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\begin{document}

\title{Dose-dependent glucosuria of DWP16001, a novel selective SGLT-2
inhibitor, in healthy subjects}



\author[1]{Jungi Hwang}%
\author[2]{SeungHwan Lee}%
\author[3]{Wan Huh}%
\author[3]{Jumi Han}%
\author[2]{Jaeseong Oh}%
\author[2]{In-Jin Jang}%
\author[2]{Kyung-Sang Yu}%
\affil[1]{Chungbuk National University Hospital}%
\affil[2]{Seoul National University College of Medicine}%
\affil[3]{Daewoong Pharmaceutical Co Ltd}%


\vspace{-1em}



  
  \date{June 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
DWP16001 is a novel sodium-glucose cotransporter-2 (SGLT2) inhibitor
under development for the treatment of type 2 diabetes mellitus. This
study was conducted to evaluate the pharmacokinetics (PK),
pharmacodynamics (PD), and safety of DWP16001 after single and multiple
doses in healthy subjects. A randomized, double-blind, placebo- and
active-controlled, single- and multiple-dose study was conducted. Twelve
subjects in each dose group received a single dose (0.2, 0.5, 1.0, 2.0,
or 5.0 mg) or multiple doses (0.1, 0.3, 0.5, 1.0, or 2.0 mg once daily
for 15 consecutive days) of DWP16001, dapagliflozin 10 mg, or placebo,
in a ratio of 8:2:2. Serial blood samples and interval urine samples
were collected for PK and PD analyses. Safety and tolerability were
assessed throughout the study period. A dose-dependent increase in
urinary glucose excretion (UGE) was observed after a single dose, and
the steady-state UGE was 50--60 g/day after multiple doses in the dose
range of 0.3 -- 2.0 mg. DWP16001 was rapidly absorbed with the time to
peak plasma concentration of 1.0 -- 3.0 hours, and eliminated with a
mean elimination half-life of 13 - 29 hours. The systemic exposure of
DWP16001 increased proportionally with the dose after multiple
administrations in the range of 0.1 -- 2.0 mg. DWP16001 was well
tolerated in all dose groups. DWP16001 caused glucosuria in a
dose-dependent manner, and systemic exposure was observed after multiple
doses. DWP16001 was well tolerated up to 5.0 mg after a single oral dose
and up to 2.0 mg after multiple oral administration%
\end{abstract}%



\sloppy


Dose-dependent glucosuria of DWP16001, a novel selective SGLT-2
inhibitor, in healthy subjects

\textbf{Jun Gi Hwang\textsuperscript{1,2}, SeungHwan
Lee,\textsuperscript{1} Wan Huh\textsuperscript{3}, Jumi
Han\textsuperscript{3}, Jaeseong Oh\textsuperscript{1}, In-Jin
Jang\textsuperscript{1}, Kyung-Sang Yu\textsuperscript{1}}

\textsuperscript{1}Department of Clinical Pharmacology and Therapeutics,
Seoul National University College of Medicine and Hospital, Seoul,
Republic of Korea

\textsuperscript{2}Department of Clinical Pharmacology and Therapeutics,
Chungbuk National University College of Medicine and Hospital,
Chungcheongbuk-do, Republic of Korea

\textsuperscript{3}Daewoong Pharmaceutical Co., Ltd., Seoul, Republic of
Korea

\textbf{* Correspondence:} Kyung-Sang Yu

Department of Clinical Pharmacology and Therapeutics, Seoul National
University College of Medicine and Hospital, 101 Daehak-ro, Jongno-gu,
Seoul 03080, Republic of Korea

Tel +82 2 2072 2343

Fax +82 2 742 9252

Email: \emph{ksyu@snu.ac.kr}

Abstract

\textbf{Aims}

DWP16001 is a novel sodium-glucose cotransporter-2 (SGLT2) inhibitor
under development for the treatment of type 2 diabetes mellitus. This
study was conducted to evaluate the pharmacokinetics (PK),
pharmacodynamics (PD), and safety of DWP16001 after single and multiple
doses in healthy subjects.

