Clinical trial NCT02964585
Conditions: Type 2 Diabetes Mellitus
Keywords: Type 2 Diabetes Mellitus Endothelial Cells Cellular Biomarker Endothelial Dysfunction CD34+ Impaired renal function
Sponsor: George Washington University
Start date: 2016-11-01; Completion date: 2020-09-01
Enrollment: 34 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The investigators hypothesize that Cana may be able to improve number and function of CD34+ endothelial progenitor cells. The investigators also propose that this expected cardiovascular benefit is independent of HbA1C reduction.

Subjects will begin taking 100 mg of Cana or placebo after initial 4 weeks. Subjects will be withdrawn from the study if the medication or placebo is not tolerated.

Detailed description:
Diabetes affects more than 11% of adults in the United States and this is projected to nearly double by 2025. Both diabetes and obesity are associated with endothelial dysfunction, oxidative stress, endothelial cell inflammation, cardiovascular pro-thrombotic states and are the most common causes of kidney disease. Use of a sodium-glucose linked transporter (SGLT-2) inhibitor has shown promise in improving glycemic control, weight reduction, hypertension and even changes in circulating Renin-angiotensin-aldosterone system (RAAS) and nitric oxide (NO). However, whether these group of drugs have any effect on cardiovascular disease (CVD) risk modification or on endothelium or endothelial progenitor cells as a surrogate of cardiovascular and renal risk outcome measure, is unclear.

The investigators have previously shown that CD34+ cells, derived from peripheral blood can act as a cellular biomarker that is more reliable than serum based markers for CVD risk estimation. Serum based inflammatory markers are not useful until the endothelium is already damaged and inflamed. Such serum based biomarkers takes several months to change and gives no preventive and predictable information as to whether a particular medication may affect future endothelium. This is why the study of endothelium progenitors is crucial. In the investigators' previous study of a prediabetes population with an aerobic exercise intervention, the investigators have demonstrated that CD34+ cells are responsive to a change in therapy or intervention within 2-4 weeks and can be used as a reliable non serum based cellular bio-marker. CD34+ cells or endothelial progenitor cells have been used clinically to improve collateral circulation and have been extensively studied as a robust cardiovascular biomarker. Therefore studying CD34+ cells in patients, with or without Canagliflozin (Cana) can give vital information about the medication and its effect on endothelium. This is particularly important as another SGLT2 inhibitor Empagliflozin has shown unparalleled positive cardiovascular effects with an oral hypoglycemic agent. Of course, the question arises whether this clinical trial effect is secondary to glucose effect or direct effect of SGLT2 inhibitor on endothelium.

Multiple glucose transporters have been identified in human cells these include GLUTs, SGLTs and even taste receptors (such as TLR2 and TLR3). The investigators know SGLT transporters are present in tubular cells and clearly blocking of SGLT2 in these cells is beneficial. Information on glucose transporter in stem or progenitor cells is almost nil. In our lab the investigators have shown presence of GLUT1, SGLTs and TLR3 on CD34+ cells. The investigators have also demonstrated that hyperglycemia is toxic to CD34+ cells, more than CD31+ positive mature endothelial cells. The investigators hypothesize that blocking SGLT2 in CD34+ cells will be beneficial rather than detrimental. As far as glucose uptake in CD34+ cells are concerned other glucose transporters should be sufficient, in fact lesser amount of glucose entry in a hyperglycemic milieu (type 2 DM patients) may be less pro-inflammatory and less pro-apoptotic.

