An Open Label, Phase IV, Mechanistic, Study to Evaluate the Natriuretic Effect of 2-Week Dapagliflozin treatment in Type 2 Diabetes Mellitus Patients with Impaired Renal Function

Dapagliflozin is a stable, reversible, highly selective, and orally active
inhibitor of human renal sodium glucose co-transporter 2 (SGLT2), the major
transporter responsible for glucose reabsorption in the kidney. Dapagliflozin*s
mechanism of action results in the direct and insulin-independent elimination
of glucose by the kidneys. Results from nonclinical and clinical studies have
shown that dapagliflozin can be used to promote urinary excretion of glucose as
a well-tolerated and effective method of reducing blood glucose levels in type
2 diabetes mellitus (T2DM) patients. The persistent loss of glucose with
associated calories in the urine, results in a consistent and maintained
reduction of the total body weight, in addition to the improved glycemic
control. Moreover, dapagliflozin also has been shown to reduce blood pressure
and albuminuria, 2 essential prognostic risk factors for progression of renal
disease.

Notably, the total amount of glucose excreted in the urine by dapagliflozin,
declines with decreasing kidney function. In a recent meta-analysis of placebo
controlled clinical trials from the dapagliflozin programme it was confirmed
that the glycosylated haemoglobin (HbA1c) lowering effect was smaller in
patients with an estimated glomerular filtration rate (eGFR) between 45 and 60
mL/min/1.73m2 compared to patients with an eGFR
> 90 mL/min/1.73m2 (Sjöström et al, 2016 [2]). Intriguingly however, the
effects of dapagliflozin on body weight, blood pressure, albuminuria, and
haematocrit were similar regardless of the eGFR level (Sjöström et al, 2016
[2]), and may therefore be independent of glycemic effects of SGLT2 inhibitors.

T2DM is associated with an elevated risk of cardiovascular morbidity and
mortality as well as renal failure. The cardiovascular benefit of dapagliflozin
was demonstrated in the DECLARE-TIMI 58 study by showing a lower rate of the
primary co-primary composite heart failure outcome in the active dapagliflozin
treatment arm (Wiviot et al, 2018 [3]). There is also a growing body of
evidence indicating that SGLT2 inhibition with dapagliflozin is
nephroprotective. Post-hoc analysis from the dapagliflozin phase II and phase
III programme have shown in T2DM patients with moderate renal impairment on top
of renin-angiotensin-aldosterone system (RAAS) blockade reduction around 40% in
albuminuria and stabilization of eGFR decline for up to 1 year (Sjöström et al,
2015 [4]) and 2 years (Fioretto et al, 2015 [5]). After an initial drop in
eGFR, kidney function was stable over time while a progressive decrease in eGFR
was seen in the placebo group. In the DECLARE TIMI 58 trial, a 53% risk
reduction was found in doubling of serum creatinine or initiation of dialysis
treatment. (Mosenzon et al, 2019 [6]).

The nephroprotective effect is thought to be achieved by mechanisms independent
of blood glucose reduction (Rajasekeran et al, 2016 [7]), such as by reduced
intra-glomerular pressure through an enhanced tubuloglomerular feedback
mechanism (De Nicola, et al 2014 [8] and Thomas, 2014 [9]), reduced glucose and
sodium transport over the proximal tubule cells (Pollock, et al 1991 [10] and
Komala, et al 2013 [11]), increased natriuresis (Heerspink et al, 2013 [12])
and reduced systemic blood pressure (Baker et al, 2015 [13]).

The central hypothesis of this study is that dapagliflozin drives a natriuretic
effect independently of renal function level. The study will therefore evaluate
average 24-hr sodium excretion during dapagliflozin treatment in patients with
T2DM with impaired renal function.
In the majority of healthy individuals blood pressure falls (dips) at night.
These individuals are classified as dippers. Those who do not exhibit this
nocturnal fall in blood pressure are referred to as non-dippers. In populations
with CKD, non-dippers are overrepresented due to reduced natriuretic capacity
(Spencer et al, 2015 and references therein [14]). Decreased natriuresis leads
to sodium retention and volume expansion with clinical consequences. Patients
with T2DM often have increased extracellular volume as a result of increased
glucose and sodium reabsorption in the kidney (Novikov et al, 2016 [15]). We
hypothesize that SGLT2 inhibition enables improved natriuresis and diuresis as
both sodium and glucose excretion contribute to osmotic diuresis. The
mechanisms downstream of natriuresis and diuresis have distinct impact on a
number of clinically important parameters. Enhanced natriuresis enables
improved systemic sodium balance, which directly impacts both volume expansion
as well as systemic sodium load. The failure to maintain natriuretic and fluid
balance also results in increased demand to drive natriuresis to a greater
extent. Consequently, blood pressure is increased as well as maintained at
raised levels during the full 24-hr period generating the non-dipping
phenotype. This has clear consequence on cardiovascular fitness via demand
placed on cardiac and vascular tissues. In particular sodium and fluid
accumulation are associated with impaired endothelial function, vascular
stiffening and consequent left ventricular hypertrophy.

