toronto (50 µL)Research were Chemicalsadded to 100 Inc. µL (North of aqueous York, ON, solution Canada) of (FigureD4-DWP160011). (IS, 20 ng/mL), and vigorously mixedPara-aminohipuric with 500 µL methyl acid tert-butyl (PAH), ether methyl (MTBE) a-D-glucopyranoside for 15 min. After (AMG), centrifugation sodium at dodecyl 16,000 g sulfate for 5 min,(SDS), samples G418, non-essentialwere kept for amino 1 h at acids, −80 °C 4-(2-hydroxyethyl)-1-piperazineethanesulfonic to make an aqueous layer freeze. An organic acid upper (HEPES), layer wasand transferred Hank’s balances to a clean salts solutiontube and (HBSS, evaporated pH 7.4) to weredryness purchased under a from gentle Sigma–Aldrich stream of nitrogen. Chemical Then, Co. the(St. dried Louis, extract MO, USA). was reconstituted [14C]Methyl- αin-D-glucopyranoside 150 µL of mobile phase, (AMG) and (290 a 3 mCi µL /aliquotmmol) wasof the purchased reconstituent from was injected into a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system. Pharmacokinetic parameters, such as the area under plasma concentration-time curve from zero to infinity (AUC), were calculated from plasma concentration vs time curves using non-compartment analysis with WinNonlin (version 5.1; Pharsights, Cary, NC, USA). The AUC ratios (i.e., ratios of kidney AUC to plasma AUC) were calculated by dividing the AUC of the three SGLT2 inhibitors in the kidney by the plasma AUC values of the three SGLT2 inhibitors.

Pharmaceutics 2020, 12, 268 3 of 16

Moravek (Brea, CA, USA). [3H]Estrone-3-sulfate (ES) (2.12 TBq/mmol) and [3H]para-aminohipuric acid (PAH)(0.13 TBq/mmol) were purchased from Perkin Elmer Inc. (Boston, MA, USA). Dulbecco’s modiﬁed Eagle’s medium (DMEM), RPMI1640 medium, fetal bovine serum (FBS), and poly-D-lysine-coated 24-well plates were purchased from Corning (Tewksbury, MA, USA). All other chemicals and solvents were reagent or analytical grade. CHO cells overexpressing SGLT1 and SGLT2 (CHO-SGLT1 and -SGLT2, respectively) and CHO-mock cells were obtained from Daewoong Pharmaceutical Co. Ltd. (Yongin, Korea). HEK293 cells overexpressing organic anion transporter 1 (OAT1) and OAT3 (HEK293-OAT1 and -OAT3, respectively) and HEK293-mock cells were purchased from Corning (Tewksbury, MA, USA).

2.2. Animals and Ethical Approval Male Institute of Cancer Research (ICR) mice (7–8-weeks-year-old, 30 35 g) were purchased from − Samtako Co. (Osan, Korea). Animals were acclimatized for 1 week in an animal facility at Kyungpook National University. Food and water were available ad libitum. All animal procedures were approved by the Animal Care and Use Committee of Kyungpook National University (Approval No. 2016-0138) and carried out in accordance with the National Institutes of Health guidance for the care and use of laboratory animals.

2.3. Pharmacokinetic Study ICR mice were randomly divided into three groups and were fasted for at least 12 h before the oral administration of DWP16001, dapagliflozin, and ipragliflozin but had free access to water. On the day of pharmacokinetic study, the mice received DWP16001, dapagliflozin, or ipragliflozin solution at a dose of 1 mg/kg (dissolved in a mixture of 10% DMSO and 90% saline) using oral gavage. Blood samples were collected at 0.5, 1, 2, 4, 8, 24, 48, and 72 h following the oral administration (1 mg/kg each) of DWP16001, dapagliflozin, or ipragliflozin and centrifuged at 12,000 g for 1 min to separate × the plasma. An aliquot (30 µL) of each plasma sample was stored at 80 C until the analysis. Kidney − ◦ samples were also isolated at 8, 24, 48, and 72 h following the oral administration of DWP16001, dapagliflozin, or ipragliflozin, minced thoroughly, and homogenized with four volumes of saline using tissue grinder. An aliquot (50 µL) of each kidney homogenate sample was stored at 80 C until − ◦ the analysis. For the analysis of SGLT2 inhibitors, aliquots of plasma (30 µL) and kidney homogenate (50 µL) were added to 100 µL of aqueous solution of D4-DWP16001 (IS, 20 ng/mL), and vigorously mixed with 500 µL methyl tert-butyl ether (MTBE) for 15 min. After centrifugation at 16,000 g for 5 min, samples were kept for 1 h at 80 C to make an aqueous layer freeze. An organic upper layer was transferred to − ◦ a clean tube and evaporated to dryness under a gentle stream of nitrogen. Then, the dried extract was reconstituted in 150 µL of mobile phase, and a 3 µL aliquot of the reconstituent was injected into a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system. Pharmacokinetic parameters, such as the area under plasma concentration-time curve from zero to infinity (AUC), were calculated from plasma concentration vs time curves using non-compartment analysis with WinNonlin (version 5.1; Pharsights, Cary, NC, USA). The AUC ratios (i.e., ratios of kidney AUC to plasma AUC) were calculated by dividing the AUC of the three SGLT2 inhibitors in the kidney by the plasma AUC values of the three SGLT2 inhibitors.

2.4. Protein Binding The protein binding of DWP16001, dapagliflozin, and ipragliflozin (1000 ng/mL) in mouse plasma and 20% kidney homogenate was determined using a rapid equilibrium dialysis kit (ThermoFisher Scientific Korea, Seoul, Korea) according to the manufacturer’s instructions. Briefly, 100 µL of mouse plasma and 20% kidney homogenate samples containing 1000 ng/mL of DWP16001, dapagliflozin, or ipragliflozin were added to the sample chamber of a semipermeable membrane (molecular weight cut-off 8000 Da) and 300 µL of phosphate buffered saline (PBS) was added to the outer buffer chamber. Pharmaceutics 2020, 12, 268 4 of 16

Four hours after incubation at 37 ◦C on a shaking incubator at 300 rpm, aliquots (50 µL) were collected from both the sample and buﬀer chambers and treated with equal volumes of fresh PBS and blank plasma or blank kidney homogenate, respectively, to match the sample matrices. The matrix-matched sample (100 µL) was added 100 µL of aqueous solution of D4-DWP16001 (IS, 20 ng/mL), and vigorously mixed with 1000 µL MTBE for 15 min. After centrifugation at 16,000 g for 5 min, samples were kept for 1 h at 80 C. An organic upper layer was transferred to a clean tube and evaporated to dryness under − ◦ a gentle stream of nitrogen. Then, the dried extract was reconstituted in 300 µL of mobile phase and a 3 µL aliquot of the reconstituent was injected into the LC-MS/MS system. Plasma protein binding was calculated using the following Equation (1) [6,7].

Drug concentration in buﬀer chamber Undiluted free drug fraction (fu) = (1) Drug concentration in plasma sample chamber

Kidney protein binding was calculated using the following equations, Equations (2) and (3), and a dilution factor (D as a value of 5) for tissue homogenates was used since we used 20% kidney homogenates [6,7].