Our preliminary data indicates that mRNA gene expression of both SGLT1 and SGLT2 are noted on human CD34+ cells however only SGLT2 mRNA gene expression is up-regulated several fold in human CD34+ cells in presence of hyperglycemia (20mM glucose). However non primary commercially obtained human endothelium (HUVEC) do not show similar results. An explanation could be SGLT2 expression decreases as the cell transitions from progenitor to mature endothelium. From these results the investigators believe SGLT2 inhibitor will be effective on progenitors and not mature endothelium. The investigators therefore hypothesize that CD34+ cells will be an ideal biomarker to study the effect of the drug. It is possible that Cana, by blocking SGLT2 receptors, may influence other CD34+ cell surface receptors including other glucose transporters and influence its function (most importantly migration). If a particular medication positively influences stem/progenitor cell migration then that medication can positively influence endothelial dysfunction and vascular complications from diabetes. The investigators are particularly interested to note effect of Canagliflozin, a SGLT2 inhibitor on other glucose transporters such as GLUT 1 and 4 while looking at SGLT 1 and 2 on CD34+ cells. It will be helpful to discern these effects particularly when choice of oral diabetic medication in a type 2 diabetes population is practically limited to metformin, DPP4 inhibitors and SGLT2 inhibitors. The investigators plan to investigate the effect of Cana on CD34+ cells, in a placebo matched study. The investigators plan to recruit subjects with type 2 diabetes with the following characteristics: 1) overweight, mild and moderately obese (BMI=25.0-39.9); 2) individuals with early type 2 diabetes (≤15 years) with inadequate control, HbA1C= 7.0 to 10.0%, on Metformin (1-2 grams/day) 3) with no history or presence of macrovascular complication and CKD no higher than stage 2. The subjects will be on Metformin as per ADA, Metformin is the 1st line of care along with life-style modification. While Metformin on its own may affect inflammatory biomarkers, the effect is minimal at best, particularly in presence of CKD and endothelial dysfunction. Also both placebo and the cana group will be on Metformin.

The investigators will recruit a total of 40 patients (20 individuals/per group) with approximately a 20% drop out rate over two years and the investigators hope to retain 32 individuals (16/group). Individuals in each group will be matched by sex, age, and race. Participants will be assessed at baseline (week 0), and at 2 and 4 months of drug intake.

Arms: arm 1: 100 mg of Canagliflozin for 16 weeks arm 2: Placebo for 16 weeks
Interventions: intervention 1: Canagliflozin intervention 2: Placebo
Intervention descriptions: intervention 1: 100 mg intervention 2: 1 tablet daily for 16 weeks

Protocol documents:
Prot_SAP_000.pdf: 
Canagliflozin-T2DM Study 
 
PI: Sabyasachi Sen, MD, FRCP, PhD, FACP, FACE 
Canagliflozin – Sen IIS 
Protocol Version: 3.0 
Date: 12/15/2017 
Title Page 
 
Role of Canagliflozin on gene expression and function 
of CD34+ endothelial progenitor cells and renal 
function in patients with type 2 diabetes  
 
Sabyasachi Sen, MD, FRCP (UK), PhD, FACP, FACE 
 
George Washington University Medical Faculty Associates 
2150 Pennsylvania Ave NW, Suite 4-403 
Washington, D.C. 20037 
Phone – (202)-994-8560 
 
Institutional Protocol Number: GW-CANA-3.25.2016 
Protocol Version: Version 3.0 
December 2017 
 
 
 
 
 
 
Sponsor: The George Washington University Medical Faculty Associates 
Funding Source: Janssen Scientific Affairs, LLC 


 
 
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PROTOCOL SYNOPSIS 
Protocol Title: 
Role of Canagliflozin on gene expression and function of CD34+ 
endothelial progenitor cells in patients with type 2 diabetes 
Site Numbers & Names 
GWU Medical Faculty Associates 
 
Research Hypothesis: 
Type 2 diabetes is associated with poor stem cell number and 
function. Poor viability and function of EPCs in diabetes affects the 
repair and regeneration of the endothelium. We hypothesize that use 
of Canagliflozin (Cana), along with certain anti-diabetic drugs may 
help reduce cardiovascular risk by improving EPC survival and 
function above and beyond adequate glucose metabolism control.   
 
Study Schema: Drugs / 
Doses / Length of 
Treatment 
We propose a 2-arm randomized, parallel group, longitudinal study 
of 16-week intervention duration. The 16-week time interval has 
been previously shown adequate to observe changes in 
biochemistry, EPCs and, importantly, pulse wave velocity (PWV) 
changes. Patients will be randomized to 2 groups:  
 
Control (n=20) Concomitant Anti-Diabetic Therapy + Placebo.  
Treatment (n=20) Concomitant Anti-Diabetic Therapy + Cana 100mg.  
 
