Clinical trial NCT02796170
Conditions: Type 2 Diabetes Hypertension
Keywords: SGLT-2 inhibitor urinary angiotensinogen
Sponsor: Tulane University School of Medicine
Start date: 2016-03-01; Completion date: 2020-08-01
Enrollment: 11 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
To assess the effect of sodium-glucose cotransporter 2 (SGLT-2) inhibitors on blood pressure and urinary angiotensinogen. This is a cross over study design, where 40 subjects will receive Dapagliflozin for 6 weeks followed by placebo for 6 weeks, or placebo for 6 weeks followed by Dapagliflozin for 6 weeks. In addition there will be an arm of 10 subjects who will receive sulfonylurea in an open label as a comparative to the cross over subjects to assess if the effect of Dapagliflozin may also be in part due to improved glycemic control.

Detailed description:
Clinical trials of two SGLT2 inhibitors, canagliflozin and dapagliflozin, have reported drops in systolic blood pressure of \~5 mmHg. Inappropriate activation of intrarenal renin-angiotensin system (RAS) is a major contributor to the increased arterial pressure and tissue injury including diabetic nephropathy. A key factor in the intrarenal RAS activation is stimulation of intrarenal angiotensinogen (AGT) which is the precursor of angiotensin peptides. From previous studies, it has been shown that high blood sugars in patients with type1 and type 2 diabetes mellitus is accompanied by elevated intrarenal AGT and urinary AGT levels. High glucose results in stimulation of AGT production. The high glucose levels augments intrarenal AGT levels in diabetes mellitus leading to the development of high blood pressure and diabetic nephropathy.

The investigators propose to conduct a single-center randomized, double blind, cross over study of the effect of Dapagliflozin over 6 weeks, followed by placebo over 6 weeks on the other treatment allocation (those getting placebo first will cross over to Dapagliflozin and vice versa). Treatment will be stratified according to the underlying presence or absence of hypertension.

1. Type 2 diabetes with hypertension and on renin-angiotensin-aldosterone system (RAAS) blocking drugs with stable blood pressure on therapy; n= 20
2. Type 2 diabetes without hypertension and not on RAAS blocking drugs n=10

If unable to recruit 10 participants without hypertension the investigators will increase the number with hypertension for a total of 30. Stratification by hypertension status will remain and is important in understanding the effect of SGLT2 inhibition in patients not on BP lowering drugs.

In addition a Sulfonylurea (SU) arm will also be included - 10 participants who are on metformin and other background therapy (with the exclusion of SGLT-2 inhibitor and sulfonylurea) will be recruited. This will be an open-labeled arm. Participants will assessed at baseline. Participants will then receive usual care for 6 weeks. At the end of 6 weeks, participants will then undergo another assessment before being provided SU for 6 weeks. At the end of 6 weeks, participants will undergo assessment again. The aim is to determine whether any effects seen with Dapagliflozin are specific to that drug or related simply to improved glycemic control.

Arms: arm 1: Participants underwent 6 weeks of Dapagliflozin or placebo (washout period for 2 weeks) and then crossed over to 6 weeks of placebo or Dapagliflozin. arm 2: Participants underwent 6 weeks of Dapagliflozin or placebo (washout period for 2 weeks) and then crossed over to 6 weeks of placebo or Dapagliflozin.
Interventions: intervention 1: Dapagliflozin intervention 2: Placebo
Intervention descriptions: intervention 1: 5mg pill taken once daily intervention 2: 5mg pill taken once daily- placebo of Dapagliflozin

Protocol documents:
Prot_SAP_000.pdf: 
 
Version: 062019 
Page 1 
 
 
 
 
 
Inhibition of Urinary 
Angiotensinogen and the Reduction 
of Blood Pressure by SGLT2 
Inhibition in patients with Type 2 
Diabetes 
 
NCT Number: NCT02796170 
 
 
Protocol date: 06/20/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
Version: 062019 
Page 2 
 
Inhibition of Urinary Angiotensinogen and the Reduction of Blood Pressure by SGLT2 Inhibition in 
patients with Type 2 Diabetes  
 
Dragana Lovre, L. Gabriel Navar 
 
Project Goals: Type 2 diabetes is a complex disease where hyperglycemia occurs as a result of the 
development of insulin resistance.  It is often associated with obesity and is characterized by hyperglycemia, 
hyperinsulinemia, as well as hyperleptinemia.  In addition to the problems associated with poor glucose control, 
type 2 diabetes is accompanied by a chronic inflammatory state.  This chronic inflammatory state leads to a 
number of associated complications, including cardiovascular disease.  Diabetes increases one’s risk for 
cardiovascular disease by 2-4 fold1, highlighting the importance of developing therapeutics that both control 
blood glucose levels and reduce one’s risk of cardiovascular disease.  The monetary impact of diabetes on our 
healthcare system continues to grow as well.  Currently 25.8 million children and adults (8.3% of the 
population) in the United States suffer from diabetes2.  In addition to established traditional treatment options, 
one new approach has focused on blocking glucose reabsorption by the renal tubules to allow substantial 
excretion of glucose in the urine.  This project will elucidate molecular and cellular mechanisms behind the 
potential cardio protective effects of a new class of antihyperglycemic, sodium glucose co-transporter 2 
(SGLT2) inhbitors.  We will also investigate clinically a potential mechanism underlying the effect of these 
drugs on lowering blood pressure (BP) and raising potassium (K). 
 