Drug concentration in buﬀer chamber Diluted free drug fraction (fu ) = , (2) 0 Drug concentration in kidney homogenate chamber

1/D fu0 0.2 Undiluted free drug fraction (fu) = = × . (3) 1 1 + 1/D 1 fu0 0.8 fu0 − − × 2.5. Substrate Specificity of DWP16001, Dapagliflozin, and Ipragliflozin for OAT1 and OAT3 HEK293-mock cells and HEK293 cells overexpressing OAT1 and OAT3 transporters (HEK293-OAT1 and -OAT3, respectively) were seeded in poly-D-lysine-coated 24-well plates at a density of 4 105 cells/well and cultured for 24 h in DMEM supplemented with 10% FBS and 5 mM non-essential × amino acids at 37 ◦C in 8% CO2 condition. For each experiment, the growth medium was discarded after 24 h, and the attached cells were washed with pre-warmed HBSS and incubated with pre-warmed HBSS for 20 min at 37 ◦C. To confirm the functionality of OAT1 and OAT3, we measured the uptake of 0.1 µM[3H]PAH and 0.1 µM [3H]ES, representative substrates for OAT1 and OAT3, respectively, into in the HEK293-mock cells and HEK293-OAT1 and -OAT3 cells, respectively, for 5 min in the presence and absence of 20 µM probenecid, a typical inhibitor for both OAT1 and OAT3 [8,9]. The cells were then washed three times with 500 µL of ice-cold HBSS immediately after placing the plates on ice. Subsequently, cells were lysed with 10% sodium dodecyl sulfate and mixed with Optiphase cocktail solution overnight. The radioactivity of the cell lysate was measured using a liquid scintillation counter (Microbeta 2; Perkin Elmer Inc., Boston, MA, USA). The uptake of DWP16001, dapagliflozin, and ipragliflozin (2 µM each) was measured for 5 min at 37 ◦C in the HEK293-mock cells and HEK293-OAT1 and -OAT3 cells, respectively, in the absence and presence of 20 µM probenecid. For the concentration dependency in the uptake of DWP16001, the uptake of DWP16001 in a concentration range of 0.5–50 µM dissolved in HBSS was measured for 5 min at 37 ◦C in the mock cells and HEK293-OAT1 and -OAT3 cells, respectively. After 5 min, the cells were washed three times with 500 µL of ice-cold HBSS immediately after placing the plates on ice. Subsequently, the cells were scraped using a cell scraper with 100 µL of PBS, and cell suspensions were transferred to a clean tube, combined with 100 µL of aqueous solution of D4-DWP16001 (IS, 20 ng/mL), and vigorously mixed with 1000 µL MTBE for 15 min. After centrifugation at 16,000 g for 5 min, samples were kept for 1 h at 80 C. An organic upper layer was transferred to a clean tube and − ◦ evaporated to dryness under a gentle stream of nitrogen. Then, the dried extract was reconstituted in 300 µL of mobile phase and a 3 µL aliquot of the reconstituent was injected into the LC-MS/MS system. Pharmaceutics 2020, 12, 268 5 of 16

In the concentration-dependent uptake studies, the transporter-mediated uptake of DWP16001 was calculated by the subtraction of the transport rates of DWP16001 into the mock cells from those of the HEK293-OAT1 and -OAT3 cells. Kinetic parameters for the OAT1- and OAT3-mediated transport of DWP16001 were determined using the Michaelis-Menten equation [V = Vmax S/(Km + S)] [10]. · 2.6. LC-MS/MS Analysis of DWP16001, Dapagliflozin, and Ipragliflozin Concentrations of DWP16001, dapagliflozin, and ipragliflozin in plasma and kidney homogenate samples were analyzed using an Agilent 6470 triple quadrupole LC–MS/MS system (Agilent, Wilmington, DE, USA). DWP16001, dapagliflozin, and ipragliflozin were separated on a Synergi Polar RP column (2.0 150 mm, 4 µm particle size; Phenomenex, Torrence, CA) using a mobile phase consisting of × water (15%) and methanol (85%) containing 0.1% formic acid at a flow rate of 0.25 mL/min. Quantification of a separated analyte peak was performed at m/z 464 131 for DWP16001 (T → R (retention time) 2.8 min), m/z 422 151 for ipragliflozin (T 2.5 min), m/z 426 167 for dapagliflozin → R → (T 2.5 min), m/z 468 135 for D4-DWP16001 (T 2.8 min), in the positive ionization mode with a R → R collision energy (CE) of 25 eV. The calibration standards of a mixture of DWP16001, dapagliflozin, and ipragliflozin in mouse plasma were 5–1000 ng/mL, and intraday and interday precision and accuracy were less than 14.7% in all samples. The calibration standards of a mixture of DWP16001, dapagliflozin, and ipragliflozin in mouse kidney homogenate were 5–1000 ng/mL, and intraday and interday precision and accuracy were less than 13.8% in all samples.

2.7. Inhibitory Effects of DWP16001, Dapagliflozin, and Ipragliflozin on the SGLT1 and SGLT2 Activities CHO cells overexpressing SGLT1 and SGLT2 cells (CHO-SGLT1 and -SGLT2) and CHO-mock cells were characterized as previously described [11]. Cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum and 200 µg/mL G418 at 37 ◦C in 5% CO2 conditions. CHO-SGLT1 and -SGLT2 cells were seeded at a density of 1 105 cells/well in 96-well plates. After × 24 h, the growth medium was discarded from the cells, and the cells were washed with pre-warmed + Na -free buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM + + + MgCl2, pH7.4) and incubated for 1 h in Na -free buffer. After replacing Na -free buffer with Na gradient buffer (10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, pH7.4) containing 10 µM[14C]AMG, the uptake of [14C]AMG into the CHO-mock cells and CHO-SGLT1 and -SLGT2 cells was measured for 0.5, 1, 1.5, 2, and 3 h. After a predetermined incubation time, cells were washed three times with 200 µL of ice-cold Na+-free buffer immediately after placing the plates on ice. Then, the cells were lysed with 10% SDS, and the cell lysates were mixed with Optiphase cocktail solution. Thereafter, the radioactivity of the cell lysates was measured using a liquid scintillation counter. The inhibitory effect of known inhibitors, such as dapagliflozin and ipragliflozin, on [14C]AMG uptake in the CHO-mock cells and CHO-SGLT1 and -SLGT2 cells was measured in the presence or absence of dapagliflozin and ipragliflozin (1, 10 µM for SGLT1; 10, 100 nM for SGLT2) for 2 h. For the 14 calculation of IC50 values, the uptake of 10 µM[ C]AMG in the CHO-mock cells and CHO-SGLT1 and -SLGT2 cells was measured for 2 h with or without DWP16001, dapagliflozin, or ipragliflozin (1 nM–50 µM for SGLT1; 0.01 nM–1 µM for SGLT2). After 2 h, cells were washed three times with 200 µL of ice-cold Na+-free buffer and the cells were lysed with 10% SDS (40 µL), followed by adding Optiphase cocktail solution (200 µL). The radioactivity of the cell lysates was measured using a liquid scintillation counter. The SGLT1 or SGLT2-mediated uptake of [14C]AMG was calculated by the subtraction of the uptake rates of [14C]AMG into the mock cells from those of the CHO-SGLT1 and -SLGT2 cells. In the inhibition studies, the percentages of the transport rate of AMG with or without SGLT2 inhibitors were calculated and the data were fitted to an inhibitory effect model. The IC50 (the concentration of the inhibitor to show half-maximal inhibition) values were calculated using Sigma Plot ver.10.0 (Systat Software, Inc.; San Jose, CA, USA) [12]. Pharmaceutics 2020, 12, 268 6 of 16

To investigate time dependency in the inhibition of SGLT2, CHO-mock and -SGLT2 cells were seeded at a density of 1 105 cells/well in 96-well plates. After 24 h, the growth medium was discarded × from the cells, and the cells were washed with pre-warmed Na+-free buffer and pre-incubated with Na+-free buffer containing various concentrations of DWP16001, dapagliflozin, or ipragliflozin (0.001 nM–100 nM) for 1 and 2 h. Then, after replacing Na+-free buffer with Na+ gradient buffer containing 10 µM[14C]AMG and various concentrations of DWP16001, dapagliflozin, or ipragliflozin (0.001 nM–100 nM), the uptake of [14C]AMG into the CHO-mock and -SGLT2 cells was measured for 2 h. After 2 h of incubation, the radioactivity of the cell lysate was measured following the same sample preparation method described above. To investigate the mode of inhibition of the three SGLT2 inhibitors, the inhibition experiments were initiated by replacing Na+-free buffer with Na+ gradient buffer containing 1, 2.5, 5, and 50 µM[14C]AMG and various concentrations of DWP16001, dapagliflozin, or ipragliflozin (0.001 nM–250 nM) and the uptake of [14C]AMG into the CHO-mock and -SGLT2 cells was measured for 2 h. The radioactivity of the cell lysate was measured following the same sample preparation method described above. Uptake rate of AMG and concentrations of DWP16001, dapagliflozin, or ipragliflozin were plotted to Dixon plots to identify the mode of inhibition [13,14]. To investigate the recovery of SGLT2 activity depending on the washout period after 24 h exposure of DWP16001, dapagliflozin, or ipragliflozin, CHO-SGLT2 cells were seeded at a density of 1 105 cells/well in 96-well plates. After 24 h, the growth medium was discarded from the cells, and the × cells were treated with RPMI1640 medium containing DWP16001, dapagliflozin, or ipragliflozin (0.2, 2, 20, and 200 nM) for 24 h. After 24 h, the RPMI1640 medium was replaced with pre-warmed fresh RPMI1640 medium and incubated for 1, 2, 3, 5, and 6 h, and proceeded another pre-incubation with Na+-free buffer for 1 h. Then, after replacing Na+-free buffer with Na+ gradient buffer containing 10 µM[14C]AMG, the uptake of [14C]AMG into the CHO-SGLT2 cells was measured for 2 h. After 2 h of incubation, the radioactivity of the cell lysate was measured following the same sample preparation method described above.