 
 
 


 
 
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Study Objectives: 
 
Primary 
 
Secondary 
The primary objective is to ascertain if the addition of Cana improves 
CD34+ cell function and gene expression in type 2 diabetes patients, 
which will be correlated to improvement in 24hr urinary protein 
estimation and creatinine clearance (obtained via micro-
albumin/creatinine ratio from a spot urine sample). 
 
The secondary objective is to correlate the cellular outcome measures 
with other measures of endothelial function such as: 
1. Arterial stiffness measures with Pulse wave analysis and pulse 
wave velocity measurements. 
2. Serum biochemistry looking at surrogates of endothelial health, 
endothelial inflammation, appetite controlling hormone levels and 
fasting glucose, insulin and lipid profile. 
3. Resting Metabolic Rate measurement 
 
Study Outcome Measures: 
Primary: 
To investigate the effect of Cana on Endothelial function in patients 
inadequately controlled (HbA1C ≥ 7.0 % to ≤10%) while being treated 
with a stable dose of certain anti-diabetic drugs.  
 
Cellular markers. We will study pre and post Cana treatment 
changes in number, function and gene expression of patients’ 
peripheral blood-derived CD34+ cells.  
 
Renal Function Estimation: 24hr urinary protein estimation, 
creatinine clearance and urine exosome study. 
 
Secondary: 
To investigate the effect of Cana treatment on: 
 
Serum endothelial inflammatory markers including high 
sensitivity C-reactive protein (hs-CRP), IL-6, TNF-alpha and 
fasting lipid profile.  
 
Glycemic control will be evaluated by measuring fasting blood 
glucose, insulin, and HbA1c levels and assessing insulin 
resistance using HOMA-IR. 
 
Adiposity, measured using a body composition analyzer scale, 
measured as percentage body fat. 
 
Creatinine clearance estimation. 
 
Vessel health will be assessed by systolic and diastolic blood 
pressure and Arterial stiffness assessed using Vascular Flow and 
wave measurement equipment, SphygmoCor CP system from 
ATCOR.  
 
Central and aortic blood pressure estimated by the sphygmocor 
system. 
 
Resting Metabolic Rate (RMR, similar to Resting Energy 
expenditure measurement) at baseline, midway point (week 8) 
and post therapy. 
 


 
 
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Study Design: 
Prospective, double-masked, randomized placebo-controlled trial 
 
Accrual Goal (Total 
number of subjects): 
N = 40 
Accrual Rate 
(Number of subjects 
expected per month): 
Recruit 40 patients in approximately 36 months 
Estimated Timeline: 
FPFV: 10-06-2016 
LPLV: 01-06-2019 
 
Co-relative studies 
(PK/PD, etc.): 
N/A 
Inclusion Criteria: 
1. Adults aged 30-70 years 
2. Diagnosis of type 2 diabetes using criteria of the American Diabetes 
Association 
3. Currently being treated with any combination of the following anti-
diabetic therapies: metformin (1-2 grams), insulin, GLP-1 agonists, 
a DPP-IV inhibitor, or sulfonylureas as therapy for their Type 2 
Diabetes 
4. HbA1C between 7.0 to 10.0 % (both inclusive) 
5. BMI 25 – 39.9 kg/m2  
Exclusion Criteria: 
Patients with: 
1. Type 1 DM 
2. History of hyperosmolar non-ketotic coma 
3. Abnormal CBC that is judged by physician to be unsafe to enroll or 
low hematocrit (<28 UNITS).   
4. History of Pancreatitis 
5. History of DKA in the past 3 months 
6. History of active cancer or cancer treatment in the past 5 years 
(except basal cell carcinoma)  
7. Heart attack or stroke within 6 months of screening  
8. Clinically significant coronary and/or peripheral vascular disease 
that would be unsafe to enroll in the study.  
9. Statin use started or dose change in the last 3 months,  
10. CKD stages 3B-5  
11. Use of oral anti-diabetic medication other than metformin, insulin,  
GLP-1 agonists, DPP-IV inhibitors, or sulfonylureas 
12. Use of consistent long-term steroid medication in the last 3 months 
(oral, inhaled, injected). 
13. Uncontrolled inflammatory disease, or current chronic use of anti-
inflammatory drugs within the last 3 months.  
14. Pre-existing  liver disease and/or ALT and AST >2.5X’s UNL 
15. Serum creatinine levels ≥2.0 
16. Estimated CrCl < 45 mL/min (measured by eGFR value) 
17. Triglycerides >450 mg/dL 
18. Implanted devices (e.g., pacemakers) that may interact with Body 
Composition scale 
19. Untreated Systolic Blood Pressure > 150 mmHg and diastolic Blood 
Pressure > 90 mmHg  


 
 
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20. Active wounds or recent surgery within 3 months.  
21. Untreated hyper/hypothyroidism 
 
Additionally, patients who are active smokers, patients who are pregnant, 
nursing women, and post-menopausal women who are on estrogen hormone 
replacement therapy (not including oral contraceptives) will be excluded. 
 