Rationale: Clinical trials of two SGLT2 inhibitors, canagliflozin and dapagliflozin, have reported drops in 
systolic blood pressure of ~5 mmHg3, 4. Inappropriate activation of intrarenal renin-angiotensin system (RAS) is 
a major contributor to the increased arterial pressure and tissue injury including diabetic nephropathy. A key 
factor in the intrarenal RAS activation is stimulation of intrarenal angiotensinogen (AGT) which is the 
precursor of angiotensin peptides5.  In animal models of type1 and type 2 diabetes mellitus, hyperglycemia is 
accompanied by elevated intrarenal AGT and urinary AGT levels6-8.  Furthermore, direct treatment of rat renal 
proximal tubular cells (PTC) with high glucose results in stimulation of AGT production9.  These findings 
suggest that hyperglycemia augments intrarenal AGT levels in diabetes mellitus leading to the development of 
high blood pressure and diabetic nephropathy.  
 
 
By inhibiting glucose reabsorption in the proximal tubule, SGLT2 inhibition has the potential to block the AGT 
elevating effect of hyperglycemia. We therefore submit the following: 
 
Hypothesis 1: SGLT2 inhibition attenuates renal AGT elevation by hyperglycemia leading to decreased blood 
pressure, renoprotection and cardio protection in diabetes mellitus. This effect can be elucidated by 
simultaneously measuring urine AGT and 24 hour ambulatory blood pressure (ABPM) 
 
We will test this hypothesis in a pilot study by: 
 
Aim 1. Exploring the effect of SGLT2 inhibition on intra-renal AGT production in subjects with diabetes 
mellitus, who are known to have augmentation of intra – renal AGT.  
 
This will be a cross-over pilot study to elucidate whether this mechanism underlies the decrease in BP, as well 
as the rise in K, in humans with diabetes.  
 


 
Version: 062019 
Page 3 
 
 
Background 
 
 
Specific Aim: Explore the effect of SGLT2 inhibition on intra-renal AGT 
production in subjects with diabetes, who are known to have augmentation of intra 
– renal AGT. 
Rationale: We have recently demonstrated that urinary AGT is increased in patients 
with diabetes, including patients with diabetes without microalbuminuria and 
hypertension (not yet published). The increase is greatest in those with established 
nephropathy and hypertension, suggesting that this activation occurs early and may contribute to both 
hypertension and proteinuria.  Since SGLT2 inhibitors are approved only for type 2 diabetes we propose 
studying only type 2 patients, stratifying them as to whether they are on RAAS blocker drugs or not. We also 
propose to study the impact of the BP reduction on vascular compliance and augmentation index – indirect 
measures of endothelial function that are non-invasive and have been shown to be predictors of CV outcomes.  
 
The RAS has been portrayed as an endocrine, paracrine, autocrine and intracrine system and thus it has been 
difficult to delineate the quantitative contributions of systemically delivered versus locally formed Ang peptides 
to the levels existing in any given tissue. Emerging evidence indicates that local formation is of major 
significance in the regulation of the Ang levels in many organs and tissues. Various studies have demonstrated 
the importance of tissue RAS in the brain, heart, adrenal glands, vasculature, as well as in the kidney. While 
every organ system in the body has elements of the RAS, the kidney is unique in having every component of the 
RAS with compartmentalization in the tubular and interstitial networks as well as intracellular accumulation.  In 
this regard, the kidneys, as well as the adrenal glands, are unique in terms of the tissue concentrations of Ang II 
which are much greater than can be explained by the concentrations delivered by the arterial blood flow. 
Indeed, the major fraction of Ang II present in renal tissues is generated locally from AGT delivered to the 
kidney as well as from AGT locally produced by proximal tubule cells.  Ang I delivered to the kidney can also 
be converted to Ang II.   
 
Additional studies demonstrated that urinary excretion rate of AGT is closely correlated with systolic blood 
pressure and kidney Ang II content, but not with plasma Ang II concentration. This increase was not just due to 
increased proteinuria, or increased filtration of AGT into the tubular fluid.  The results indicate that urinary 
AGT originates primarily from the kidneys and not from the plasma.   
 
The principal action of ACE inhibition is a disruption of the conversion of Ang I to Ang II and consequently, 
inhibition of the Ang II effects such as vasoconstriction.  Angiotensin converting enzyme inhibition may trigger 
additional events as a result of protection of other peptides. Inhibition of ACE also prevents degradation of 
kinins into inactive peptides.  Kinins act as endogenous vasodilators via stimulation of nitric oxide and release 
vasodilatory prostaglandins; thus kinins contribute to renal vasodilatory actions of ACE inhibitors, particularly 
in the medullary circulation.  Indeed, blockade of ACE or deletion of kidney ACE reduces the severity of the 
hypertension that develops with chronic Ang II or Ang I infusions showing that ACE mediated de novo 
formation of Ang II is an essential step in the full development of hypertension.  Ang II receptor blockers 
(ARB) are a specific class of antagonists that block AT1 receptors at the tissue level.  Angiotensin II, generated 
by non-ACE mechanisms, could be blocked at the receptor level by an ARB.  However, increases in Ang II 
concentration after ARB therapy may stimulate AT2 receptors if receptors are not fully blocked.   
 


 
Version: 062019 
Page 4 
 
The use of ARBs and ACE inhibitors has become common practice in treating patients with diabetes. Because 
RAS activation plays such a central role in the development and progression of DN, there has been extensive 
interest in the potential hope for reduction in morbidity and mortality by using agents that block 1 or other ore 
steps in the RAS. Accordingly, the assessment of urinary AGT as an early biomarker of the status of the 
intrarenal RAS may be of substantial importance. It may be particularly helpful in serving as a means to 
determine efficacy of the treatment to reduce intrarenal angiotensin II levels.  
 
In our experience urinary AGT remains elevated in patients treated with ACEI or ARBs and present a novel 
treatment target (with an easily measurable biomarker) 
 
 
Hypothesis 2:  SGLT2 inhibition will reduce urinary AGT in subjects with 2 diabetes, and this reduction will 
correlate with reduction in BP.  
 