\textbf{Methods}

A randomized, double-blind, placebo- and active-controlled, single- and
multiple-dose study was conducted. Twelve subjects in each dose group
received a single dose (0.2, 0.5, 1.0, 2.0, or 5.0 mg) or multiple doses
(0.1, 0.3, 0.5, 1.0, or 2.0 mg once daily for 15 consecutive days) of
DWP16001, dapagliflozin 10 mg, or placebo, in a ratio of 8:2:2. Serial
blood samples and interval urine samples were collected for PK and PD
analyses. Safety and tolerability were assessed throughout the study
period.

\textbf{Results}

A dose-dependent increase in urinary glucose excretion (UGE) was
observed after a single dose, and the steady-state UGE was 50--60 g/day
after multiple doses in the dose range of 0.3 -- 2.0 mg. DWP16001 was
rapidly absorbed with the time to peak plasma concentration of 1.0 --
3.0 hours, and eliminated with a mean elimination half-life of 13 - 29
hours. The systemic exposure of DWP16001 increased proportionally with
the dose after multiple administrations in the range of 0.1 -- 2.0 mg.
DWP16001 was well tolerated in all dose groups.

\textbf{Conclusion}

DWP16001 caused glucosuria in a dose-dependent manner, and systemic
exposure was observed after multiple doses. DWP16001 was well tolerated
up to 5.0 mg after a single oral dose and up to 2.0 mg after multiple
oral administrations.

\textbf{What is already known about this subject} :

Type 2 diabetes is the chronic metabolic disorder which prevalence has
been increasing worldwide.

SGLT-2 inhibitor, the antidiabetic agent which inhibits reabsorption of
glucose in the renal tubule, recommended in T2DM accompanied by
cardiovascular disease.

\textbf{What this study adds} :

We studied the pharmacodynamics and pharmacokinetics of DWP16001, a
novel SGLT-2 inhibitor under development.

This study proved the effect of DWP16001 as a potent SGLT-2 inhibitor.
This finding may provide information for clinical research and
combination with other antidiabetic agents.

\section*{Introduction}

{\label{introduction}}

Type 2 diabetes mellitus (T2DM) is a major chronic metabolic disorder.
Over 463 million people were diagnosed with T2DM in 2019 and the
prevalence of T2DM has been increasing steadily worldwide {[}1{]}. T2DM
is characterized by insulin insensitivity, resistance, and secretion
defects caused by pancreatic beta cell dysfunction {[}2{]}. Metformin is
the preferred initial pharmacologic agent for the treatment of
T2DM{[}3,4{]}. However, sulfonylureas, thiazolidinedione, DPP-4
inhibitors, GLP-1 receptor agonists, and SGLT-2 inhibitors are
prescribed as add-on drugs when target HbA1c levels are not achieved
with metformin therapy {[}5,6{]}. Among these, SGLT-2 inhibitor is
recommended in T2DM accompanied by cardiovascular disease such as heart
failure {[}7{]}.

Sodium glucose cotransporters (SGLT) are of two types. SGLT-1 is
distributed throughout organs such as the intestine and kidneys and has
low capacity and high affinity. SGLT-2 is primarily present in the
kidneys and has a relatively high capacity and low affinity. Under
normal physiological conditions, both transporters reabsorb glucose in
the renal proximal tubule (\textless{}180 g/day), but the glucose uptake
of SGLT-2 is over 97\% in the renal proximal tubule {[}8,9{]}. When
SGLT-2 is dysfunctional---as a result of mutations in the SLC5A2
gene---the renal capacity for absorbing glucose decreases to the
residual capacity of SGLT-1 (80 g/day) {[}10{]}.

