*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. 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].

$$\text{Undiluted free drug fraction } (\text{fu}) = \frac{\text{Drug concentration in buffer chamber}}{\text{Drug concentration in plasma sample chamber}} \tag{1}$$

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].

$$\text{Diluted free drug fraction } (\text{f}\omega) = \frac{\text{Drug concentration in buffer chamber}}{\text{Drug concentration in kidney homogenate chamber}'} \tag{2}$$

$$\text{Undiduted free drug fraction (fu)} = \frac{1/\text{D}}{\left(\frac{1}{\text{fu}'} - 1\right) + 1/\text{D}} = \frac{\text{fu}' \times 0.2}{1 - \text{fu}' \times 0.8}.\tag{3}$$

#### *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 × 10<sup>5</sup> 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% CO<sup>2</sup> 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 [ <sup>3</sup>H]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.

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* = *V*max·S/(*K*<sup>m</sup> + 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<sup>R</sup> (retention time) 2.8 min), *m*/*z* 422 → 151 for ipragliflozin (T<sup>R</sup> 2.5 min), *m*/*z* 426 → 167 for dapagliflozin (T<sup>R</sup> 2.5 min), *m*/*z* 468 → 135 for D4-DWP16001 (T<sup>R</sup> 2.8 min), in the positive ionization mode with a 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 E*ff*ects 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% CO<sup>2</sup> conditions. CHO-SGLT1 and -SGLT2 cells were seeded at a density of 1 × 10<sup>5</sup> 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<sup>+</sup> 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 calculation of IC<sup>50</sup> values, the uptake of 10 µM [14C]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 IC<sup>50</sup> (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].

To investigate time dependency in the inhibition of SGLT2, CHO-mock and -SGLT2 cells were seeded at a density of 1 × 10<sup>5</sup> 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<sup>+</sup> 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<sup>+</sup> 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 × 10<sup>5</sup> 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<sup>+</sup> 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**