Rationale: 
Progression of hypertension and nephropathy in diabetes mellitus (DM). Elevated levels of angiotensinogen 
(AGT) are found in the urine of patients with type 1 DM and RAS blockade is commonly used to slow or 
prevent the development of diabetic nephropathy. AGT is upregulated in proximal tubular cells (PTC) exposed 
to high glucose conditions in response to the accumulation of reactive oxygen species (ROS). AGT forms 
angiotensin II (Ang II) which further upregulates AGT via angiotensin II type 1 receptor (AT1R) 
activation.AT1R signaling is another major source of ROS generation in PTC11 and AT1R blockade attenuates 
high glucose-induced AGT expression.12 Intrarenal RAS activation is strongly associated with hypertension 
and kidney injury.1, 10 However, the mechanisms underlying intrarenal RAS activation in DM have not been 
delineated. Plasma glucose is filtered at the glomerulus and almost completely reabsorbed in the proximal 
tubules. Sodium-glucose co-transporter 2 (SGLT2) is located in the early segment of the proximal tubule and is 
responsible for the majority of glucose reabsorption. Increased expression of SGLT2 and glucose reabsorption 
are observed in DM, but the mechanisms remain unclear. Ang II increases SGLT2 expression in hypertensive 
rats, but it is not known if AT1R activation in PTC is responsible. Recently, pharmacological blockade of 
SGLT2 has been developed as a treatment strategy for DM which normalizes blood glucose levels by reducing 
glucose reabsorption. SGLT2 inhibition also leads to decreased blood pressure and renoprotective effects.22, 
23, 24. These findings suggest a potential role for SGLT2 in the activation of the intrarenal RAS. However, it is 
not known if glucose entry via SGLT2 mediates high glucose-induced AGT expression in PTC. 
 
 
Preliminary Studies 
Immortalized mouse proximal tubule cells consists of cells originating from the early segment of the mouse 
proximal tubule.To demonstrate the effects of high glucose on AGT expression in this cell line, mPTCs were 
seeded on 6-well plates at a density of 5.0 x 105 cells per well. Before treatment in high glucose, the cells were 
rinsed with PBS and treated in 1.5 mL serum-free DMEM (5 mM glucose) for 8 hours. For high glucose 
treatment, serum-free Dulbecco Modified Eagle’s Medium (DMEM) was supplemented with glucose to a final 
concentration of 5 mM (control), 15 mM, or 25 mM (n = 4). The cells were incubated at 37 ºC for 24 hours. 
After treatment, cell lysates were prepared. AGT protein was detected via western blot analysis. 
The results of this experiment showed that our mPTC cell line exhibits a significant increase in AGT expression 
under high glucose conditions (15 mM and 25 mM glucose) compared to normal glucose (5 mM glucose). 
To test the effects of high glucose on AGT mRNA, cells were cultured as previously described in serum-free 
DMEM with 5 mM or 25 mM glucose (n = 3) and incubated at 37 ºC. 


 
Version: 062019 
Page 5 
 
After 24 hours, AGT mRNA was quantified via qRT-PCR. AGT mRNA relative to GAPDH mRNA was found 
to be significantly higher after treatment in 25 mM glucose. 
 
The results of these preliminary experiments demonstrate that our mPTC line exhibits very low expression of 
AGT under normal glucose conditions (5 mM).  
 
The impact of treatment with an SGLT2 inhibitor on intra –renal AGT production is unknown. Additionally, it 
is unclear what the impact of having significant glycosuria will have on AGT production. Finally, it is important 
to determine if there are differential effects on AGT production between SGLT2 inhibition and other methods 
of improving glycemic control, such as Sulfonylurea therapy 
 
 
Proposed Studies  
 
We propose to conduct a single-center randomized, double blind, cross over study of the effect of Dapagliflozin 
over 6 weeks, followed by placebo over 6 weeks on the other treatment allocation (those getting placebo first 
will cross over to Dapagliflozin and vice versa). Treatment will be stratified according to the underlying 
presence or absence of hypertension.  
1. Type 2 diabetes with hypertension and on RAAS blocking drugs with stable blood pressure on therapy 
for at least 30 days;  n= 20 
2. Type 2 diabetes without hypertension and not on RAAS blocking drugs n=10 
If we are unable to recruit 10 patients without hypertension we will increase the number with hypertension for a 
total of 30. Stratification by hypertension status will remain and is important in understanding the effect of 
SGLT2 inhibition in patients not on BP lowering drugs 
 
In addition a Sulfonlyurea (SU) arm will also be included – 10 patients who are on metformin and other 
background therapy (with the exclusion of SGLT-2 inhibitor and sulfonylurea) will be recruited. This will be an 
open-labeled arm. Patients will assessed at baseline. They will then receive usual care for 6 weeks. At the end of 
6 weeks, patients will then undergo another assessment before being provided SU for 6 weeks. At the end of 6 
weeks, patients will undergo assessment again.  The aim is to determine whether any effects seen with 
Dapagliflozin are specific to that drug or related simply to improved glycemic control. 
 
 
Inclusion criteria; 
As per the current approved indications for Dapagliflozin. In addition; 
1. Type 2 diabetes with hypertension (history of hypertension in clinical records and on medication for 
management of hypertension) and on RAAS blocking drugs; or 
2. Type 2 diabetes without hypertension and not on RAAS blocking drugs 
3. Hemoglobin A1c between 7% - 10% inclusive 
4. Background therapy for diabetes to include maximally tolerated dose of metformin. 
5. For the dapagliflozin/placebo arm, in addition to metformin, sulfonylureas and/or insulin is allowed 
6. Estimated glomerular filtration rate (eGFR) > = 60 ml/min 
7. Between the ages of 18 -75   
8. Capacity to understand and sign the informed consent. 
 