2.8. Statistics The statistical signiﬁcance was assessed by t-test using SPSS for Windows (version 24.0; IBM Corp., Armonk, NY, USA).

3. Results

3.1. LC-MS/MS Analysis of DWP16001, Dapagliflozin, and Ipragliflozin To compare the pharmacokinetics and tissue distribution of DWP16001, dapagliflozin, ipragliflozin in mice, analyses of the three SGLT2 inhibitors using LC-MS/MS were applied. Figure2 shows the selected precursor and product ions of DWP16001, dapagliflozin, ipragliflozin, and D4-DWP16001 (IS). The selected precursor and product ions of dapagliflozin and ipragliflozin were consistent with previously published findings [12,15]. Representative multiple reaction-monitoring (MRM) chromatograms of DWP16001, D4-DWP16001 (IS), dapagliflozin, and ipragliflozin (Figure3) showed that all the analyte peaks obtained using the liquid-liquid extraction method using MTBE were well separated with no interfering peaks at their respective retention times. Pharmaceutics 2020, 12, x 7 of 17

Pharmaceutics 2020, 12, 268 7 of 16 Pharmaceutics 2020, 12, x 7 of 17

Figure 2. Product ion spectra of (A) DWP16001, (B) D4-DWP16001 (IS), (C) dapagliflozin, and (D) ipragliflozin.

Representative multiple reaction-monitoring (MRM) chromatograms of DWP16001, D4-DWP16001 (IS), dapagliflozin, and ipragliflozin (Figure 3) showed that all the analyte peaks obtained using the liquid-liquid extraction method using MTBE were well separated with no Figure 2. ProductProduct ion ion spectra spectra of of(A ()A DWP16001,) DWP16001, (B) ( BD4-DWP16001) D4-DWP16001 (IS), (IS), (C) (dapagliflozin,C) dapagliﬂozin, and and(D) interfering peaks at their respective retention times. (ipragliflozin.D) ipragliﬂozin.

Figure 3. A Figure 3.Representative Representative multiple multiple reaction-monitoring reaction-monitoring (MRM)(MRM) chromatograms of of ( (A)) DWP16001, DWP16001, (B) (B) D4-DWP16001 (IS), (C) dapagliflozin, and (D) ipragliflozin in mouse double-blank plasma, blank D4-DWP16001 (IS), (C) dapagliflozin, and (D) ipragliflozin in mouse double-blank plasma, blank plasma spiked with DWP16001, dapagliflozin, ipragliflozin at the lower limit of quantification (LLOQ) plasma spiked with DWP16001, dapagliflozin, ipragliflozin at the lower limit of quantification (5 ng/mL), and plasma samples at 1 h following single oral administration of DWP16001, dapagliflozin, (LLOQ) (5 ng/mL), and plasma samples at 1 h following single oral administration of DWP16001, or ipragliflozin at a dose of 1 mg/kg. dapagliflozin, or ipragliflozin at a dose of 1 mg/kg. .

Figure 3. Representative multiple reaction-monitoring (MRM) chromatograms of (A) DWP16001, (B) D4-DWP16001 (IS), (C) dapagliflozin, and (D) ipragliflozin in mouse double-blank plasma, blank plasma spiked with DWP16001, dapagliflozin, ipragliflozin at the lower limit of quantification (LLOQ) (5 ng/mL), and plasma samples at 1 h following single oral administration of DWP16001, dapagliflozin, or ipragliflozin at a dose of 1 mg/kg. .

Pharmaceutics 2020, 12, x 8 of 17

3.2. Pharmacokinetics and Kidney Distribution of DWP16001, Dapagliflozin, and Ipragliflozin in Mice To compare the pharmacokinetic profile of DWP16001 with those of dapagliflozin and ipragliflozin, the same dose of the three SGLT2 inhibitors was orally administered to ICR mice (1 mg/kg each), and the concentrations of DWP16001, dapagliflozin, and ipragliflozin in plasma and kidney samples were analyzed. The PK profiles of DWP16001, dapagliflozin, and ipragliflozin are Pharmaceuticsshown in Figure2020, 12 ,4 268 and the PK parameters were summarized in Table 1. 8 of 16 As shown in Figure 4, plasma concentrations of DWP16001 were similar to those of ipragliflozin and greater than those of dapagliflozin. Consequently, the AUC and Cmax values of DWP16001 were 3.2. Pharmacokinetics and Kidney Distribution of DWP16001, Dapagliflozin, and Ipragliflozin in Mice similar to those of ipragliflozin and higher than those of dapagliflozin. However, the concentrations of DWP16001To compare in the the pharmacokinetic kidney were maintained profile of DWP16001at higher concentrations with those of dapagliflozin for 72 h compared and ipragliflozin, with those theof ipragliflozin same dose of and the dapagliflozin, three SGLT2 suggesting inhibitors wasthe prolonged orally administered efficacy of toDWP16001 ICR mice via (1 mgthe /inhibitionkg each), andof SGLT2, the concentrations which is located of DWP16001, in the renal dapagliflozin, proximal andtubule ipragliflozin [3]. Because in plasmaof the andhigh kidney and prolonged samples wereconcentration analyzed. of The DWP16001, PK profiles the of AUC, DWP16001, AUC dapagliflozin,ratio, and t1/2 values and ipragliflozin of DWP16001 are shownwere significantly in Figure4 andgreater the PKthan parameters those of dapagliflozin were summarized and ipragliflozin in Table1. (Table 1).

