Methods

Subjects

Prior to any study-related procedures, the subjects provided written informed consent during the screening period. Healthy Korean men aged 19 to 50 years, with a body mass index of 18 – 27 kg/m2were selected. Subjects were excluded from the study if they had a clinically significant history or suffered from a concurrent disease that could have affected the safety, PK, or PD of DWP16001. Exclusion criteria also included a GFR under <90 mL/min as calculated from the MDRD equation, AST or ALT > 1.5 times the upper limit of normal, fasting serum glucose >110 mg/dL or <70 mg/dL, or a glycosylated hemoglobin A1c (HbA1c) level over 6.5 %.

Study Design

This randomized, double-blind, active- and placebo-controlled, single- and multiple-dose study was conducted at the Seoul National University Hospital Clinical Trials Center in accordance with the Declaration of Helsinki and the rules of Korean Good Clinical Practice. The study protocol was reviewed by the Ministry of Food and Drug Safety (ClinicalTrials.gov: NCT03364985).
Twelve subjects in each dose group were orally administered a single dose or multiple doses for 15 consecutive days of DWP16001, dapagliflozin 10 mg (active comparator), or placebo in a ratio of 8:2:2. The investigated dose levels were 0.2, 0.5, 1.0, 2.0 and 5.0 mg in the single-dose arm, and 0.1, 0.3, 0.5, 1.0 and 2.0 mg in the multiple-dose arm. Meals were provided 2, 6, and 10 hours post-dose; breakfast was not provided on the days of intensive pharmacokinetic evaluation or oral glucose tolerance test. In addition, the food effect was explored in the 2.0 mg dose group in the single dose arm in a crossover manner. The subjects in this arm received the study drugs in a fasted state and received them once again in a fed state after a two-week washout. In the fed status, a high-fat meal of 900 kcal was provided at 30 minutes prior to study drug administration.

Pharmacodynamic (PD) evaluation

In the single-dose arm, urine samples for urinary glucose excretion (UGE) analysis were collected up to 168 h (collection interval: 0–4, 4–8, 8–12, 12–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 h post-dose). In the multiple-dose arm, urine collection was conducted at Day 1 and Day 15 at the same time intervals as the single-dose arm. Urine samples for PD analysis were separated in a 10 mL conical tube and stored at −20 °C until analysis. Urine glucose was measured using an automated analyzer (TBA‐120FR; Toshiba Medical Systems, Tochigi, Japan). Urine PD parameters were defined as UGE from 0 to the last measurement (UGElast) and UGE within 24 hours after the first and last dose (UGE0-24h, UGE15d).
An oral glucose tolerance test (75 g OGTT) was performed in the multiple-dose period before administration as the baseline (Day -2) and during the steady state (Day 14). Blood samples for serum glucose were obtained at pre-dose and 0.5, 1, 1.5, 2, 3, and 4 hours post-dose on those days. Plasma insulin concentration samples were collected for up to 6 hours on the same day as serum glucose. Each blood sample collected in a serum separator tube was temporarily left at room temperature for approximately 30 minutes, and then centrifuged for 10 minutes at 1820 ×g. The supernatant was collected in Eppendorf tubes and stored at −20 °C until analysis. Serum concentrations of glucose were measured using an automated analyzer (automatic chemical analyzer: TBA-FX8, Toshiba, Japan). The plasma concentrations of insulin were analyzed with the immunoradiometric assay (IRMA) method (gamma counter: Dream Gamma-10, Shin Jin, Republic of Korea)
The maximum serum glucose concentration (Emax) was determined from the individual profiles, and the area under the 4-hour glucose-time curve (AUEC0-4h) was calculated.