 


 
Version: 062019 
Page 6 
 
Exclusion criteria: 
1. Current use of Dapagliflozin or any SGLT2 inhibitor  
2. History of intolerability to Dapagliflozin 
3. Patients with current and past history of bladder cancer  
4. Patients with unexplainable baseline hematuria 
5. All current contraindications of Dapagliflozin as per the label 
6. Severe hepatic insufficiency and/or significant abnormal liver function defined as aspartate 
aminotransferase (AST) >3x upper limit of normal (ULN) and/or alanine aminotransferase (ALT) >3x 
ULN 
7. Total bilirubin >2.0 mg/dL (34.2 µmol/L) 
8. Estimated glomerular filtration rate (eGFR): <60 mL/min (calculated by Cockcroft-Gault formula) 
9. Recent Cardiovascular Events in a patient: 
10. Acute Coronary Syndrome (ACS) within 2 months prior to enrolment 
11. Hospitalization for unstable angina or acute myocardial infarction within 2 months prior to enrolment 
12. Acute Stroke or TIA within two months prior to enrolment 
13. Less than two months post coronary artery revascularization 
14. Congestive heart failure defined as New York Heart Association (NYHA) class IV, unstable or acute 
congestive heart failure. Note: eligible patients with congestive heart failure, especially those who are on 
diuretic therapy, should have careful monitoring of their volume status throughout the study. 
15. Pregnant or breastfeeding patients 
16. Patients who, in the judgment of the investigator, may be at risk for dehydration 
17. Blood pressure: At enrolment: Systolic BP ≥165 mmHg and/or diastolic BP ≥110 mmHg 
a. At randomization: Systolic BP ≥160 mmHg and/or diastolic BP ≥100 mmHg 
18. For patients in the dapagliflozin/placebo arm, use of SGLT-2 inhibitor class of drugs is an exclusion. 
Patients may continue the background therapy.  
a. For patients that will be included in the sulfonylurea/placebo arm – use of SLGT-2 inhibitor and 
sulfonylurea class of drugs in an exclusion. Patients can continue the rest of the background 
therapy.   
Methods:  
Once the subjects have been provided with the informed consent procedure and have signed the 
approved consent form and HIPAA form, the screening procedure will commence.  Screening will 
include a completed medical history and list of current medications to determine eligibility for 
participation in this study. Details of the procedures are listed in the visit schedule section (see below).  
 
 
Blinding and unblinding:  
The research study participants will be blinded throughout the study in the dapagliflozin-placebo arm.  
Unblinding will be done only in case of emergency for patient safety. In this case, unblinding will be 
done by the sponsor or the research site as the randomization code will be with the sponsor.  
 
The SU arm will be open label arm.  
 
Storage of investigational product:  
All study drugs should be kept in a secure place and under appropriate storage conditions. The Investigational 
Product label on the bottle specifies the appropriate storage  
 
Accountability:  


 
Version: 062019 
Page 7 
 
Storage of IP will be handled by the Investigational Pharmacist, who will maintain both accountability 
for the IP as well as maintenance of the IP, according to the manufacturer’s requirements and 
recommendations for product safety. 
 
Labeling of drug:  
The IP and placebo will be labeled identically. The labels will be prepared according to Good 
Manufacturing Practice (GMP) and local regulatory guidelines. AZ will provide with prefilled bottles of 
Dapagliflozin 5 mg and the matching placebo. Labelling will be done by the sponsor.  
 
Randomization:  
The randomization code will be generated and provided by the sponsor to the site which the research site 
personnel will follow.  
 
Dose and treatment regimens: After the subject has started the 5 mg dose of Dapagliflozin or the 
medication in test period 1, the subject will be called 1 week after initiation of study drug. If the subject 
is tolerating the medication, the dose will be increased to 2 tabs of placebo or 10mg of Dapagliflozin. If 
for some reason, the subject is not tolerating the medication the subject will continue on 5 mg daily, 
particularly if dizziness is present. For the sulfonylurea arm, titration of the sulfonylurea and the placebo 
will be at the principal investigator’s discretion as per usual care. For all the patients, titration of the 
background medications that have the potential of causing hypoglycemia, titration of the background 
medications will be at the principal investigator’s discretion. The purpose of titration will be to prevent 
hypoglycemia while the patient is in the study.  
 
Discontinuation of IP:  
The investigational drug will be discontinued according to the following criteria:  
Discontinuation procedures 
Patients with a central laboratory ALT and/or AST >3x ULN will be scheduled for a follow-up visit 
within 3 days following the receipt of the result.  Patients will be discontinued from study if the initial 
and repeat laboratory tests meet any of the following criteria: 
1. ALT and/or AST are >3 x ULN and TB >2 x ULN 
2. ALT and/or AST are >5 x ULN for ≥ 14 consecutive days, at any time after initial confirmatory 
results 
3. ALT and /or AST are > 8 x ULN 
4. Study drug will be discontinued when eGFR is persistently <60 ml/min/1.73m2. Clinical judgement 
by the Principal Investigator will be used to determine if the observed decrease in eGFR < 60 
ml/min/1.73m2 warrants discontinuation after the eGFR is repeated after the initial decrease  in eGFR is 
noted.  
5. Pregnancy confirmed by a positive pregnancy test or otherwise verified  
 
Sample size calculation and statistical analysis:  
This is a pilot study. Data from this study can be used to derive the sample size for an adequately 
powered study. Exploratory data analysis: All data will be cleaned and logically checked.  
Exploratory analysis will be conducted to examine the distributions of each variable by means of plots 
(bar charts, box plots, and normal probability plots) and goodness-of-fit test statistics. Means and 
percentages of baseline variables will be compared between the groups. Possible outliers will be 
detected. Data transformation will be used to normalize the distribution of continuous variables and to 
improve linearity in the linear regression analyses if needed.  
 