(A) Plasma (B) Kidney

1000 10000

DWP16001 Ipragliflozin Dapagliflozin 100 1000

10 100

Concentration (ng/mL) DWP16001 Concentration (ng/g tissue) Ipragliflozin Dapagliflozin 1 10 0 5 10 15 20 25 0 10203040506070 Time (h) Time (h) Figure 4. (A) Plasma and (B) kidney concentration vs. time profile of DWP16001 ( ), dapagliflozin (H), • andFigure ipragliflozin 4. (A) Plasma ( ) after and a(B single) kidney oral concentration administrations vs. oftime DWP16001, profile of dapagliflozin,DWP16001 (● and), dapagliflozin ipragliflozin (▼), atand a dose ipragliflozin of 1 mg/ #kg(○ in) after Institute a single of Canceroral administrations Research (ICR) of mice,DWP16001, respectively. dapagliflozin, Data are and expressed ipragliflozin as at mean SD from five mice. a dose± of 1 mg/kg in Institute of Cancer Research (ICR) mice, respectively. Data are expressed as Tablemean±SD 1. Pharmacokinetic from five mice. parameters of DWP16001, dapagliflozin, and ipragliflozin after a single oral administrations of DWP16001, dapagliflozin, and ipragliflozin at a dose of 1 mg/kg in ICR mice,Table respectively. 1. Pharmacokinetic parameters of DWP16001, dapagliflozin, and ipragliflozin after a single oral administrations of DWP16001, dapagliflozin, and ipragliflozin at a dose of 1 mg/kg in ICR mice, respectively. Parameters DWP16001 Dapagliflozin Ipragliflozin Cmax (ng/mL) 371.4 108.6 274.8 143.6 409.6 59.1 Parameters DWP16001± Dapagliflozin± Ipragliflozin± Tmax (h) 0.7 0.3 0.5 0.0 0.6 0.2 ± ± ± Plasma CmaxAUC (ng/mL)(µg h/mL) 371.4 1.69 ± 0.34108.6 0.48 274.80.18 ± 143.6 * 1.96 409.60.41 ± 59.1 72h · ± ± ± AUCTmax (h)(µ g h/mL) 1.73 0.7 ± 0.340.3 0.55 0.5143.8 ± 0.0 * 1.97 0.60.41 ± 0.2 ∞ · ± ± ± t (h) 3.8 1.4 2.1 0.2 * 3.1 0.5 Plasma AUC72h (µg1/∙2h/mL) 1.69 ±± 0.34 0.48± ± 0.18 * ± 1.96 ± 0.41 AUCAUC∞ 72h(µg(∙µh/mL)g h/g tissue) 139.2 1.73 ± 0.345.19 26.30 0.55 ±3.34 143.8 * * 73.65 1.977.05 ± *0.41 · ± ± ± Kidney AUC ratio 85.0 16.1 64.6 31.8 38.4 5.3 * t1/2 (h) 3.8 ±± 1.4 2.1± ± 0.2 * ± 3.1 ± 0.5 t1/2 (h) 125.5 80.4 24.9 5.4 * 24.3 5.7 * AUC72h (µg∙h/g tissue) 139.2 ±± 5.19 26.30± ± 3.34 * 73.65± ± 7.05 * Area under curve (AUC) ratio: Ratios of Kidney AUC to plasma AUC; Data expressed as mean SD from five mice; Kidney*: p < 0.05, compared withAUC DWP16001 ratio group. 85.0 ± 16.1 64.6 ± 31.8 ± 38.4 ± 5.3 * t1/2 (h) 125.5 ± 80.4 24.9 ± 5.4 * 24.3 ± 5.7 * Area under curve (AUC) ratio: Ratios of Kidney AUC to plasma AUC; Data expressed as mean ± SD As shown in Figure4, plasma concentrations of DWP16001 were similar to those of ipragliflozin from five mice; * : p < 0.05, compared with DWP16001 group. and greater than those of dapagliflozin. Consequently, the AUC and Cmax values of DWP16001 were similar to those of ipragliflozin and higher than those of dapagliflozin. However, the concentrations of DWP16001 in the kidney were maintained at higher concentrations for 72 h compared with those of ipragliflozin and dapagliflozin, suggesting the prolonged efficacy of DWP16001 via the inhibition of SGLT2, which is located in the renal proximal tubule [3]. Because of the high and prolonged concentration of DWP16001, the AUC, AUC ratio, and t1/2 values of DWP16001 were significantly greater than those of dapagliflozin and ipragliflozin (Table1). Pharmaceutics 2020, 12, 268 9 of 16

3.3. Substrate Specificity of DWP16001, Dapagliflozin, and Ipragliflozin for OAT1 and OAT3 The AUC ratios of these three SGLT2 inhibitors were much greater than unity, suggesting that DWP16001, dapagliflozin, and ipragliflozin are distributed in the kidney, in which tissue protein binding ability to the specific protein or drug transporters may be involved. Therefore, firstly, we measured the protein binding of DWP16001, dapagliflozin, and ipragliflozin in the plasma and kidney homogenate. The plasma and kidney tissue protein binding of DWP16001, dapagliflozin, and ipragliflozin were high and comparable and the logP values of three compound were also comparable (i.e., in the range of 2.27–2.65) (Table2). It suggests that the large distribution of DWP16001, dapagliflozin, and ipragliflozin in the kidney was not necessarily reliant on plasma/tissue-specific binding ability or lipophilicity of these compounds.

Table 2. Protein binding of DWP16001, dapagliﬂozin, and ipragliﬂozin in mouse plasma and kidney homogenates.

Protein Binding (% Bound) b Compounds logP a Plasma Kidney DWP16001 2.65 98.50 0.20 97.24 0.08 ± ± Dapagliflozin 2.27 94.19 1.07 95.83 0.68 ± ± Ipragliflozin 2.56 96.58 0.75 96.50 1.01 ± ± a logP values were obtained from the partition coefficient of n-octanol/water; b Data expressed as mean SD of triplicate experiments. ±

We conducted further investigations into the tissue-specific transport of DWP16001, dapagliflozin, and ipragliflozin by using cell systems overexpressing OAT1 and OAT3 since OAT1 and OAT3 are exclusively distributed in the kidney and contribute to the drug distribution to the kidney [16]. The functionality of the OAT1 and OAT3 transport system was confirmed by the significantly greater uptake rates of the probe substrates in HEK293-OAT1 and -OAT3 cells than that in HEK293-mock cells (i.e., 14.7-fold increase in PAH uptake for OAT1 and 16.4-fold increase in ES uptake for OAT3) and by the decreased uptake rate of each probe substrate by the addition of a representative inhibitor, probenecid (Figure5A,B) [ 17]. The uptake of DWP16001 in HEK293-OAT1 and OAT3 cells was significantly higher than that in HEK293-mock cells, and it was significantly decreased by the presence of probenecid (Figure5C). However, dapagliflozin and ipragliflozin were not substrates for both OAT1 and OAT3 (Figure5D,E). Next, we investigated the concentration dependency in the OAT1 and OAT3-mediated DWP16001 uptake. As shown in the figure, the OAT1 and OAT3-mediated uptake of DWP16001 showed saturable kinetics, and the kinetic parameters, such as Km and Vmax, calculated from the concentration-dependent uptake of DWP16001 in HEK293-OAT1 and –OAT3 cells are shown in Figure6. Collectively, OAT1 and OAT3-mediated uptake of DWP16001 could contribute to the highest kidney distribution of DWP16001 among three SGLT2 inhibitors. However, these processes were not dominant over the cellular uptake of the three SGLT2 inhibitors into HEK293-mock cells (i.e., passive diffusion), which is evident from two-fold increases in HEK293-OAT1 or -OAT3 cells compared with mock cells (Figure5C). Therefore, OAT1- and OAT3-mediated active transport and passive diffusion all contributed to the highly permeable absorptive process of DWP16001 in the kidney, and dapagliflozin and ipragliflozin also showed higher uptake levels into HEK293 cells via passive diffusion. Pharmaceutics 2020, 12, 268 10 of 16 Pharmaceutics 2020, 12, x 10 of 17

25 25 Control (A) Control * (B) Probecid * Probecid 20 Pharmaceutics 2020, 12, x 20 10 of 17

15 15 25 25 Control (A) Control * (B) Probecid * Probecid 10 20 10 20

15 15 5 ES uptake (pmol/min) 5 PAHuptake (pmol/min) + + 10 10 0 0 mock OAT1 mock OAT3

5 ES uptake (pmol/min) 5 PAHuptake (pmol/min) + + 15 15 15 0 0 Control Control Control mock OAT1(C) mock OAT3 (D) (E) Probecid Probenecid Probenecid

15 * 15 15 10 Control * (C) 10 Control (D) 10 Control (E) Probecid Probenecid Probenecid + * 10 +* 10 10 5 + 5 5 + 5 5 5 DWP16001 uptake (pmol/min) Ipragliflozin uptake (pmol/min) Dapagliflozin uptake (pmol/min) 0 0 0 DWP16001 uptake (pmol/min) Ipragliflozin uptake (pmol/min) mock OAT1 OAT3 Dapagliflozin uptake (pmol/min) mock OAT1 OAT3 mock OAT1 OAT3 0 0 0 mock OAT1 OAT3 mock OAT1 OAT3 mock OAT1 OAT3 Figure 5. (A) Uptake of 0.1 µM[3H]para-aminohippuric acid (PAH) was measured in HEK293-mock Figure 5. (A) Uptake of 0.1 µM [3H]para-aminohippuric acid (PAH) was measured in HEK293-mock 3 µ µ and –OAT1and –OAT1Figure cells cells in5. (A the ) inUptake presencethe presence of 0.1 µM and and[ H]para-aminohippuric absence absence of of 20 20 µMM acid probenecidprobenecid (PAH) was for formeasured 5 5min. min. in(B HEK293-mock ()B Uptake) Uptake of 0.1 of µM 0.1 M 3 and –OAT1 cells in the presence and absence of 20 µM probenecid for 5 min. (B) Uptake of 0.1 µM [ H]Estrone-3-sulfate[3H]Estrone-3-sulfate (ES) (ES) was measuredwas measured in HEK293-mock in HEK293-mock and and –OAT3 –OAT3 cells cells in the in presencethe presence and absenceand [3H]Estrone-3-sulfate (ES) was measured in HEK293-mock and –OAT3 cells in the presence and of 20 µabsenceM probenecid. of 20 µM Uptakeprobenecid. of ( CUptake) DWP16001, of (C) DWP16001, (D) dapagliﬂozin, (D) dapagliflozin, and (E) and ipragliﬂozin (E) ipragliflozin (2 µM (2 each) absence of 20 µM probenecid. Uptake of (C) DWP16001, (D) dapagliflozin, and (E) ipragliflozin (2 into HEK293-mockµM each)µM each)into andHEK293-mockinto HEK293-mock HEK293 cells and and expressingHEK293 HEK293 cellscells OAT1 expressingpressing and OAT3OAT1 OAT1 and was and OAT3 measured OAT3 was measuredwas for measured 5 min.for 5 Each for 5 data point representsmin. Eachmin. dataEach the datapoint mean point representsstandard represents the deviationthe mean±stand mean±stand of triplicateard deviationdeviation experiments. of oftriplicate triplicate experiments. *: experiments.p < 0.05, *: p compared< 0.05,*: p < 0.05, with ± mockcompared cells; +compared: p with< 0.05, withmock compared mock cells; cells; +: p +: with< p 0.05, < 0.05, control compared compared group. with control control group. group. (A) (A) (B)(B) 100 200 100 200