Pharmacokinetic (PK) evaluation

Blood samples for single-dose PK evaluation were obtained pre-dose and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hours after administration. In the multiple dose arm, blood samples were obtained up to 24 and 96 hours post-dose after the first and last administration (Day 1 and Day 15) in a manner equal to that of the single-dose PK. In addition, pre-dose PK samplings (Day 1, Day 2, Day 3, Day 4, Day 5, Day 7, Day 10, Day 13, and Day 15) were conducted to check whether the steady-state was reached. At each blood sampling point, 12 mL of blood was collected in a sodium heparin tube and centrifuged for 10 min at 1820 g . The supernatant was separated in Eppendorf tubes and stored at −70 °C until analysis.
Urine samples for PK evaluation were also collected up to 96 hours post-dose after a single dose in the single-dose arm. In the multiple-dose arm, urine samples were collected up to 24 hours after the first dose (Day 1) and up to 96 hours post-dose after the last dose (Day 15). Urine samples were separated and stored in the same way as the plasma samples.
The plasma concentrations of DWP16001 and metabolites were determined using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using a SCIEX API 5000 system (SCIEX, Framingham, MA, USA) in positive-ion electrospray mode. The concentration of the parent molecules and metabolites in the urine was measured using LC-MS/MS in negative-ion electrospray mode. The lower limits of quantification of DWP16001 in the plasma and urine were 0.1 μg/L and 0.05 μg/L, respectively. The corresponding values of M1 and M2 were 0.04 and 0.05 μg/L in plasma, and 0.04 and 0.1 μg/L in urine, respectively.
Single-dose and multiple-dose pharmacokinetic parameters of DWP16001 and its metabolites were determined using a noncompartmental method with Phoenix® WinNonlin® 7.1 (Certara, L.P., St. Louis, MO, USA). The PK parameters of DWP16001 and M1/M2 included the maximum plasma concentration (Cmax) and time to reach Cmax (Tmax) and were directly determined from the observed individual plasma concentration-time profiles. The terminal elimination half-life (t1/2) was calculated as the natural logarithm of 2 divided by λz, which is the terminal elimination rate constant estimated in the linear portion of the decline of the natural logarithmic-transformed individual plasma concentrations. Areas under the concentration-time curve (AUClast, AUCinf and AUCtau) were calculated using the linear-up log-down trapezoidal rules. Apparent clearance (CL/F) was derived as the administered dose divided by the AUC. The renal clearance (CLR) and the fraction of the dose excreted into the urine (fe) were calculated through urine analysis. The accumulation ratio (R) was calculated as the ratio of AUCtau after the last dose after multiple doses (Day 15) to that after a single dose (Day 1). The metabolic ratio (MR) was determined as the ratio of the AUCtau of the metabolites to that of the parent molecules after multiple administrations.

PK-PD Relationship

The AUCtau after a single dose in both the single dose arm and multiple dose arm, and the UGE0-24h were selected as PK and PD parameters, respectively. A sigmoid Emax model was selected to show the relationship, and half of the maximum effect (EC50), maximum effect (Emax), and Hill coefficient (γ) were calculated using the following equation:
\begin{equation} E=\frac{E_{\max}\bullet C^{r}}{{\text{EC}_{50}}^{r}+C^{r}}\nonumber \\ \end{equation}
Spearman’s correlation coefficients and p-values were calculated to assess the PK/PD relationship.

Safety Evaluation

Safety was assessed based on adverse events (AEs), vital signs, physical examinations, electrocardiograms, and clinical laboratory tests, including urinalysis, throughout the study. The severity of the AE was classified as mild, moderate, or severe, and the causal relationship of the AE was evaluated to determine whether the AE was related or not to DWP16001. An adverse drug reaction (ADR) was defined as an AE that could not be ruled out as unrelated to DWP16001.
N-acetyl-b-D-glucosaminidase (NAG) and beta 2-microglobulin (B2M) levels were measured at baseline (Day -1) and steady-state (Day 15) to evaluate the extent of proximal tubule (SGLT-2 receptor -presented) damage as exploratory safety markers. Urine NAG and B2M were measured using commercial radioimmunoassay kits (Beckman Coulter, Fullerton, CA, USA).

Statistical analysis

Statistical analyses were performed using SAS 9.4 software (SAS Institute, Inc., Cary, NC, USA). All descriptive data were summarized as the mean and standard deviation for continuous variables, and frequencies and percentages for categorical data. With regard to the Cmax and AUC of DWP16001, dose proportionality in the plasma was evaluated through power model analysis using SAS 9.4 software. To compare the pharmacodynamic effect between DWP16001 and dapagliflozin, geometric mean ratios (GMRs, DWP16001 to dapagliflozin) and the 2-sided 90% confidence interval (CIs) of UGE0-15d were calculated. The effect of food was assessed through the GMR and 90% CIs of the PK parameters (log-transformed Cmax and AUC) and PD parameter (UGE0-24h) were calculated. The incidences of AEs and ADRs were compared among the treatment groups using the Kruskal-Wallis test.