 
Version: 062019 
Page 8 
 
Comparisons of continuous variables between groups will be performed using t-tests. Chi-square tests 
will be employed for comparisons between categorical data. Linear regression models will be fit to 
examine the association between a continuous dependent variables and possible independent co-
variables. Data analyses will be performed using SAS version 9 (SAS Institute Inc., SAS/STAT User’s 
Guide, Version 9. Cary, NC: SAS Institute Inc. 2007). 
 
 
Patient Safety:  
A copy of the MedWatch/AdEERs report will be faxed to AstraZeneca at the time the event is reported to 
the FDA.  It will be the responsibility of the investigator to compile all necessary information and ensure 
that the FDA receives a report according to the FDA reporting requirement timelines and to ensure that 
these reports are also submitted to AstraZeneca at the same time. When reporting to AstraZeneca, a cover 
page will accompany the MedWatch/AdEERs form indicating the following: 
- 
Investigator Sponsored Study (ISS) 
- 
The investigator IND number assigned by the FDA 
- 
The investigator’s name and address 
- 
The trial name/title and AstraZeneca ISS reference number 
The collection of AEs should start after the signing of the informed consent.  Investigative site will also 
indicate, either in the SAE report or the cover page, the causality of events in relation to all study 
medications and if the SAE is related to disease progression, as determined by the principal investigator. 
The site research personnel will send SAE report and accompanying cover page by way to AstraZeneca: 
AEMailboxClinicalTrialTCS@astrazeneca.com or fax number is 302-886-4114.  
Serious adverse events that do not require expedited reporting to the FDA will be reported to AstraZeneca 
preferably using the MedDRA coding language for serious adverse events.  In the case of blinded trials, 
AstraZeneca will request that the Sponsor either provide a copy of the randomization code/ code break 
information or unblind those SAEs which require expedited reporting. All SAEs have to be reported to 
AstraZeneca, whether or not considered causally related to the investigational product.  All SAEs will be 
documented.  The investigator is responsible for informing the IRB and/or the Regulatory Authority of the 
SAE as per local requirements. 
 
Visit Schedule: 
Following is the schedule of the study visits: 
 
1. Visit 1(screening visit): Screening and informed consent will be conducted during this visit.  If subjects 
meet the inclusion criteria they will come back within 4 weeks for randomization.  If the required 
laboratory tests to establish whether the subject meets the inclusion criteria are available and have been 
drawn within the past ninety days, no laboratory tests will be required at this visit.  However, if not, 
laboratory tests will be drawn at this visit for the purpose of establishing that the subject meets the 
criteria for inclusion in the study. The patient will be asked to keep a log of the blood sugars while 
participating in the study.  
 
2. Visit 2 (baseline visit): During this fasting baseline visit, subject will undergo history and physical, 
blood draw, urine sample, EndoPAT test and given a bottle for 24 hour urine collection. Subjects will 


 
Version: 062019 
Page 9 
 
be given the monitor for 24 hr. BP monitoring.  The patient will return the monitor along with a 24 hour 
urine sample 
 
If however during the screening visit, the laboratory results are available for review and the  
patient qualifies for the study, the screening and the baseline visits will be combined. 
 
3. Visit 3 (randomization visit) - subject will return the ABPM monitor and urine bottle and will be 
randomized to either dapagliflozin or placebo in a double-blind fashion. The first arm of the study will 
be labeled test period 1 and the second arm of the study will be labeled test period 2. All subjects will 
be educated on the recognition and management of hypoglycemia at this visit. 
 
4. Test period 1: Test period 1 will start once the subject has taken the first dose of the drug to which the 
subject was randomized. Test period 1 will continue for 6 weeks.  
 
5. Visit 4 (Telephone visit). After the subject has started the 5 mg dose of the medication in test period 1, 
the subject will be called 1 week after initiation of study drug. If the subject is tolerating the 
medication, the dose will be increased to 2 tabs (10mg). If for some reason, the subject is not tolerating 
the medication the subject will continue on 5 mg daily, particularly if dizziness is present. The patients 
will also be asked if they have had any low blood sugars and what time the low blood sugars were. The 
blood sugars will be relayed to the PI in case there is any hypoglycemia. The PI will then make the 
necessary changes to the patient’s background therapy to prevent further hypoglycemia. 
 
6. Visit 5 (telephone visit): Visit 5 will be a telephone visit that will occur 2 weeks after the visit 4 
telephone visit. The purpose of the telephone visit is to ensure that the subject is tolerating the 
medication. This is particularly important if the dose had been increased to the maximum dose of 2 
tablets during visit 4. The patients will also be asked if they have had any low blood sugars and what 
time the low blood sugars were. The blood sugars will be relayed to the PI in case there is any 
hypoglycemia. The PI will then make the necessary changes to the patient’s background therapy to 
prevent further hypoglycemia. 
 
 
7. Visit 6 (cross over visit) –Visit 6 will occur 6 weeks +/- 7 days after randomization. The subject will 
come in fasting for this visit. During this visit, subject will undergo a brief physical examination. Blood 
and urine sample will be collected and the EndoPAT procedure will be conducted. Subject will take the 
last dose of the first lot of medication on the day of visit 6. Subject will start the24 hr. AMBP and 24 
hour urine collection on the day of visit 6 after taking the last dose of the medication for test period 1.  
 
8. Visit 7: Next day. Return AMBP monitor and then start the washout period which will last at least 14-28 
days. 
 
9. Test period 2: Test period 2 will start with visit 8 
 
10. Visit 8: After the washout period of 14-28 days, the subject will come to the research suite in a fasting 
state and undergo blood and urine sample collection. Patient will receive the second medication and 
enter test period 2. The subject will start the new lot of medication- dapagliflozin at 5 mg (1 tablet) or 
placebo. During this visit, the subject will undergo EndoPAT procedure and will be started on ABPM. 