SGLT-2 inhibitors are considered potential strategies for T2DM treatment
because of the following advantages: (1) the mechanism of action of
SGLT-2 inhibitors is independent of insulin {[}11{]}; (2) SGLT-2
inhibitors lower the risk of cardiovascular disease---a major
complication of T2DM {[}12,13{]}; (3) SGLT-2 inhibitors protect kidney
function by decreasing glomerular filtration rate (GFR) reversibly and
preserving GFR in the long-term in patients with T2DM {[}14{]}; (4)
SGLT-2 inhibitors promote weight loss, which lowers the risk of
complications related to T2DM and can compensate for the weight gain
that is a side effect of insulin and sulfonylureas {[}15{]}.
Canagliflozin (Invokana\selectlanguage{ngerman}\textsuperscript{®}), dapagliflozin
(Farxiga\textsuperscript{®}), empagliflozin
(Jardiance\textsuperscript{®}), and ertugliflozin
(Steglatro\textsuperscript{TM}) have recently been approved by the US
FDA and EMA for the treatment of T2DM {[}16{]}.

DWP16001 is a novel SGLT-2 selective inhibitor under clinical
development. In an \emph{in vitro} study, DWP16001 selectively inhibited
SGLT2 and showed high potency for SGLT2 inhibition---more than 1000
times that of SGLT1. DWP16001 is mainly metabolized by CYP3A4 and
CYP2C19 into active metabolites M1 and M2 or via glucuronidation by
UGT2B7, UGT1A4, and UGT1A9 {[}17{]}. The inhibitory potency of these
metabolites for SGLT-2 was 10 times weaker than that of parent molecule.
{[}18{]}.

The purpose of this study was to evaluate the safety, pharmacokinetics
(PKs), and pharmacodynamics (PDs) of DWP16001 after single and multiple
doses in healthy Korean subjects.



\section*{Methods}

{\label{methods}}

\subsection*{Subjects}

{\label{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/m\textsuperscript{2}were 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 \textless{}90
mL/min as calculated from the MDRD equation, AST or ALT \textgreater{}
1.5 times the upper limit of normal, fasting serum glucose
\textgreater{}110 mg/dL or \textless{}70 mg/dL, or a glycosylated
hemoglobin A1c (HbA1c) level over 6.5 \%.

\subsection*{Study Design}

{\label{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.

\subsection*{Pharmacodynamic (PD)
evaluation}

{\label{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 \selectlanguage{english}-20 degC until analysis. Urine glucose was
measured using an automated analyzer (TBA\selectlanguage{english}-120FR; Toshiba Medical
Systems, Tochigi, Japan). Urine PD parameters were defined as UGE from 0
to the last measurement (UGE\textsubscript{last}) and UGE within 24
hours after the first and last dose (UGE\textsubscript{0-24h},
UGE\textsubscript{15d}).

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
x\emph{g.} The supernatant was collected in Eppendorf tubes and stored
at \selectlanguage{english}-20 degC 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 (E\textsubscript{max}) was
determined from the individual profiles, and the area under the 4-hour
glucose-time curve (AUEC\textsubscript{0-4h}) was calculated.

\subsection*{Pharmacokinetic (PK)
evaluation}

{\label{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 \emph{g} . The supernatant was separated in Eppendorf
tubes and stored at \selectlanguage{english}-70 degC 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
\selectlanguage{greek}μ\selectlanguage{english}g/L and 0.05 \selectlanguage{greek}μ\selectlanguage{english}g/L, respectively. The corresponding values of M1 and M2
were 0.04 and 0.05 \selectlanguage{greek}μ\selectlanguage{english}g/L in plasma, and 0.04 and 0.1 \selectlanguage{greek}μ\selectlanguage{english}g/L in urine,
respectively.

Single-dose and multiple-dose pharmacokinetic parameters of DWP16001 and
its metabolites were determined using a noncompartmental method with
Phoenix\selectlanguage{ngerman}\textsuperscript{®} WinNonlin\textsuperscript{®} 7.1 (Certara,
L.P., St. Louis, MO, USA). The PK parameters of DWP16001 and M1/M2
included the maximum plasma concentration (C\textsubscript{max}) and
time to reach C\textsubscript{max} (T\textsubscript{max}) and were
directly determined from the observed individual plasma
concentration-time profiles. The terminal elimination half-life
(t\textsubscript{1/2}) was calculated as the natural logarithm of 2
divided by \selectlanguage{greek}λ\selectlanguage{english}\textsubscript{z}, 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 (AUC\textsubscript{last},
AUC\textsubscript{inf} and AUC\textsubscript{tau}) 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
(CL\textsubscript{R}) 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 AUC\textsubscript{tau} 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
AUC\textsubscript{tau} of the metabolites to that of the parent
molecules after multiple administrations.