75 150 75 150

50 100

Vmax 267.9 pmol/min 25 Vmax 119.2 pmol/min 50 μ K 31.1 μM OAT1-mediated uptake K 38.0 M m OAT3-mediated uptake of DWP16001 (pmol/min) of DWP16001 m V 119.2 pmol/min of DWP16001 (pmol/min) Vmax 267.9μ pmol/min 25 maxCL 3.1 μL/min 50 CLint 8.6 L/min int μ 0 μ 0 K 31.1 M OAT1-mediated uptake K 38.0 M m OAT3-mediated uptake of DWP16001 (pmol/min) of DWP16001 m 0 102030405060 of DWP16001 (pmol/min) 0 102030405060μ CL 3.1 μL/min CLint 8.6 L/min int μ μ 0 DWP16001 ( M) 0 DWP16001 ( M) 0 102030405060 0 102030405060 Figure 6. (AFigure) OAT1- 6. (A and) OAT1- (B) and OAT3-mediated (B) OAT3-mediated uptake uptake ofof DWP16001. DWP16001. Concentration Concentration dependent dependent uptake uptake of DWP16001 was measured for 5 min into HEK293-mock cells and HEK293 cells expressing OAT1 of DWP16001 was measuredDWP16001 for 5 ( minμM) into HEK293-mock cells and HEK293DWP16001 cells expressing (μM) OAT1 and and OAT3. The transporter-mediated uptake rate was obtained by subtracting the uptake in OAT3.Figure The transporter-mediatedHEK293-mock 6. (A) OAT1- cells and (passive (B) OAT3-mediated uptake diffusion rate) from was thoseuptake obtained in HEK293 of DWP16001. by cells subtracting expressing Concentration OAT1 the uptake and dependent OAT3 in (total HEK293-mock uptake cells (passiveof DWP16001uptake). diffusion) Eachwas datameasured from point those represents for in5 min HEK293 the into means HEK293- cells ± standard expressingmock deviation cells OAT1and from HEK293 triplicate and OAT3 cellsexperiments. expressing (total uptake). OAT1 Each data pointand OAT3. represents The thetransporter-mediated means standard uptake deviation rate fromwas triplicateobtained by experiments. subtracting the uptake in 3.4. Inhibition Potential of DWP16001,± Dapagliflozin, Ipragliflozin on SGLT1 and SGLT2 Activities HEK293-mock cells (passive diffusion) from those in HEK293 cells expressing OAT1 and OAT3 (total 3.4. Inhibitionuptake). Potential Each data of DWP16001, point represents Dapagliflozin, the means ± standard Ipragliflozin deviation on SGLT1from triplicate and SGLT2 experiments. Activities

3.4.1. Comparison3.4. Inhibition Potential of IC50 Valuesof DWP16001, of DWP16001, Dapagliflozin, Dapagliﬂozin, Ipragliflozin andon SGLT Ipragliﬂozin1 and SGLT2 on Activities SGLT1 and SGLT2

To conﬁrm the functionality of CHO-SGLT1 cells, we measured the time-dependent uptake of AMG, a representative substrate for SGLT1 [11,18]. As shown in Figure7A, AMG uptake was increased with incubation time, and the optimal incubation time was selected as 2 h from the linear phase of the slope. AMG uptake into CHO-SGLT1 cells was 42-fold greater than that into CHO-mock cells and Pharmaceutics 2020, 12, x 11 of 17

3.4.1. Comparison of IC50 Values of DWP16001, Dapagliflozin, and Ipragliflozin on SGLT1 and SGLT2 To confirm the functionality of CHO-SGLT1 cells, we measured the time-dependent uptake of AMG, a representative substrate for SGLT1 [11,18]. As shown in Figure 7A, AMG uptake was Pharmaceuticsincreased2020, 12with, 268 incubation time, and the optimal incubation time was selected as 2 h from the linear11 of 16 phase of the slope. AMG uptake into CHO-SGLT1 cells was 42-fold greater than that into CHO-mock cells and significantly inhibited by the presence of known inhibitors, such as significantlydapagliflozin inhibited and by ipragliflozin the presence [3,5] of known (Figure inhibitors, 7B). With such the as same dapagliflozin experimental and ipragliflozincondition, the [3 ,5] (Figureinhibitory7B). With theeffect same of experimentalDWP16001, dapagliflozin, condition, the and inhibitory ipragliflozin e ffect on of DWP16001,the AMG uptake dapagliflozin, into and ipragliflozinCHO-SGLT1 on cells the AMGwas measured uptake into in a CHO-SGLT1 concentration cells range was of measured1–50,000 nM in aand concentration IC50 values rangewere of calculated (Figure 7C–E; Table 3). 1–50,000 nM and IC50 values were calculated (Figure7C–E; Table3).

20000 6000 (A) (B) 5000 15000 4000

10000 3000

2000

5000 (pmol) uptake AMG AMG uptakeAMG (pmol) * 1000 * * * 0 0 l 01234 ck T1 o M M M M o L tr μ μ μ μ m G on 1 0 1 0 S c + +1 + +1 Time (h) X Data Dapagliflozin Ipragliflozin

120 120 120 (C) (D) (E)

80 80 80 % of control) %

40 40 40 uptake ( SGLT1-mediated AMG

0 0 0 100 101 102 103 104 105 100 101 102 103 104 105 100 101 102 103 104 105 DWP16001 (nM) Dapagliflozin (nM) Ipragliflozin (nM) FigureFigure 7. (A )7. Uptake (A) Uptake of 10 ofµ 10M[ µM14C]methyl-a-D-glucopyranoside [14C]methyl-a-D-glucopyranoside (AMG) (AMG) was was measured measured in CHO- in CHO- sodium-glucosesodium-glucose cotransporter cotransporter 1 (SGLT1) 1 (SGLT1) cells cells with with a variousa various incubation incubation time (0.5–3 (0.5–3 h). h). (B ()B Inhibitory) Inhibitory effect ofeffect dapagliflozin of dapagliflozin and ipragliflozinand ipragliflozin (1, (1, 10 10µM) µM) on on the the uptake uptake ofof 1010 µµMM[ [14C]AMGC]AMG in inCHO-mock CHO-mock and -SGLT1and -SGLT1 cells wascells measuredwas measured for 2for h. 2 h. Concentration Concentration dependentdependent inhibition inhibition of ofDWP16001 DWP16001 (C), ( C), dapagliflozin (D), and ipragliflozin (E) on the SGLT1-mediated uptake of [14C]AMG. dapagliflozin (D), and ipragliflozin (E) on the SGLT1-mediated uptake of [14C]AMG. SGLT1-mediated SGLT1-mediated uptake of [14C]AMG was calculated by subtracting the uptake of 10 µM [14C]AMG uptake of [14C]AMG was calculated by subtracting the uptake of 10 µM[14C]AMG for 2 h in CHO-mock for 2 h in CHO-mock cells from that in CHO-SGLT1 cells in a concentration range of 1–50,000 nM of cells from that in CHO-SGLT1 cells in a concentration range of 1–50,000 nM of DWP16001, dapagliflozin, DWP16001, dapagliflozin, and ipragliflozin. Each data point represents the mean ± standard and ipragliflozin. Each data point represents the mean standard deviation of three independent deviation of three independent experiments. *: p < 0.05, ±compared with control group. experiments. *: p < 0.05, compared with control group. Similarly, the functionality of CHO-SGLT2 cells was confirmed from the time-dependent Table 3. Inhibition potential of DWP16001, dapagliflozin, and ipragliflozin on SGLT1 and SGLT2 activities. uptake of AMG, a representative substrate for SGLT2 [3,5]. As shown in Figure 8A, AMG uptake