 
Version: 062019 
Page 10 
 
 
11. Visit 9 (the day after visit 8):  The subject will return the ABPM. 
 
12. Visit 10 (telephone visit): After the subject has started the medication in test period 2, the subject will 
be called 1 week later. If the subject is tolerating the medication, the dose will be increased to 2 tablets 
daily (10 mg). If for some reason, the subject is not tolerating the medication the subject will continue 
on 5 mg daily, particularly if dizziness is present. The patients will also be asked if they have had any 
low blood sugars and what time the low blood sugars were. The blood sugars will be relayed to the PI 
in case there is any hypoglycemia. The PI will then make the necessary changes to the patient’s 
background therapy to prevent further hypoglycemia. 
 
 
13. Visit 11 (telephone visit): will be a telephone visit that will occur 2 weeks after visit 10 which is also a 
telephone visit. The purpose of the telephone visit is to ensure that the subject is tolerating the 
medication. This is particularly important if the dose had been increased to the maximum dose of 2 
tablets (10 mg) during visit 7. The patients will also be asked if they have had any low blood sugars and 
what time the low blood sugars were. The blood sugars will be relayed to the PI in case there is any 
hypoglycemia. The PI will then make the necessary changes to the patient’s background therapy to 
prevent further hypoglycemia. 
 
14. Visit 12; (end of treatment visit): This visit will conclude test period 2 six weeks after the washout 
period. During this fasting visit, a brief physical examination will be done, blood and urine samples 
will be collected, and the EndoPAT procedure will be conducted. Subjects will take the last dose of the 
second lot of medication on the day of visit 11. Subject will start the 24 hr. AMBP and urine collection 
after taking the last dose of the medication for test period 2. 
 
  
15. Visit 13: The day after visit 12, the subject will return the AMBP monitor. This concludes the study.  
 
 
NOTE: In order to give patients flexibility all visit will have a window of +/ - 7 days for visits 
Study Flow chart: 
 
 
 
A: For the study comparing Dapagliflozin and placebo: 
 
 
 
 
 
 
 
 
 
 
Screening visit  
(Visit 1) 
Excluded  
Included 
Test Period 1 


 
Version: 062019 
Page 11 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1-  (Visit 2 is the baseline visit: within 4 
weeks of the screening visit) History and 
physical examination 
2- Fasting laboratory work up  
 (Collection of fasting blood and urine 
samples) 
3- Subject is started on ABPM 
4- EndoPAT procedure conducted 
Visit 3 (Randomization visit)- 
(the day after visit 2) 
Subject returns the ABPM and 24 hour 
urine 
No blood or urine collection 
Visit 4 (Telephone visit- 1 week +/- 7 days 
after visit 3) 
To see if subject is tolerating the medication 
If subject is tolerating medication –  
Then the dose will be increased to maximum dose 
Visit 5 (Telephone visit- 2 weeks +/- 7 days 
after visit 4) 
To see if subject is tolerating the maximum dose 
of the medication  
Visit 6 (cross over visit- 6 weeks +/- 7 days 
after visit 2) 
1- physical examination 
2- Fasting laboratory work up  
 (Collection of fasting blood and urine samples) 
3- Subject is started on ABPM.  Subject takes 
the last dose of the medication given in test 
period 1 
4- EndoPAT procedure conducted. 


 
Version: 062019 
Page 12 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Visit 7 (the day after visit 6) 
Subject returns the ABPM and urine 
No physical exam 
No blood or urine collection 
Subject enters the washout period for 14-28 days 
Washout period (14-28 days)  
Test Period 2  
Visit 8(on the last day +/- 7 days of the washout period)  
1- Subject will be given the medication for test period 2 
2- Subject will start the same visit schedule as from Visit 3 
3- Collection of fasting blood and urine samples 
4- Patient is started on ABPM 
5- EndoPAT procedure conducted  
Visit 10(Telephone visit- 1 week +/- 7 days 
after visit 8) 
To see if subject is tolerating the medication 
If subject is tolerating medication –  
Then the dose will be increased to maximum dose 
 
Visit 11 (Telephone visit- 2 weeks +/- 7 days 
after visit 10) 
To see if subject is tolerating the maximum dose 
of the medication  
Visit 9 (the day after visit 8) 
Patient returns the ABPM and 24 hour urine collection 


 
Version: 062019 
Page 13 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Protocol for the sulfonylurea arm:  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening visit  
(Visit 1) 
Excluded  
Included  
Test Period 1 
 (Visit 2- within 4 weeks of the screening 
visit)  
1- History and physical examination 
2- Fasting laboratory work up  
 (Collection of fasting blood and urine 
samples) 
3- Subject is started on ABPM 
Visit 12 (cross over visit- 6 weeks +/- 7 days 
after visit 8) 
5- physical examination 
6- Fasting laboratory work up  
 (Collection of fasting blood and urine samples) 
7- Subject is started on ABPM.  Subject takes 
the last dose of the medication given in test 
period 2 
8- EndoPAT procedure conducted. 
Visit 13 (the day after visit 12) 
 
Subject returns the ABPM and 24 hour urine 
collection 
No physical exam 
No blood or urine collection 


 
Version: 062019 
Page 14 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Visit 3  
Patient will receive usual care 
(the day after visit 2) 
Subject returns the ABPM and 24 hour urine 
No blood or urine collection 
Visit 4 (Telephone visit- 1 week +/- 7 days 
after visit 3) 
Patients’ medications will be adjusted according 
to usual care. Usual care will be at the physician’s 
discretion. Sulfonylurea class of drugs and 
SGLT2 inhibitor class of drugs will be prohibited 
in this part of the arm 
Visit 5 (Telephone visit- 2 weeks +/- 7 days 
after visit 4) 
To assess patients’ blood sugars and make 
adjustments according to usual care 
Visit 6 (cross over visit- 6 weeks +/- 7 days after visit 2) 
 