Detailed Inclusion / exclusion criteria are listed on pages 12-15 of the 
protocol. 
Criteria for 
Evaluation:  
(Efficacy, safety,  
stopping rules, etc.) 
Subjects will be phone screened to determine initial eligibility. If the phone-
screening indicates possible eligibility, then the subjects will come to an in-
person screening visit at week -2, then evaluated for end points at weeks 0, 8 
and 16. Follow up phone call will be done 30 days after last dose of study 
drug to assess for adverse effects.  
 
Study participation will be stopped for adverse effects at the discretion of the 
study investigator. Subjects may stop study participation at any time and for 
any reason. 
 
Statistics: 
The total sample size is requested, after accounting for attrition over the 16-
week period, is 20 subjects per group or 40 subjects total. Sample size 
estimates were based on the effects of exercise on CD34+/KDR+ cells as 
described in the literature.  The effect of a single session, as well as extended 
training, on healthy subjects or those with existing cardiovascular conditions 
appears to increase the CD34+/KDR+ cells.  
   
This is a pilot study; as a result appropriate power calculation is not feasible. 
We however did conduct power analysis, which provided us with 73% power 
(see statistics section).  A p-value of less than 0.05 will be considered 
statistically significant. 
 
 
 
 


 
 
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INTRODUCTION 
Diabetes affects more than 11% of adults in the United States and this is projected to nearly double 
by 2025.1 Both diabetes and obesity are associated with endothelial dysfunction, oxidative stress, 
endothelial cell inflammation, cardiovascular pro-thrombotic states and are the most common causes of 
kidney disease2,3,4. Use of a sodium-glucose linked transporter (SGLT-2) inhibitor has shown promise in 
improving glycemic control, weight reduction, hypertension and even changes in circulating Renin-
angiotensin-aldosterone system (RAAS) and nitric oxide (NO)5,6 . However, whether these group of drugs 
have any effect on cardiovascular disease (CVD) risk modification or on endothelium or endothelial 
progenitor cells as a surrogate of cardiovascular and renal risk outcome measure, is unclear5.  
We have previously shown that CD34+ cells, derived from peripheral blood can act as a cellular 
biomarker that is more reliable than serum based markers7 for CVD risk estimation. Serum based 
inflammatory markers are not useful until the endothelium is already damaged and inflamed4. Such serum 
based biomarkers takes several months to change and gives no preventive and predictable information as 
to whether a particular medication may affect future endothelium. This is why the study of endothelium 
progenitors is crucial. In our previous study of a prediabetes population with an aerobic exercise 
intervention, we have demonstrated that CD34+ cells are responsive to a change in therapy or 
intervention within 2-4 weeks and can be used as a reliable non serum based cellular bio-marker7. CD34+ 
cells or endothelial progenitor cells have been used clinically to improve collateral circulation and have 
been extensively studied as a robust cardiovascular biomarker 8,9,7. Therefore studying CD34+ cells in 
patients, with or without Canagliflozin (Cana) can give vital information about the medication and its 
effect on endothelium. This is particularly important as another SGLT2 inhibitor Empagliflozin has 
shown unparalled positive cardiovascular effects with an oral hypoglycemic agent10. Of course, the 
question arises whether this clinical trial effect is secondary to glucose effect or direct effect of 
SGLT2 inhibitor on endothelium.  
Multiple glucose transporters have been identified in human cells these include GLUTs, 
SGLTs and even taste receptors (such as TLR2 and TLR3). We know SGLT transporters are 
present in tubular cells and clearly blocking of SGLT2 in these cells is beneficial. Information on 
glucose transporter in stem or progenitor cells is almost nil. In our lab we have shown presence 
of GLUT1, SGLTs and TLR3 on CD34+ cells. We have also demonstrated that hyperglycemia is 
toxic to CD34+ cells, more than CD31+ positive mature endothelial cells. We hypothesize that 
blocking SGLT2 in CD34+ cells will be beneficial rather than detrimental. As far as glucose 
uptake in CD34+ cells are concerned other glucose transporters should be sufficient, in fact 