\subsection*{PK-PD Relationship}

{\label{pk-pd-relationship}}

The AUC\textsubscript{tau} after a single dose in both the single dose
arm and multiple dose arm, and the UGE\textsubscript{0-24h} were
selected as PK and PD parameters, respectively. A sigmoid
E\textsubscript{max} model was selected to show the relationship, and
half of the maximum effect (EC\textsubscript{50}), maximum effect
(E\textsubscript{max}), and Hill coefficient (\selectlanguage{greek}γ\selectlanguage{english}) 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.

\subsection*{Safety Evaluation}

{\label{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).

\subsection*{Statistical analysis}

{\label{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
C\textsubscript{max} 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
UGE\textsubscript{0-15d} were calculated. The effect of food was
assessed through the GMR and 90\% CIs of the PK parameters
(log-transformed C\textsubscript{max} and AUC) and PD parameter
(UGE\textsubscript{0-24h}) were calculated. The incidences of AEs and
ADRs were compared among the treatment groups using the Kruskal-Wallis
test.

\section*{Results}

{\label{results}}

\subsection*{Demographics}

{\label{demographics}}

In the single-dose arm, a total of 61 subjects were enrolled, and 60
subjects completed the study. The mean (\selectlanguage{ngerman}± standard deviation) age, body
weight and BMI of the randomized subjects were 30.75 ± 6.89 years, 71.06
± 7.48 kg, and 23.43 ± 2.06 kg/m\textsuperscript{2}, respectively.

In the multiple-dose arm, 62 subjects were enrolled, and 60 subjects
completed the study. The mean (± standard deviation) age, body weight
and BMI of the randomized subjects were 30.84 ± 5.43 years, 70.72 ± 7.15
kg, and 23.47 ± 2.07 kg/m\textsuperscript{2}, respectively. The
demographic characteristics were comparable among the dose groups for
both single and multiple arms.

\subsection*{Pharmacodynamics}

{\label{pharmacodynamics}}

In the single dose arm, UGE\textsubscript{0-24h} and
UGE\textsubscript{last} increased in a dose-dependent manner in the
range of 0.2 to 1.0 mg. UGE\textsubscript{0-24h} tended to be saturated
in the range of 1.0 mg to 5.0 mg of DWP16001, which was similar to that
of dapagliflozin 10 mg. Meanwhile, UGE\textsubscript{last} was higher
over 1.0 mg of DWP16001 than that of dapagliflozin (Figure 1,2 Table 1).

In the multiple dose arm, daily UGE reached a plateau at 50 - 60 g/day
at the steady state. The UGE was higher with all doses of DWP16001 and
dapagliflozin 10 mg compared with placebo (Table S1). The mean UGE over
24 hours at steady state of 0.1 mg, 0.3 mg, 0.5 mg, 1.0 mg, and 2.0 mg
DWP16001 were 36.1 g, 48.3 g, 55.1 g, 56.8 g, and 53.4 g, respectively,
and the corresponding value of dapagliflozin 10 mg was 40.5 g (Figure
1,2, Table 1).

In the OGTT after dosing for 14 consecutive days of DWP16001, the
E\textsubscript{max} of the plasma glucose was lower than that of
baseline. The mean E\textsubscript{max} changed from baseline of 0.1 mg,
0.3 mg, 0.5 mg, 1.0 mg and 2.0 mg DWP16001 were -27.5, -32.8, -7.8,
-23.9 and -17.4 mg/dL, respectively. These values were different from
-2.2 mg/dL of placebo, but similar to -21.6 mg/dL of dapagliflozin 10
mg. A similar pattern was observed for AUEC\textsubscript{0-4h} after
OGTT. The mean AUEC\textsubscript{0-4h} changed from baseline was
positive in placebo (4.3 mg*h/dL) , whereas 0.1 mg, 0.3 mg, 0.5 mg, 1.0
mg, 2.0 mg DWP16001 were -46.8, -55.6, -40.1, -54.0 and -21.9 mg*h/dL,
respectively. Postprandial insulin showed a decreasing tendency after
multiple doses, but it was not statistically significant (Figure S1,
Table S2).