As described in the background there is an apparent disconnect between HbA1c
lowering and the other effects of SGLT2 inhibition by dapagliflozin (e.g.,
blood pressure, body weight) which may be in part consequent on volume
contraction or changes in tubuloglomerular feedback. Tubuloglomerular feedback
is the process whereby the macular densa which sits downstream of the proximal
tubule senses the delivery of sodium and chloride and provides feedback to the
glomerulus to alter afferent and efferent pressure via several mechanisms
including renin activity and adenosine production. This will be addressed in
the study by assessing the impact of treatment on factors of the RAAS and urine
adenosine. At the same time diuresis leads to decrease arterial pressure and
volume. Dapagliflozin is hypothesized to work through both these mechanisms to
reduce renal renin activity with concomitant reductions in angiotensin II and
its metabolites and alter the production of adenosine. Recent studies have also
suggested a cross-talk between the SGLT2 transporter and the sodium-hydrogen
exchanger-3 (NHE3) (Novikov et al 2016 [15]). NHE3 is located in the proximal
tubule and responsible for approximately 30% of the reabsorption of sodium in
the kidney. Inhibition of SGLT2 results in down regulation of NHE3 sodium
transport activity, which may also contribute to the natriuretic effects of
dapagliflozin. As a result of the interaction between SGLT2 and NHE3, as well
as potential effects (both direct and indirect, e.g., via altered renal RAAS
activity) on other transporters, the effect of dapagliflozin on electrolyte and
fluid related parameters may persist to a significant extent in patients with
lower eGFR levels. By analysing urinary exosomes using mass spectrometry it
will be possible to evaluate these effects on other transporter levels (Schey
et al, 2015 [16]).

This study will further clarify the dapagliflozin mechanisms of action by
investigating if and how the effect of dapagliflozin on natriuresis, blood
pressure regulation, plasma volume, extracellular volume, hormones, biochemical
variables and electrolytes are impacted by T2DM status and level of kidney
function.

This study is an open label study to evaluate the changes in average 24-hr
sodium excretion during dapagliflozin treatment in patients with T2DM with
impaired renal function.

Optimization of Patient Population:
The study will include patients with T2DM with impaired renal function (an eGFR
by Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI] between >=25 and
<=50 mL/min/1.73m2); Details of eGFR estimation using CKD-EPI are provided in
Section 3.1.
All patients will be required to be on patient specific optimal
antihypertensive doses of an angiotensin converting enzyme inhibitor (ACEi) or
angiotensin receptor blocker (ARB) (as per Investigator*s judgement) prior to
being considered for the study. During the study, all patients will be provided
with food in food boxes which contain in total 150 mmol sodium per day,
starting in the Run-in Period prior to treatment initiation and to be continued
throughout the duration of the study. This will allow a homogenous stable
population to be enrolled in the study for evaluation of the study objective.
If required patients will be using a stable insulin dosing (intermediate, long*
acting, premixed insulin, basal bolus insulin) for the last 12 weeks prior to
start of treatment with dapagliflozin. Metformin, sulphonylurea, Di-Peptidyl
Peptidase 4 inhibitors (DPP4i), or combinations of these agents with or without
insulin would be accepted but is not mandatory. If used, stable dose of
metformin, sulphonylurea, DPP4 inhibitors, GLP1 receptor agonists or their
combination as anti-diabetic therapy for the last 12 weeks prior to start of
treatment with dapagliflozin is required. Other oral antidiabetic drugs,
including pioglitazone, are not allowed (as per Investigator's judgement).

Dapagliflozin Dose and Regimen:
Patients will receive one tablet dapagliflozin 10 mg per day for a total period
of 14±1 days. This dose is the recommended dose for monotherapy and for add-on
combination therapy with other glucose-lowering medicinal products including
insulin to improve glycaemic control in T2DM.