 
 
Canagliflozin – Sen IIS 
Protocol Version: 3.0 
Date: 12/15/2017 
 
 
Page 7 of 35 
 
lesser amount of glucose entry in a hyperglycemic milieu (type 2 DM patients) may be less pro-
inflammatory and less pro-apoptotic. 
Our preliminary data indicates that mRNA gene expression of both SGLT1 and SGLT2 
are noted on human CD34+ cells however only SGLT2 mRNA gene expression is up-regulated 
several fold in human CD34+ cells in presence of hyperglycemia (20mM glucose). However non primary 
commercially obtained human endothelium (HUVEC) do not show similar results. An explanation could 
be SGLT2 expression decreases as the cell transitions from progenitor to mature endothelium. From these 
results we believe SGLT2 inhibitor will be effective on progenitors and not mature endothelium. We 
therefore hypothesize that CD34+ cells will be an ideal biomarker to study the effect of the drug. It is 
possible that Cana, by blocking SGLT2 receptors, may influence other CD34+ cell surface receptors 
including other glucose transporters and influence its function (most importantly migration). If a 
particular medication positively influences stem/progenitor cell migration then that medication can 
positively influence endothelial dysfunction and vascular complications from diabetes. We are 
particularly interested to note effect of Canagliflozin, a SGLT2 inhibitor on other glucose transporters 
such as GLUT 1 and 4 while looking at SGLT 1 and 2 on CD34+ cells. It will be helpful to discern these 
effects particularly when choice of oral diabetic medication in a type 2 diabetes population is practically 
limited to metformin, DPP4 inhibitors and SGLT2 inhibitors2. We plan to investigate the effect of Cana 
on CD34+ cells, in a placebo matched study. We plan to recruit subjects with type 2 diabetes with the 
following characteristics: 1) overweight, mild and moderately obese (BMI=25.0-39.9); 2) individuals 
with type 2 diabetes with inadequate control, HbA1C= 7 to 10.0%, on any combination of the 
following anti-diabetic therapies: metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-IV 
inhibitor, or sulfonylureas 3) with no current macrovascular complications. As per the ADA [2], 
Metformin, insulin, GLP-1 agonists, DPP-IV inhibitors, and sulfonylureas are all 1st line of care along 
with life-style modification. While Metformin on its own may affect inflammatory biomarkers, the effect 
is minimal at best, particularly in presence of endothelial dysfunction. For accurate comparison, both 
placebo and the Canagliflozin group will be on any combination of the following anti-diabetic 
therapies: metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-IV inhibitor, or sulfonylureas 
We will recruit a total of 40 patients (20 individuals/per group) with approximately a 20% drop out rate 
over two years and we hope to retain 32 individuals (16/group). Individuals in each group will be matched 
by sex, age, and race. Participants will be assessed at baseline (week 0), and at 2 and 4 months of drug 
intake.  
 
 


 
 
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1 
RESEARCH HYPOTHESIS 
We hypothesize that Cana may be able to improve number and function of CD34+ endothelial 
progenitor cells. We also propose that this expected cardiovascular benefit is independent of HbA1C 
reduction11,12. 
In this proposed study we plan to recruit patients with type 2 diabetes, who are on any combination 
of the following anti-diabetic therapies: metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-IV 
inhibitor, or sulfonylureas, but still remain inadequately controlled with HbA1C between 7.0% and 10% 
(both values inclusive), but not overtly out of control (> 10% of HbA1C).  
Subjects will begin taking 100 mg of Cana or placebo after initial 4 weeks. Subjects will be 
withdrawn from the study if the medication or placebo is not tolerated.  
 
2 
STUDY OBJECTIVES 
PRIMARY OBJECTIVE 
 
To determine whether 4 months of Canagliflozin modifies CD34+ cell number, gene 
expression and migration function. 
 