was increased with incubation time,IC 50and(nM) the optimal incubation time Selectivity was selected as 2 h from the linear phase of the slope. AMG uptake into CHO-SGLT1 cells was 9.8-fold greater than that into SGLT1 SGLT2 (IC50 Ratio of SGLT1/SGLT2) CHO-mock cells and significantly inhibited by the presence of known inhibitors, such as DWP16001 549.3 139.6 0.8 0.3 667 dapagliflozin and ipragliflozin± [3] (Figure 8B).± With the same experimental conditions, the Ipragliﬂozin 2328.7 377.6 8.9 1.7 261 ± ± inhibitoryDapagliﬂozin effect of DWP16001, 803.0 96.8 dapagliflozin, 1.6 0.3 and ipragliflozin 493on the AMG uptake into ± ± Data were expressed as mean SD from triplicate measurement. ±

Similarly, the functionality of CHO-SGLT2 cells was confirmed from the time-dependent uptake of AMG, a representative substrate for SGLT2 [3,5]. As shown in Figure8A, AMG uptake was increased with incubation time, and the optimal incubation time was selected as 2 h from the linear phase of the slope. AMG uptake into CHO-SGLT1 cells was 9.8-fold greater than that into CHO-mock cells and significantly inhibited by the presence of known inhibitors, such as dapagliflozin and ipragliflozin [3] (Figure8B). With the same experimental conditions, the inhibitory e ffect of DWP16001, dapagliflozin, and ipragliflozin on the AMG uptake into CHO-SGLT2 cells was measured in a concentration range of 0.001–100 nM, and IC50 values were calculated (Figure8C–E; Table3). Pharmaceutics 2020, 12, x 12 of 17 Pharmaceutics 2020, 12, 268 12 of 16 CHO-SGLT2 cells was measured in a concentration range of 0.001–100 nM, and IC50 values were calculated (Figure 8C–E; Table 3). DWP16001DWP16001 showed showed greater greater affi affinitynity for for SGLT2 SGLT2 comparedcompared with with dapagliflozin dapagliflozin and and ipragliflozin. ipragliflozin. Moreover,Moreover, the a ffithenity affinity to SGLT1 to SGLT1 was was also also greater greater with with DWP16001DWP16001 compared compared with with dapagliflozin dapagliflozin and and ipragliflozin. When compared with selectivity, which was calculated from the SGLT1/SGLT2 IC50 ipragliflozin. When compared with selectivity, which was calculated from the SGLT1/SGLT2 IC50 ratio, ratio, the selectivity of DWP16001 was high relative to that of dapagliflozin and ipragliflozin (Table 3). the selectivity of DWP16001 was high relative to that of dapagliflozin and ipragliflozin (Table3).

4000 1400 (A) (B) 1200

3000 1000

800 2000 600

400 * AMG uptake (pmol) uptake AMG 1000 (pmol) uptake AMG * * * 200

0 0 01234 k 2 l c T ro nM nM nM nM o L nt 0 0 0 0 m SG o 1 0 1 0 Time (h) c + +1 + +1 X Data Dapagliflozin Ipragliflozin

120 120 120 (C) (D) (E)

80 80 80 % % of control) 40 40 40 uptake ( SGLT2-mediated AMG

0 0 0 -3 -2 -1 0 1 2 3 10-3 10-2 10-1 100 101 102 103 10-3 10-2 10-1 100 101 102 103 10 10 10 10 10 10 10 DWP16001 (nM) Dapagliflozin (nM) Ipragliflozin (nM) FigureFigure 8. (A) The8. (A uptake) The ofuptake 10 µM[ of 1410C]methyl-a-D-glucopyranoside µM [14C]methyl-a-D-glucopyranoside (AMG) was(AMG) measured was measured in CHO-SGLT2 in cells withCHO-SGLT2 a various incubationcells with timea various (0.5, 1,incubation 1.5, 2, 3 h). time (B) Inhibitory(0.5, 1, 1.5, effect 2, 3 of h). dapagliflozin (B) Inhibitory and effect ipragliflozin of 14 (10, 100dapagliflozin nM) on the and uptake ipragliflozin of 10 µM[ (10,14 100C]AMG nM) on in the CHO-mock uptake of 10 and µM –SGLT2 [ C]AMG cells in wasCHO-mock measured and for– 2 h. RepresentativeSGLT2 cells concentration was measured dependent for 2 h. Representative inhibition of concentration DWP16001 (C dependent), dapagliflozin inhibition (D), of and DWP16001 ipragliflozin (C), dapagliflozin (D), and ipragliflozin (E) on the SGLT2-mediated uptake of [14C]AMG. (E) on the SGLT2-mediated uptake of [14C]AMG. SGLT2-mediated uptake of [14C]AMG was calculated SGLT2-mediated uptake of [14C]AMG was calculated by subtracting the uptake of 10 µM [14C]AMG by subtracting the uptake of 10 µM[14C]AMG for 2 h in CHO-mock cells from that in CHO-SGLT2 cells for 2 h in CHO-mock cells from that in CHO-SGLT2 cells in a concentration range of 1–100 nM of in a concentrationDWP16001, dapagliflozin, range of 1–100 and nM ipragliflozin. of DWP16001, Each dapagliflozin, data point andrepresents ipragliflozin. the mean±standard Each data point represents the mean standard deviation of three independent experiments. *: p < 0.05, compared with deviation of three± independent experiments. *: p < 0.05, compared with control group. control group. Table 3. Inhibition potential of DWP16001, dapagliflozin, and ipragliflozin on SGLT1 and SGLT2 3.4.2. Modeactivities. of Inhibition

To investigate whether the inhibitionIC50 (nM) of SGLT2 was time-dependent, Selectivity the inhibitory effects of DWP16001, dapagliflozin, and ipragliflozinSGLT1 on SGLT2-mediatedSGLT2 (IC50 uptake Ratio of of SGLT1/SGLT2) AMG were measured with or without pretreatmentDWP16001 with DWP16001, 549.3 ± 139.6 dapagliflozin, 0.8 ± 0.3 and ipragliflozin, 667 respectively. Figure9 shows that the inhibitoryIpragliflozin potential of 2328.7 DWP16001, ± 377.6 dapagliflozin, 8.9 ± 1.7 and ipragliflozin 261 on SGLT2 did not change with differentDapagliflozin pretreatment times, 803.0 suggesting ± 96.8 that 1.6 ± all 0.3 the DWP16001, dapagliflozin, 493 and ipragliflozin inhibited SGLT2 functionData in a were time-independent expressed as mean manner.± SD from triplicate measurement. To3.4.2. determine Mode of the Inhibition mode of inhibition on SGLT2, the inhibitory effects of DWP16001, dapagliflozin, and ipragliflozin on the AMG uptake into CHO-SGLT2 cells were measured with different substrate concentrations, and the results were expressed as Dixon plots (Figure 10A–C) and a replot of the Dixon slopes (Figure 10D–F) [13,19,20]. Dixon transformation of inhibition profile by DWP16001 (0.001–250 nM) for different concentrations of AMG indicated competitive inhibition of DWP16001 on SGLT2 activity (Figure 10A). A replot of the Dixon slopes vs 1/S produced a straight line converging on the zero (Figure 10D), suggesting competitive and reversible inhibition of DWP16001. Similar results were shown in case of dapagliflozin and ipragliflozin (Figure 10B,C,E,F). The results collectively Pharmaceutics 2020, 12, x 13 of 17

To investigate whether the inhibition of SGLT2 was time-dependent, the inhibitory effects of DWP16001, dapagliflozin, and ipragliflozin on SGLT2-mediated uptake of AMG were measured Pharmaceutics 2020, 12, 268 13 of 16 with or without pretreatment with DWP16001, dapagliflozin, and ipragliflozin, respectively. Figure 9 shows that the inhibitory potential of DWP16001, dapagliflozin, and ipragliflozin on SGLT2 did not suggestedchange with that different the inhibition pretreatment of SGLT2 times, by DWP16001, suggesting dapagliﬂozin, that all the DWP16001, and ipragliﬂozin dapagliflozin, is reversible and andipragliflozin competitive. inhibited SGLT2 function in a time-independent manner.