Study Endpoints:
The primary endpoint of the study is the average change in 24-hr sodium
excretion during dapagliflozin treatment from average Baseline to average
values at Day 2 to 4. This endpoint will provide information on acute changes
in sodium. Additionally, urinary sodium excretion at Days 12 to 14 will provide
information on the effect of 10 mg dapagliflozin on steady state natriuresis,
and Days 15 to 17 will provide data on the effect of treatment withdrawal.
Additionally, changes in urinary glucose excretion, urine albumin:creatinine
ratio (UACR), plasma volume, extracellular volume, and
24-hr systolic blood pressure will also be evaluated. Pharmacokinetics of
dapagliflozin will also be studied.

As exploratory endpoints, changes in hormones of RAAS; N-terminal pro B-type
natriuretic peptide [NT-ProBNP] and B-type natriuretic peptide [BNP], urinary
adenosine and plasma co-peptin; changes in 24-hr urinary volume, uric acid,
creatinine, cortisol, isoprostanes, and electrolytes; changes in serum/plasma
biomarkers of metabolism, renal function, electrolytes, uric acid, and
haematocrit; changes in calculated intracellular red blood cell concentrations
of electrolytes; change in intracellular volume; change in total body water;
and changes in extracellular volume and intracellular volume over a 4-hr time
course in relation to pharmacokinetics measurements will be evaluated.
Additionally, changes in day:night blood pressure ratios, and changes in body
weight will be assessed. These parameters will provide more insight into the
potential effects of dapagliflozin (both direct and indirect, e.g., via altered
renal RAAS activity). Urinary exosome analysis will be performed using mass
spectrometry and will allow analysis of dapagliflozin influence on other
transporters.

The study consists of a 2-week, open label, Treatment Period. Patients will be
provided with one bottle of dapagliflozin tablets on Day 1 (Visit 4) to last
for the 14±1 days of the Treatment Period. The tablet is taken orally once
daily in the morning and at approximately the same time of the day.

The first dose of dapagliflozin will be administered at Visit 4 (Day 1) at the
study site after all baseline assessments (including laboratory tests, plasma
volume assessments, and bioimpedance spectroscopy measurement) have been
performed. On days of study site visits where blood sampling is scheduled,
i.e., Visit 5 and Visit 8, the patients will be required to bring the bottle of
dapagliflozin tablets to site. At Visit 5 (Day 4), the patient will consume the
tablet together with breakfast after fasting blood sampling is completed at the
site. At Visit 8 (Day 14), the patient will consume the tablet together with
breakfast after pre-dose bioimpedance spectroscopy measurements,
pharmacokinetics blood sampling, and other fasting blood sampling is completed.

Dapagliflozin has global market approval and based on global cumulative sale
figures up to March 2016 it is estimated that dapagliflozin has been
administered during >1000000 patient years.

Potential risks
The potential risks for the treatment with dapagliflozin and other SGLT2
inhibitors are described in the Investigator*s Brochure (IB). Due to its mode
of action resulting in increased urinary glucose excretion an increased risk of
urinary tract infections (slightly higher compared to placebo in the phase III
studies) and genital infections has been seen. Higher proportions of patients
with marked laboratory abnormalities of hyperphosphataemia were reported in
dapagliflozin vs placebo. The magnitude and clinical significance of this in
patients with CKD is unclear.
There have been reports of ketoacidosis, including diabetic ketoacidosis (DKA),
in patients with type 2 diabetes mellitus taking FORXIGA and other SGLT2
inhibitors. Patients presenting signs and symptoms consistent with
ketoacidosis, including nausea, vomiting, abdominal pain, malaise, and
shortness of breath, should be assessed for ketoacidosis, irrespective of blood
glucose levels. If ketoacidosis is suspected by the Investigator,
discontinuation or temporary interruption of dapagliflozin should be considered
and the patient should be promptly evaluated.
Predisposing factors to ketoacidosis include a low beta-cell function reserve
resulting from pancreatic disorders (e.g., T1DM, history of pancreatitis, or
pancreatic surgery), insulin dose reduction, reduced caloric intake, or
increased insulin requirements due to infections, illness or surgery and
alcohol abuse. Dapagliflozin should be used with caution in these patients.
Patients on sulphonylurea and/or insulin at the onset of the study treatment
have an increased risk of experiencing hypoglycaemic events. Blood glucose is
therefore monitored at Day 4. Once patients on insulin enter the study, they
will be carefully followed once starting on food boxes. Additionally, insulin
dosing will be adjusted, if needed, to avoid hypoglycaema/hyperglycaemia.
In this study, indocyanine green will be used to assess changes in plasma
volume. Indocyanine green has been used for decades for this purpose and has a
short half-life of only approximately 3 min (Jacob et al, 2007 [17]). The risk
profile of indocyanine green is considered good (Jacob et al, 2007 [18]), but
there are reports of allergic reactions including anaphylactic reactions
(Speich et al, 1988 [18] and Garski et al, 1978 [19]). In patients with
terminal renal insufficiency, the possibility that an anaphylactic reaction
occurs seems to be increased (Summary of Product Characteristics [SPC] Verdye
[20]).
Assessment of body composition (including extracellular and intracellular
volume) will be conducted with bioimpedance spectroscopy. This is an easy to
use and non-invasive technique not considered to cause any risk to the patient
if following the manufacturer*s instructions.
No other study procedure is considered putting the patients at risk beyond
those ordinarily encountered during the performance of routine medical
examinations or routine tests.