\subsection*{Pharmacokinetics}

{\label{pharmacokinetics}}

DWP16001 was rapidly absorbed after a single dose with
T\textsubscript{max} between 1 and 3 h post-dose. After reached a
T\textsubscript{max}, plasma concentration of DWP16001 fell in a
biphasic manner with the elimination half-life of 11 to 28 hours (Figure
3). The t\textsubscript{1/2} was tended to decrease as the dose of
DWP16001 increased (Table 2).

After multiple doses, the time-plasma concentration was similar to that
of the single-dose arm. The mean T\textsubscript{max} and
t\textsubscript{1/2} at steady state were within a range of 1 to 2 hours
and 17 to 23 hours, respectively. After multiple administrations, the
95\% CIs of the slope of the log-transformed C\textsubscript{max,ss} and
AUC\textsubscript{tau,ss} included 1.0 (0.98--1.10 and 0.98--1.09,
respectively), thus considered dose-proportional. The mean accumulation
index was approximately 1.6 to 1.9 over the range of 0.1 mg to 2.0 mg of
DWP16001. Less than 3 \% of DWP16001 was excreted unchanged in the urine
at all doses (Table 2). The metabolic ratio of M1 ranged from 0.20 to
0.25. M2 was detected in very low amounts compared to the parent
molecules in all dose groups (Table S3).

\subsection*{PK-PD relationship}

{\label{pk-pd-relationship-1}}

Drug exposure and UGE showed a clear exposure-response relationship. The
AUC\textsubscript{tau} and UGE over 24 h after the last dose were
correlated, with a Spearman correlation coefficient of 0.4061
(p=0.0002). The PK-PD relationship was well-explained by a sigmoid
E\textsubscript{max} model, and the E\textsubscript{0},
E\textsubscript{max}, EC\textsubscript{50}, EC\textsubscript{90}, and \selectlanguage{greek}γ
\selectlanguage{english}were calculated to be 0.1 g, 58.8 g, 31.8 \selectlanguage{greek}μ\selectlanguage{english}g\selectlanguage{ngerman}·h/L, 58.4 \selectlanguage{greek}μ\selectlanguage{english}g\selectlanguage{ngerman}·h/L, and 3.6,
respectively (Figure 4).

\subsection*{Effect of Food}

{\label{effect-of-food}}

After a high-fat meal was provided, the median T\textsubscript{max} was
prolonged from 1.3 to 2.5 hours, and the C\textsubscript{max} was
decreased approximately 43 \% (GMR {[}90\% CIs{]}: 0.57
{[}0.45--0.72{]}), compared to that of fasting state (Figure 5).
However, the AUC\textsubscript{inf} was similar between fed and fasting
state (GMR {[}90\% CIs{]}: 0.97 {[}0.82--1.16{]}). The UGE was
comparable between fed and fasting state; GMR {[}90\% CIs{]} of
UGE\textsubscript{0-24h} and UGE\textsubscript{last} were 0.80
{[}0.66--0.97{]} and 0.90 {[}0.68--1.19{]}, respectively.