1- physical examination 
2- Patient continues background therapy 
3- Fasting laboratory work up  
 (Collection of fasting blood and urine samples) 
4- Subject is started on ABPM.   
Visit 7 (the day after visit 6) 
Subject returns the ABPM and urine 
No physical exam 
No blood or urine collection 


 
Version: 062019 
Page 15 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Test Period 2  
Visit 8  
1- Subject will be given SU class of drug for test period 2 
according to physician’s discretion 
2- Subject will start the same visit schedule as from Visit 3 
3- Subject will continue the background therapy as before 
Visit 9(Telephone visit- 1 week +/- 7 days 
after visit 8) 
To see if subject is tolerating the medication and assess patients’ blood sugars 
 
Then the dose of SU will be according to the blood sugars and physician’s discretion for 
glycemic control 
Visit 10 (Telephone visit- 2 weeks +/- 7 days after visit 9) 
To assess patient’s blood sugars. Adjustment to dose of SU will be adjusted according 
to the blood sugars at physician’s discretion 
Visit 11 (cross over visit- 6 weeks +/- 7 days after visit 8) 
1- physical examination 
2- Fasting laboratory work up  
 (Collection of fasting blood and urine samples) 
3- Subject is started on ABPM.  Subject takes the last dose of 
the SU given in test period 2 


 
Version: 062019 
Page 16 
 
 
 
 
 
 
 
 
 
 
 
 
Measurements:  
1- Blood pressure will be measured as per clinical practice and we will also do a 24 hr. ambulatory 
blood pressure monitoring before and after 6 weeks of treatment in the Dapagliflozin/ placebo arm 
as well as before and 6 weeks after treatment in the Sulfonylurea/placebo arm 
2- We will also measure vascular compliance/ endothelial function using the EndoPAT test at the same 
visits as the ABPM only in the Dapagliflozin/placebo arm. 
3- Spot and 24 hour urine will be collected. Urine samples will be obtained, before and after treatment. 
After appropriate processing, the samples will be quickly stored at -20°C, and analyzed for 
concentrations of AGT, albumin, and creatinine within one week of collection as previously 
described. The urinary AGT concentrations and urinary albumin concentrations will be normalized 
by urinary creatinine concentrations. Urine creatinine will also be measured and urine AGT/ 
creatinine calculated to correct the AGT for volume of excretion. Plasma K, renin and AGT will be 
measured at these visits.  
4- Fasting plasma glucose will be measured for the Dapagliflozin/placebo and sulfonylurea arm at the 
following time points: a) at baseline and end of period 1 and b) at baseline and end of period 2. 
Blood and urine will be stored at -70°C, for future testing of other biomarkers of renal and 
cardiovascular disease. 
 
 
 
 
 
 
 
The table below summarizes the timings of the measurements:  
Measurements 
Timings 
ABPM and 24 hour urine collection  
Dapagliflozin/Placebo arm  
- 
Before randomization visit (in test period 1)  
- 
At the end of test period 1 (before entering washout 
period)  
- 
At the beginning of test period 2 
- 
At the end of test period 2 (at the end of the study) 
 
 
Sulfonylurea arm 
- 
Before usual care arm visit (ie visit 2 in test period 1)  
Visit 12 (the day after visit 11) 
 
Subject returns the ABPM and 24 hour urine 
collection 
No physical exam 
No blood or urine collection 


 
Version: 062019 
Page 17 
 
- 
At the end of test period 1  
- 
At the end of test period 2 (at the end of the study) 
 
Fasting glucose, blood and urine 
samples 
Dapagliflozin/placebo  
- 
At the beginning of test period 1 
- 
Before the end of test period 1 
- 
At the beginning of test period 2 
- 
At the beginning of test period 2 
 
Sulfonylurea arm 
- 
Before the usual care arm visit (in test period 1)  
- 
At the end of test period 1  
- 
At the end of test period 2 (at the end of the study) 
 
EndoPAT Procedure 
- 
Dapagliflozin/placebo arm only 
- 
Before randomization visit (in test period 1) 
- 
At the end of test period 1 
- 
At the beginning of test period 2 
- 
At the end of test period 2 
 
 
The primary end point will be reduction in urine AGT comparing the AGT before and after treatment 
using a paired ANOVA Test.  
Exploratory endpoints will be: 
1. Examine the relationship between change in plasma K and change in urine AGT 
2. Examine the relationship between change in BP and change in endothelial function/vascular 
compliance using the EndoPAT procedure. 
 
 Depending on results of this pilot study we will plan further definitive studies of the effect of SGLT2 
inhibition on urine AGT, in a study with appropriate statistical power.  
 
 
Forecasted key milestones dependent on IRB/IEC approval and contracting  
 
1. Research Agreement executed 
 
Feb 28, 2016 
2. Projected IRB/IEC approval 
 
Feb 28, 2016 
3. First Subject In 
 
 
 
Apr 29, 2016 
4. Last Subject In (100% enrollment)    
Apr 30, 2017 
5. Last Subject Last Visit (Treatment end)   Jun 30, 2017 
6. Publication                    
 
 
Jan 30, 2018 
 
 
 
 
 
 
 


