**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.

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 (Figure 1), a selective SGLT2 inhibitor, is under development by Daewoong Pharmaceutical Co. Ltd. (Yongin, Korea) and is currently undergoing phase 2 clinical trials (Registration No. NCT04014023).

**Figure 1.** Structure of (**A**) DWP16001, (**B**) D4-DWP16001 (IS), (**C**) dapagliflozin, and (**D**) ipragliflozin.

− Recently, Tahara et al. [5] compared the pharmacokinetics, pharmacodynamics, and pharmacological characteristics of six SGLT2 inhibitors, such as ipragliflozin, dapagliflozin, tofogliflozin, canagliflozin, empagliflozin, and luseogliflozin. The study showed that all the SGLT2 inhibitors induced urinary glucose excretion in a dose-dependent manner but the duration of action differed among the six drugs. Ipragliflozin and dapagliflozin showed persistent duration of action; these two drugs exhibited increased urinary glucose excretion even 18 h post dose but the others showed about 12 h of duration. In addition, ipragliflozin and dapagliflozind showed the lowest blood glucose and insulin level 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 kidney and elimination half-life [5], suggesting the importance of kidney distribution and elimination profile is prerequisite in the efficacy of SGLT2 inhibitors, as well as the potent SGLT2 inhibition. Therefore, this study aimed to compare the pharmacokinetic properties and kidney distribution of DWP16001 with those of dapagliflozin and ipragliflozin, representative SGLT2 inhibitors that showed potent and long duration efficacy [5] and to compare the selectivity and mode of inhibition of DWP16001 on SGLT2 with dapagliflozin and ipragliflozin to evaluate the potency of DWP16001 over other SGLT2 inhibitors.

#### − **2. Materials and Methods**

#### *2.1. Materials*

− DWP16001 and D4-DWP16001 (for internal standard (IS)), were obtained from Daewoong Pharmaceutical Co. Ltd. (Yongin, Korea). Dapagliflozin and ipragliflozin were obtained from Toronto Research Chemicals Inc. (North York, ON, Canada) (Figure 1).

− Para-aminohipuric acid (PAH), methyl a-D-glucopyranoside (AMG), sodium dodecyl sulfate (SDS), G418, non-essential amino acids, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and Hank's balances salts solution (HBSS, pH 7.4) were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). [14C]Methyl-α-D-glucopyranoside (AMG) (290 mCi/mmol) was purchased from

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.