\subsection*{Safety and Tolerability}

{\label{safety-and-tolerability}}

DWP16001 was found to be safe and tolerable in all dose groups, both in
single- and multiple-dose administration. In the single-dose arm,
treatment-emergent adverse events (TEAEs) were reported in 12 cases by 5
subjects who received DWP16001. In the multiple-dose arm, 27 cases were
reported in 13 subjects who received DWP16001, and four cases were
reported in three subjects who received placebo. Adverse drug reactions
(ADRs) that were determined to have a causal relationship with the
investigational product were reported in two cases from one subject in
the single-dose group and 22 cases from 12 subjects in the multiple-dose
group (Table 3). The most frequently reported ADRs were abdominal pain,
headache, and rhinorrhea. Abdominal pain was reported by three subjects
(1 in the 0.1 mg group and 2 in the 1.0 mg group). All TEAEs were mild
and spontaneously resolved. There were no serious adverse events, and no
subject withdrew from the study owing to adverse events. No other
clinically significant changes were observed in the clinical laboratory
tests, vital signs, ECGs, or physical examinations.

Renal tubular marker {[}N-acetyl-\selectlanguage{greek}β-\selectlanguage{english}D-glucosaminidase (NAG) and
\selectlanguage{greek}β\selectlanguage{english}2-microglobulin (B2M){]} tests were conducted in the multiple-dose arm.
Only one subject (DWP16001 2.0 mg group) had an increased B2M by more
than 0.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL compared to the baseline, but it was found to be closer
to the baseline level in a post-study visit. The mean change in NAG and
B2M concentrations showed no significant increase across the different
dose groups (Table S4).

\section*{Discussion}

{\label{discussion}}

In this study, the amount of UGE showed maximum values of \textgreater{}
60 g/day in groups treated 1.0 mg or higher after a single dose. The
steady state daily UGE seemed to peak at \textgreater{} 50 g/day in the
groups treated with 0.5 mg or higher after multiple dosing. The UGE over
24 h seemed to be saturated at higher doses. This was considered to be
compensation for glucose reabsorption in the proximal tubule by the
SGLT1 receptor under normoglycemic conditions and by a decrease in renal
clearance {[}19,20{]}.

In previous clinical studies of SGLT-2 inhibitors in healthy subjects,
the UGE after multiple doses was 33.8 g/day with canagliflozin 100 mg
and 35.5 g/day with dapagliflozin 10 mg {[}20,21{]}. The corresponding
values for canagliflozin 100 mg and dapagliflozin 10 mg after multiple
doses in T2DM patients both exceeded 70 g/day and were demonstrated to
show clinical efficacy {[}22,23{]}. Considering this relationship
between the results in healthy subjects and patients, DWP16001 doses
above 0.1 mg are expected to show clinical efficacy in T2DM patients.

Urine glucosuria induced by administration of DWP16001 lasted up to 168
h after a single dose. DWP16001 maintained a longer inhibition of SGLT2
than dapagliflozin and ipragliflozin in an \emph{in vitro} test
{[}18{]}. In addition, DWP16001 showed a better kidney distribution and
longer elimination half-life than dapagliflozin and ipragliflozin at the
ICR mouse and these results could have accounted for the long duration
of UGE of DWP16001 {[}18{]}. Considering the elimination half-life and
the usage of most type2 DM treatments (daily medication), DWP16001 seems
to be desirable to be administered as a once daily therapy.
Additionally, it is expected that long-lasting UGE and
anti-hyperglycemic effect suppress glucose excursion to achieve
desirable treatment target even in T2DM patients with low medication
adherence.

The C\textsubscript{max} and AUC\textsubscript{tau} of DWP16001 after
multiple administrations showed dose-proportional increments in the
range of 0.1 mg to 2.0 mg. Meanwhile, the t\textsubscript{1/2} and
T\textsubscript{max} were independent of the dose. A high-fat meal
delayed the absorption of DWP16001 and decreased C\textsubscript{max},
but drug exposure was almost unaffected. The fraction of DWP16001
excreted in an unchanged form in the urine was less than 2.5\%. This
finding is consistent with nonclinical data that show that DWP16001 is
extensively metabolized and mainly eliminated through a non-renal
pathway in humans {[}17{]}. SGLT2 inhibitors have controversial effects
on kidney injuries, but the PK characteristics of DWP16001 might be less
affected by renal impairment. Further clinical pharmacology studies in
subjects with decreased renal function will be needed to elucidate the
PK characteristics of DWP16001.