PROTOCOL FOR OBTAINING CD34+ CELLS:  
We will obtain a total of approximately 95 mL of peripheral blood per visit. Of these 95 mL, 
60-70 mL will be used to obtain CD34+ cells from mononuclear cell (MNC) population and 25-
35 mL for biochemistry and plasma ELISA assays. MNC will be obtained from whole blood 
similar to protocols described before13,14. MNCs will be put through CD34 magnetic bead column 
to obtain CD34+ cells (Miltenyi Biotec). Purity of CD34+ cells, post sort, usually is above 90%, 
to be verified by FACS analysis. 
 
A. From Mononuclear (MNC) population (pre CD34+ magnetic column sorting): We will 
assess the proportion of various cell types, progenitor stems and mature cells in MNCs to 
note if Cana influences stem vs non stem cell pool such as percentage of : CD34, CD309 
(VEGFR2-KDR), CD184 (CXCR4), receptor substrate for SDF1 alpha, CD31 (PECAM-
1, a mature circulating endothelial cell marker), CD133 (a progenitor marker), CD144 
(Vascular Endothelial cadherin), Sytox Blue OR Propidium Iodide staining (to detect 
apoptotic cells).  
B. MNC cells (prior to CD34 magnetic bead sorting) will be also used to assay CFU Hill 
Colony forming unit (Stem Cell Technologies- Cat#05900) media for Colony Forming unit 
(CFU) Assay. CFU will be counted on 5-7 days post plating (from initial plating).  
C. From CD 34+ cell population (Post Sorting): 
a. Migration Assay using SDF1Alpha concentrations of 0, 10, 100ng/ml and VEGF-
A concentrations of 20 and 50 ng/ml, using 150,000 cells in 300ul serum free cell 
suspension media per insert.  
b. Gene expression studies. Genes to be assessed on sorted CD34 positive cells: 


 
 
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i. Endothelial lineage cell surface markers: CD34, VEGFR2 (KDR), CD31, 
CD144 
ii. For Anti-oxidant gene expression: Superoxide dismutase (SOD) 1, 2 and 3, 
catalase, glutathione- peroxidase. 
iii. Apoptosis pathway: p53, p21, Bcl2, caspase-3 
iv. Endothelial Function Assay Gene: endothelial Nitric Oxide Synthase, von-
Willebrand’s Factor 
v. Genes associated with progenitor cell chemotaxis: VEGF-A, SDF1 alpha, 
CXCR4. 
 
SECONDARY OBJECTIVE 
 
To determine whether use of Canagliflozin alters other cardio-metabolic health indicator 
parameters such as: arterial stiffness measures, resting metabolic rate, body composition 
measures, hs-CRP, IL-6, TNF-alpha (inflammatory markers), fasting lipid profile, levels of 
insulin, glucose appetite controlling hormones and urine levels of microalubumin 
(Microalbumin/Creatinine ratio). 
 
We expect to corroborate our findings of the Primary Objective (cellular outcome measures) 
with traditional markers of endothelial/ vascular function such as serum biochemistry and arterial 
stiffness. 
 
Effect of Cana on Serum Biochemistry:  
While we believe cell based biomarkers are superior to traditional serum biomarkers, we believe 
the outcome report will be stronger if one can show positive co-relation between the two 
outcome measures. We therefore will be looking at:  
I. 
Inflammation, apoptosis and anti-oxidant protein levels: Highly selective C-reactive 
protein (hs-CRP), IL-6, TNF-alpha, fasting glucose, insulin and lipid profile. 
a. Glycemic control will be evaluated by measuring fasting blood glucose, insulin 
levels and HbA1c. Fasting blood glucose, insulin and lipid profile will be used to 
assess insulin resistance15,16 (32, 37) 
II. 
Plasma SDF1 alpha (ELISA) and GLP-1 (ELISA) will be estimated to assess endothelial 
health and factors that may influence CD34+ cell chemotaxis 
III. 
Estimation of Creatinine Clearance and proteinuria: 24hr urinary protein estimation and 
creatinine clearance (obtained via micro-albumin/creatinine ratio from a spot urine 
sample). 
a. Subjects will have the option of opting into a sub-study that is being run by Dr. 
Eric Nylen at the Veterans Affairs (VA) Maryland Health Care System. 10-20 mL 
of the excess fresh spot urine that has been collected would be sent to the VA for 
exosome analysis. This is optional, and is not required of subjects. For details of 
urine-exosome study please see page 31.  
IV. 
The glomerular filtration rate (GFR) will be estimated using the CKD-EPI (Chronic 
Kidney Disease Epidemiology Collaboration) equation. The CKD-EPI equation, 
expressed as a single equation, is: 
a. GFR = 141 X min (Scr/κ,1)α X max(Scr/κ,1)-1.209 X 0.993Age X 1.018 [if female] 
X 1.159 [if black]; where Scr is serum creatinine (mg/dL), κ is 0.7 for females and 