(A) (B) (C)

150 150 150 IC : 0 h 1.1 nM IC : 0 h 0.7 nM 50 IC50 : 0 h 10.6 nM 50 1 h 1.0 nM 1 h 0.7 nM 1 h 11.0 nM 2 h 1.0 nM 2 h 0.8 nM 2 h 9.9 nM 100 100 100

50 50 50 AMG uptake (% of control) uptake (% AMG

0 0 0 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 DWP16001 (nM) Dapagliflozin (nM) Ipragliflozin (nM) FigureFigure 9.9. Inhibitory eeffectffect ofof ((AA)) DWP16001,DWP16001, ((BB)) dapagliflozin,dapagliflozin, andand ((CC)) ipragliflozinipragliflozin onon thethe 14 SGLT2-mediatedSGLT2-mediated uptakeuptake ofof 10 10µ M[µM [14C]methyl-a-D-glucopyranosideC]methyl-a-D-glucopyranoside (AMG)(AMG) waswas measuredmeasured withwith preincubationpreincubation time time for for 1 and 1 2and h or without2 h or preincubationwithout preincubation of DWP16001, of DWP16001, dapagliflozin, dapagliflozin, and ipragliflozin, and respectively,ipragliflozin, in respectively, a concentration in a range concentration of 0.001–100 range nM. of Each 0.001–100 data point nM. representsEach data thepoint mean representsstandard the Pharmaceutics 2020, 12, x ± 14 of 17 deviationmean±standard of three deviation independent of three experiments. independent experiments.

400 (A) 400 (B) 400 (C) To determine the mode of inhibition on SGLT2, the inhibitory effects of DWP16001, dapagliflozin,300 and ipragliflozin on the300 AMG uptake into CHO-SGLT2300 cells were measured with different substrate concentrations, and the results were expressed as Dixon plots (Figure 10A–C) 200 200 200 and a 1/V replot of the Dixon slopes (Figure1/V 10D–F) [13,19,20]. Dixon1/V transformation of inhibition profile by100 DWP16001 (0.001-250 nM) for100 different concentrations of100 AMG indicated competitive inhibition of DWP16001 on SGLT2 activity (Figure 10A). A replot of the Dixon slopes vs 1/S 0 0 0 produced a0 straight 50 100 150line 200 converging 250 300 on the0 zero 50 100(Figure 150 200 10D), 250 300 suggesting0 competitive 50 100 150 200 and 250 reversible 300 inhibition-100 of DWP16001.DWP16001 (nM) Similar results-100 wereDapagliflozin shown (nM) in case of dapagliflozin-100 Ipragliflozin and (nM) ipragliflozin (Figure 10B,C,E,F). The results collectively suggested that the inhibition of SGLT2 by DWP16001,

dapagliflozin,4 (D) and ipragliflozin is reversible4 and(E) competitive. 4 (F)

3.4.3. Retained3 Inhibition Potential of DWP160013 Compared to Dapagliflozin3 and Ipragliflozin Dixon Dixon Dixon The recovery2 of SGLT2 activities depending2 on the washout period2 after 24 h exposure of Slope Slope Slope

DWP16001,1 ipragliflozin, and dapagliflozin1 in CHO-SGLT2 cells was 1investigated. As shown in Figure 11, the activity of reduced SGLT2 by treatment with DWP16001, dapagliflozin, and 0 0 0 ipragliflozin-0.5 0.0 for 0.524 h in 1.0 CHO-SGLT2 1.5 -0.5 cells 0.0revealed 0.5 differences 1.0 1.5 in the-0.5 recovery 0.0 0.5of SGLT2 1.0 depending 1.5 on the time-1 of drug1/S removal from the medium,-1 as well1/S as SGLT2 inhibitors.-1 In the case1/S of DWP16001, the activity tended to gradually recover as the washout time increased. When treated at a high FigureFigure 10. 10.Dixon Dixon plot plot for thefor inhibitorythe inhibitory effect ofeffect (A) DWP16001, of (A) DWP16001, (B) dapagliflozin, (B) dapagliflozin, and (C) ipragliflozin and (C) concentration (200 14nM), the activity did not recover even 8 h after the drug was removed from the onipragliflozin the uptake ofon [ theC]methyl-a-D-glucopyranoside uptake of [14C]methyl-a-D-glucopyranoside (AMG) in CHO-SGLT2 (AMG) cells. in CHO-SGLT2 Each symbol representscells. Each medium (Figure 11A). 14However, when dapagliflozin and ipragliflozin were used, SGLT2 activity thesymbol concentration represents of the [ C]AMG:concentration, 1 µ ofM; [14C]AMG:, 2.5 µM; ●H, ,1 5 µM;µM; ○, ,2.5 50 µM;µM. ▼ Replot, 5 µM; of  the, 50 slopes µM. ofReplot Dixon of was recovered to the control level• 4 h after the removal of4 dapagliflozin and ipragliflozin (Figure plotthe (slopesslopes vsof 1Dixon/[S]) was plot shown (slopes for vs (D 1/[S])) DWP16001,# was shown (E) dapagliflozin, for (D) DWP16001, and (F) ( ipragliflozin.E) dapagliflozin, V indicates and (F) 11B,C). The results indicate that the14 dissociation of DWP16001 to SGLT214 is slower and more theipragliflozin. SGLT2-mediated V indicates uptake ratethe ofSGLT2-mediated [ C]AMG and S indicatesuptake rate the concentrationof [14C]AMG of and [ C]AMG. S indicates The data the incompleteareconcentration the means than ofthatstandard [14 C]AMG.of dapagliflozin deviations The data of are triplicate and the ipraglif means±standard measurements.lozin and deviations that the ofbinding triplicate affinity measurements. to SGLT2 is stronger than that± of dapagliflozin and ipragliflozin. 3.4.3. Retained Inhibition(A) Potential of DWP16001 Compared(B) to Dapagliflozin and Ipragliflozin(C) The150 recovery of SGLT2 activities depending150 on the washout period150 after 24 h exposure of DWP16001, ipragliflozin,0.2 nM and dapagliflozin in CHO-SGLT2 cells was investigated. As shown in 2 nM Figure 11, the activity20 nM of reduced SGLT2 by treatment with DWP16001, dapagliflozin, and ipragliflozin 200 nM 100 100 100 for 24 h in CHO-SGLT2 cells revealed differences in the recovery of SGLT2 depending on the time of drug removal from the medium, as well as SGLT2 inhibitors. In the case of DWP16001, the activity

50 50 50 0.2 nM 0.2 nM 2 nM 2 nM AMG AMG uptake activity)(% 20 nM 20 nM 200 nM 200 nM

0 0 0 02468 02468 02468 Time (h) Time (h) Time (h) Figure 11. Recovery of SGLT2 transport function with different washout time following 24 h of incubation with (A) DWP16001, (B) dapagliflozin, and (C) ipragliflozin (0.2–200 nM) was measured through the uptake of [14C]methyl-a-D-glucopyranoside (AMG) in CHO-SGLT2 cells. Each data point represents the mean±standard deviation of three independent experiments.