 
Version: 062019 
Page 18 
 
References 
1. 
McFarlane SI. In: Izzo JL, Black HR, eds. Hypertension primer: The essentials of high blood pressure. 
Lippincott Williams & Wilkins; 2003:488. 
2. 
National diabetes fact sheet. 2011 
3. 
List JF, Woo V, Morales E, Tang W, Fiedorek FT. Sodium-glucose cotransport inhibition with 
dapagliflozin in type 2 diabetes. Diabetes Care. 2009;32:650-657 
4. 
Foote C, Perkovic V, Neal B. Effects of sglt2 inhibitors on cardiovascular outcomes. Diab Vasc Dis Res. 
2012;9:117-123 
5. 
Navar LG, Prieto MC, Satou R, Kobori H. Intrarenal angiotensin ii and its contribution to the genesis of 
chronic hypertension. Curr Opin Pharmacol. 2011;11:180-186 
6. 
Kamiyama M, Zsombok A, Kobori H. Urinary angiotensinogen as a novel early biomarker of intrarenal 
renin-angiotensin system activation in experimental type 1 diabetes. J Pharmacol Sci. 2012;119:314-323 
7. 
Miyata K, Ohashi N, Suzaki Y, Katsurada A, Kobori H. Sequential activation of the reactive oxygen 
species/angiotensinogen/renin-angiotensin system axis in renal injury of type 2 diabetic rats. Clin Exp 
Pharmacol Physiol. 2008;35:922-927 
8. 
Park S, Bivona BJ, Kobori H, Seth DM, Chappell MC, Lazartigues E, Harrison-Bernard LM. Major role 
for ace-independent intrarenal ang ii formation in type ii diabetes. American journal of physiology. 
Renal physiology. 2010;298:F37-48 
9. 
Hsieh TJ, Zhang SL, Filep JG, Tang SS, Ingelfinger JR, Chan JS. High glucose stimulates 
angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubular 
cells. Endocrinology. 2002;143:2975-2985 
10. 
Leiter EH, Reifsnyder PC, Xiao Q, Mistry J. Adipokine and insulin profiles distinguish diabetogenic and 
non-diabetogenic obesities in mice. Obesity (Silver Spring). 2007;15:1961-1968 
11. 
Cho YR, Kim HJ, Park SY, Ko HJ, Hong EG, Higashimori T, Zhang Z, Jung DY, Ola MS, Lanoue KF, 
Leiter EH, Kim JK. Hyperglycemia, maturity-onset obesity, and insulin resistance in noncnzo10/ltj 
males, a new mouse model of type 2 diabetes. Am J Physiol Endocrinol Metab. 2007;293:E327-336 
12. 
Brosius FC, 3rd, Alpers CE, Bottinger EP, Breyer MD, Coffman TM, Gurley SB, Harris RC, Kakoki M, 
Kretzler M, Leiter EH, Levi M, McIndoe RA, Sharma K, Smithies O, Susztak K, Takahashi N, 
Takahashi T, Animal Models of Diabetic Complications C. Mouse models of diabetic nephropathy. J 
Am Soc Nephrol. 2009;20:2503-2512 
13. 
Zhao D, Zhang J, Blaustein MP, Navar LG. Attenuated renal vascular responses to acute angiotensin ii 
infusion in smooth muscle-specific na+/ca2+ exchanger knockout mice. Am J Physiol Renal Physiol. 
2011;301:F574-579 
14. 
Cervenka L, Mitchell KD, Oliverio MI, Coffman TM, Navar LG. Renal function in the at1a receptor 
knockout mouse during normal and volume-expanded conditions. Kidney Int. 1999;56:1855-1862 
15. 
Kanai Y, Lee WS, You G, Brown D, Hediger MA. The human kidney low affinity na+/glucose 
cotransporter sglt2. Delineation of the major renal reabsorptive mechanism for d-glucose. The Journal of 
clinical investigation. 1994;93:397-404 
16. 
Kolb RJ, Woost PG, Hopfer U. Membrane trafficking of angiotensin receptor type-1 and 
mechanochemical signal transduction in proximal tubule cells. Hypertension. 2004;44:352-359 
17. 
Liang Y, Arakawa K, Ueta K, Matsushita Y, Kuriyama C, Martin T, Du F, Liu Y, Xu J, Conway B, 
Conway J, Polidori D, Ways K, Demarest K. Effect of Dapagliflozin on renal threshold for glucose, 
glycemia, and body weight in normal and diabetic animal models. PloS one. 2012;7:e30555 
18. 
Lightell DJ, Jr., Moss SC, Woods TC. Loss of canonical insulin signaling accelerates vascular smooth 
muscle cell proliferation and migration through changes in p27kip1 regulation. Endocrinology. 
2011;152:651-658 


 
Version: 062019 
Page 19 
 
19. 
Lightell DJ, Jr., Woods TC. Relative resistance to mammalian target of rapamycin inhibition in vascular 
smooth muscle cells of diabetic donors. Ochsner J. 2013;13:56-60 
20. 
Coleman CB, Lightell DJ, Jr., Moss SC, Bates M, Parrino PE, Woods TC. Elevation of mir-221 and -
222 in the internal mammary arteries of diabetic subjects and normalization with metformin. Molecular 
and cellular endocrinology. 2013 
 
 
 
Budget 
We will need support in the form of  
1. Study drug and placebo pills 
2. Funding –  


 
Version: 062019 
Page 20 
 
 
Appendix A: Identity of investigational product(s) 
 
 
 
Investigational 
Product 
Manufacturer 
IMP/ 
NIMP 
Type 
Route of 
administration 
Formulation 
Comment 
Dapagliflozin 5 
mg  
AstraZeneca 
IMP 
Active 
Drug  
Oral  
Film Coated 
Tablet   
Green, plain, 
diamond shaped  
Matching placebo 
for Dapagliflozin 
5 mg  
AstraZeneca 
IMP 
Placebo  
Oral 
Film Coated 
Tablet   
Green, plain, 
diamond shaped 
AstraZeneca will provide the Dapagliflozin and matching placebo tablets. Dapagliflozin and matching placebo 
tablets will be supplied in bottles, each containing 35 tablets. The tablets contain lactose, which may cause 
discomfort in lactose-intolerant individuals.