The possibility of DWP16001-related kidney damage is expected to be
relatively low considering the results obtained in healthy subjects.
There are positive reports that the long-term use of SGLT-2 inhibitors
decreases the burden on the glomerulus and shows a renoprotective effect
{[}25,26,27{]}. In contrast, there is a suspicion that long-term use of
SGLT2 inhibitors could lead to tubular injury depending on the mechanism
of action {[}28,29{]}. This has been observed through the elevation of
renal tubular markers in some previous studies {[}30,31{]}. Therefore,
the levels of NAG and B2M were monitored in this study throughout the
multiple dose period. After administration of 0.1 mg DWP16001, NAG
levels slightly increased at steady state compared to the baseline.
However, this tendency was not dose-related, and the values returned to
the respective baseline level during follow-up visits. B2M levels were
not statistically different at all doses, including those in the
placebo. Likewise, there was a similar pattern of the estimated GFR
between the baseline and post-dose.

DWP16001 was generally safe and well tolerated among all doses in this
study. The incidence of AEs after single and multiple doses of DWP16001
did not appear to be dose-related in either study. Genitourinary
infection is known to be a major adverse event following the use of
SGLT2 inhibitors {[}32{]}, but only one subject in the 1.0 mg multiple
DWP16001 dose group suffered balanoposthitis; there were no other
occurrences of genitourinary infections. In addition, only a few
subjects experienced gastrointestinal adverse events, reflecting the
inhibition of SGLT1 in the GI tract. This can be explained by the
selective inhibitory effect of DWP16001 on SGLT2.

\section*{Conclusions}

{\label{conclusions}}

In summary, DWP16001 showed clear dose- and exposure-response
relationships, exhibited linear PK characteristics and was
well-tolerated in healthy subjects. These findings support further
evaluation of DWP16001 as a treatment option for T2DM.

\section*{Acknowledgments}

{\label{acknowledgments}}

The authors certify that this manuscript represents valid work and that
neither this manuscriptnor one with substantially similar content has
been published or is being considered for publication elsewhere. Part of
the data were previously partially presented at the 55th European
association for the study of diabetes (EASD) Annual Meeting, 16-20
September 2019. The data that support the findings of this study are
available from the corresponding author, Kyung-Sang Yu, upon reasonable
request. This study was funded by Daewoong Pharmaceutical Co., Ltd.



\section*{Conflicts of Interest}

{\label{conflicts-of-interest}}

Wan Huh and Jumi Han are employees of Daewoong Pharmaceutical Co., Ltd.
The other authors report no conflicts of interest.

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{\label{references}}

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\section*{Legend to figures}

{\label{legend-to-figures}}

Figure 1. Mean urinary glucose excretion-time profiles after a single or
multiple oral administration of DWP16001 or dapagliflozin. (A)
single-dose, B)multiple-doses)

Figure 2. Mean (SD) urinary glucose excretion over 24 hours after single
and multiple oral administration of DWP16001 (A) single-dose,
B)multiple-doses)

Figure 3. Mean plasma concentration-time profiles after single and
multiple oral administration of DWP16001. (A) single-dose,
B)multiple-doses)

Figure 4. Relationship between AUC\textsubscript{tau} of DWP16001 after
a single dose (from both the single-dose arm \& multiple-dose arm) and
urinary glucose excretion over 24 h after first administration
(UGE\textsubscript{0-24h})

Figure 5. a) Mean plasma DWP16001 concentration-time profiles and b)
cumulative UGE after a single administration at fasted or fed state.

\section*{Supplementary appendix}

{\label{supplementary-appendix}}

Figure S1. a)Mean serum glucose and b)insulin profiles before and after
multiple oral administration of DWP16001.

\textbf{Hosted file}

\verb`DWP16001_CFT_bjcp.docx`  available at \url{https://authorea.com/users/448298/articles/547105-dose-dependent-glucosuria-of-dwp16001-a-novel-selective-sglt-2-inhibitor-in-healthy-subjects}

\selectlanguage{english}
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\end{document}