4. Discussion DWP16001 is a candidate SGLT2 inhibitor that is currently under development. As a first step, the pharmacokinetic properties and in vitro SGLT2 inhibition were compared with currently used SGLT2 inhibitors. Dapagliflozin and ipragliflozin were selected based on their high kidney distributions and long elimination half-lives (t1/2) in the kidney [5], which are thought to be important for the efficacy and duration of action of SGLT2 inhibitors. DWP16001 showed higher kidney distributions compared with dapagliflozin and ipragliflozin. DWP16001 also had longer t1/2 in the kidney than dapagliflozin and ipragliflozin, as well as a comparable plasma profile with ipragliflozin (Table 1). Moreover, the kidney concentration of DWP16001 was maintained over 72 h following oral administration of 1 mg/kg DWP16001 (Figure 4). Taken together, these

Pharmaceutics 2020, 12, x 14 of 17

400 (A) 400 (B) 400 (C)

300 300 300

200 200 200 1/V 1/V 1/V

100 100 100

0 0 0 0 50 100 150 200 250 300 0 50 100 150 200 250 300 0 50 100 150 200 250 300

-100 DWP16001 (nM) -100 Dapagliflozin (nM) -100 Ipragliflozin (nM)

4 (D) 4 (E) 4 (F)

3 3 3 Dixon Dixon Dixon 2 2 2 Slope Slope Slope

1 1 1 Pharmaceutics 2020, 12, 268 14 of 16

0 0 0 -0.5 0.0 0.5 1.0 1.5 -0.5 0.0 0.5 1.0 1.5 -0.5 0.0 0.5 1.0 1.5 tended-1 to gradually1/S recover as the washout-1 time1/S increased. When-1 treated at1/S a high concentration (200Figure nM), 10. theDixon activity plot for did the not inhibitory recover eveneffect 8of h (A after) DWP16001, the drug (B was) dapagliflozin, removed from and the(C) medium (Figureipragliflozin 11A). on However, the uptake when of [14 dapagliflozinC]methyl-a-D-glucopyranoside and ipragliflozin (AMG) were used, in CHO-SGLT2 SGLT2 activity cells. wasEach recovered tosymbol the control represents level the 4 concentration h after the removal of [14C]AMG: of dapagliflozin ●, 1 µM; ○, 2.5 and µM; ipragliflozin ▼, 5 µM;  (Figure, 50 µM. 11 ReplotB,C). of The results indicatethe slopes that of Dixon the dissociation plot (slopes vs of 1/[S]) DWP16001 was shown to SGLT2for (D) DWP16001, is slower and(E) dapagliflozin, more incomplete and (F) than that ofipragliflozin. dapagliflozin V indicates and ipragliflozin the SGLT2-mediated and that theuptake binding rate aofffi nity[14C]AMG to SGLT2 and isS strongerindicates thanthe that of dapagliflozinconcentration of and [14C]AMG. ipragliflozin. The data are the means±standard deviations of triplicate measurements.

(A) (B) (C)

150 150 150

0.2 nM 2 nM 20 nM 200 nM 100 100 100

50 50 50 0.2 nM 0.2 nM 2 nM 2 nM AMG AMG uptake activity)(% 20 nM 20 nM 200 nM 200 nM

0 0 0 02468 02468 02468 Time (h) Time (h) Time (h)

FigureFigure 11. Recovery 11. Recovery of SGLT2 of SGLT2 transport transport function function with withdifferent different washout washout time timefollowing following 24 h 24of h of incubationincubation with with(A) DWP16001, (A) DWP16001, (B) dapagliflozin, (B) dapagliflozin, and ( andC) ipragliflozin (C) ipragliflozin (0.2–200 (0.2–200 nM) nM)was measured was measured 14 throughthrough the theuptake uptake of of[ C]methyl-a-D-glucopyranoside [14C]methyl-a-D-glucopyranoside (AMG) (AMG) in in CHO-SGLT2 CHO-SGLT2 cells. EachEach datadata point pointrepresents represents the the mean mean±standardstandard deviation deviation of of three three independent independent experiments. experiments. ± 4. Discussion4. Discussion DWP16001DWP16001 is a candidate is a candidate SGLT2 SGLT2 inhibitor inhibitor that thatis currently is currently under under development. development. As a Asfirst a firststep,step, the pharmacokineticthe pharmacokinetic properties properties and in and vitroin vitro SGLT2SGLT2 inhibition inhibition were were compared compared with with currently currently used used SGLT2SGLT2 inhibitors. inhibitors. Dapagliflozin Dapagliflozin and and ipragliflozinipragliflozin werewere selected selected based based on their on hightheir kidney high distributionskidney distributionsand long and elimination long elimination half-lives (thalf-lives1/2) in the (t kidney1/2) in the [5], whichkidney are[5], thought which toare be thought important to forbe the importantefficacy for and the duration efficacy ofand action duration of SGLT2 of acti inhibitors.on of SGLT2 DWP16001 inhibitors. showed DWP16001 higher kidneyshowed distributions higher kidneycompared distributions with dapagliflozincompared with and dapagliflozin ipragliflozin. and DWP16001 ipragliflozin. also DWP16001 had longer also t1/2 inhad the longer kidney t1/2 than in thedapagliflozin kidney than and dapagliflozin ipragliflozin, and as ipragliflozin, well as a comparable as well plasmaas a comparable profile with plasma ipragliflozin profile with (Table 1). ipragliflozinMoreover, (Table the kidney 1). Moreover, concentration the kidney of DWP16001 concentration was maintained of DWP16001 over 72 was h following maintained oral administrationover 72 h followingof 1 mg /oralkg DWP16001 administration (Figure 4)of. Taken 1 mg/kg together, DWP16001 these pharmacokinetic (Figure 4). results Taken show together, that the durationthese of action of DWP16001 is greater than that of dapagliflozin and ipragliflozin and that the oral therapeutic dose of DWP16001 could be reduced compared with both dapagliflozin and ipragliflozin. To investigate the underlying mechanisms of highest kidney distribution and maintained concentration of DWP16001 in the kidney, we measured the kidney tissue binding and involvement of OAT1 and OAT3 transporters of the three SGLT2 inhibitors. All three SGLT2 inhibitors showed high protein binding, but the kidney tissue binding performances of these three SGLT2 inhibitors were not different from their plasma protein binding capabilities (Table2). However, DWP16001 was a substrate for both OAT1 and OAT3, which are dominantly expressed in the kidney, whereas dapagliflozin and ipragliflozin were not (Figures5 and6). Although this could not solely explain the high kidney distribution, OAT1- and OAT3-mediated transport process may contribute to the high kidney distribution. Next, we compared the in vitro SGLT2 inhibition and selectivity of SGLT2 inhibition over SGLT1. All three SGLT2 inhibitors inhibited SGLT2 and SGLT1 activity in a concentration-dependent manner and IC50 values of dapagliflozin and ipragliflozin were in the range of previous reports (1.0–1.3 nM for dapagliflozin; 6.75–8.07 nM for ipragliflozin) [21,22]. IC50 values of DWP16001 to SGLT2 and SGLT1 were lower than those of dapagliflozin and ipragliflozin, suggesting a greater affinity to SGLT2 Pharmaceutics 2020, 12, 268 15 of 16 inhibition for DWP16001 with a higher selectivity over SGLT1 than dapagliflozin and ipragliflozin. The mode of inhibition of DWP16001 was not different from the other SGLT2 inhibitors. They all showed reversible and competitive inhibition (Figure 10), which is consistent with other SGLT2 inhibitors [23,24]. However, the affinity to SGLT2 inhibition seemed to be different among the three SGLT2 inhibitors (Table3). In addition, the recovery of SGLT2 transport activity following the pretreatment of DWP16001, dapagliflozin, and ipragliflozin for 24 h was retained at a higher concentration (200 nM) of DWP16001 compared with dapagliflozin and ipragliflozin. These results suggested that DWP16001 had the highest SGLT2 inhibition potential and that this inhibition potential retained for a longer time compared with dapagliflozin and ipragliflozin. Combined with the higher kidney distribution of DWP16001, retained SGLT2 inhibition with a high concentration of DWP16001 could also potentiate the efficacy of DWP16001 compared with dapagliflozin and ipragliflozin. The comparative pharmacokinetics and in vitro SGLT2 inhibition findings suggest that DWP16001 might be a superior alternative to dapagliflozin and ipragliflozin; however, we should note that comparisons of the in vivo pharmacologic properties of these agents using therapeutic doses in animals and humans need to be further undertaken.

Author Contributions: Conceptualization, M.-K.C., H.-Y.J and I.-S.S.; Investigation, M.-K.C., S.J.N., H.-Y.J., M.J.P., J.-S.C. and I.-S.S.; Writing-Original Draft Preparation, M.-K.C.; Supervision, I.-S.S.; Writing-Review & Editing, I.-S.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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