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.