Protection against risks
This study has been designed with appropriate measures in place to monitor and
minimise any of the potential health risks to participating patients.
Based on the mechanism of action of dapagliflozin there may be a potential risk
for this compound to cause hypovolaemia or electrolyte imbalance. As a
precaution, patients who, in the judgement of the Investigator, may be at risk
for dehydration or volume depletion due to co-existing conditions or
concomitant medications, such as loop diuretics, should have careful monitoring
of their volume status. For this study, patients on a stable dose for at least
4 weeks of loop or thiazide diuretics will be allowed to participate. In
hypovolaemic patients starting treatment with dapagliflozin, there is a
potential risk for increased serum creatinine levels. Patients who show a
greater than 50% increase in serum creatinine should therefore be discontinued
on the study drug. In patients already receiving dapagliflozin who develop
conditions that may cause hypovolaemia or electrolyte imbalance, decisions to
interrupt or discontinue dapagliflozin therapy and management of patients
should be based on clinical judgement.

Safety signal detection will include the integration of all available sources
of safety information, including clinical study data, AE reports, pre-clinical
data, epidemiological studies and literature reports, to identify and
characterise unrecognised safety risks or changes in those which are currently
expected Adverse Drug Reactions. Any information that may affect the
benefit-risk profile of dapagliflozin will be immediately communicated to
relevant Health Authorities and appropriate actions will be taken regarding the
clinical programme as needed. Thus real-time, active safety surveillance will
be conducted during the entire duration of this study.
In addition, all dapagliflozin studies are subject to a carefully designed
patient risk management plan that includes the temporary and if necessary
permanent discontinuation of the investigational product in individual patients
in whom a potential health risk or a laboratory abnormality of clinical concern
has been identified.
Due to a potential risk of allergic reactions, the use of indocyanine green
should be performed under supervision of a physician. Symptoms related to an
allergic reaction may include unrest, feeling of warmth, pruritus, urticarial,
acceleration of heart rate, fall in blood pressure, shortness of breath,
bronchospasm, flush, cardiac arrest, laryngospasm, facial oedema, and nausea.
Patients with a known hypersensitivity to indocyanine green, sodium iodide, or
iodine, or patients who have poorly tolerated indocyanine green in the past
should not use indocyanine green again (see Section 9 Exclusion Criteria).
Additionally, patients with hyperthyroidism or with autonomic thyroid adenomas
are excluded (see SPC [21]). Some medicinal products and substances can reduce
or increase absorption of indocyanine green and should thus be avoided (see
Section 9, Exclusion Criteria). Due to the risk of allergic reactions including
anaphylactic reactions, emergency equipment should be available to immediately
start treatment of an allergic reaction if needed. For the bioimpedance
spectroscopy measurements, patients should not be pregnant, have a pacemaker or
other implanted electronic devices (see Section 9, Exclusion Criteria).

Potential benefits to patients
In this study, the dose of dapagliflozin 10 mg was chosen based on previous
clinical experience. This mechanistic study is non-therapeutic; therefore, it
has limit or no direct clinical benefit for the subjects. In studies of longer
duration, in patients randomised to active drug, dapagliflozin is expected to
reduce progression of renal failure and reduce cardiovascular mortality.
Dapagliflozin is known to decrease body weight (or prevent weight gain) as well
as lower blood pressure. Patients are also expected to receive some benefit in
the form of increased medical care/attention when participating in study
procedures.