 
 
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0.9 for males, α is –0.329 for females and –0.411 for males, min indicates the 
minimum of Scr/κ or 1, and max indicates the maximum of Scr/κ or 117. 
 
Effect of Cana on Body Habitus (Determination of body composition and visceral fat) AND 
Resting Metabolic Rate.   
As we plan to study cardio-metabolic effect of Cana we believe the RMR and body composition 
data is useful. 
I. 
Using body composition scale:  
a. Height and weight will be measured and the body mass index (BMI=kg•m-2) 
used as an indicator of relative weight.  
b. The body composition scale calculates body fat%, total body water%, fat free 
mass, etc. in addition to BMI.  
II. 
Resting Metabolic Rate (RMR): at weeks 0, 8 and 16 of the protocol using ReeVue 
(trademark) machine, with or without SGLT2 inhibitor therapy to ascertain if Cana has 
any effect on RMR. Other related trials have shown weight loss but effect on metabolic 
rate has not been studied18. 
 
 
 
Effect of Cana on Assays of Arterial Stiffness:  
I. 
We will acquire Pulse Wave Analysis and Vascular Flow using SphygmoCor CP system 
from ATCOR as a measure of central arterial pressure and arterial stiffness. Again 
SGLT2 inhibitors have been shown to reduce systolic blood pressure but effect on 
stiffness has not been looked at, except with Empagliflozin17,18 
II. 
Vessel health will be assessed by degree of arterial stiffness, using arterial tonometry.  
III. 
The central and the aortic pressure is assessed by pulse wave analysis (PWA) and pulse 
wave velocity (PWV). 
IV. 
Arterial stiffness will be assessed using Vascular Flow and wave form analysis 
equipment, SphygmoCor CP system from ATCOR19. 
 
3 
INVESTIGATIONAL PLAN 
 
STUDY DESIGN AND DURATION 
 
40 Type 2 diabetic Subjects Aged 30-70 on stable dose of certain anti-diabetic therapies  


 
 
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+/- 6 day window for visits 
*Assessed at week 0, 8 and 16: Biochemical and cellular markers of endothelial function and Pulse 
wave analysis and velocity (PWA and PWV), REE and Adiposity. 
 
Week 20 -  A telephone call to subjects will be made 30 days after last dose of study medication 
to determine if there have been any adverse events.  
 
AVAILABLE RELATED STUDIES AND EXPECTED RESULTS: 
Studies measuring changes to EPCs in type 2 diabetes patients are very limited.  
 
Use of CD34 positive cells as a cardiovascular risk surrogate has been reported in the past. Various 
studies20,21, have shown that various disease states, including those seen in diabetes, can deplete 
and damage EPC, thereby diminishing their regenerative potential.  The inability to maintain or 
repair damaged endothelial tissue leads to cumulative vascular dysfunction and cardiovascular 
disease.  
 
We hypothesize that monitoring number, function and gene expression of endothelial 
progenitors will allow us to quantify cardiovascular disease risk at the onset and regenerative 
potential post intervention at a cellular level. It will also help us to identify and correlate the 
best endothelial function bio-chemical inflammatory marker as an early indicator of 
cardiovascular disease progression in early type 2 diabetes and identify patients that are 
responders. 
   
Cana can influence the glucose transportation and modify intercellular glucose and cell energetics, 
and so it can influence the migration of the cell. SDF-1alpha, being a chemotactic agent, will also 
influence migration depending on its level.  
  
Cana 100 mg or placebo will be added to patients who have type 2 diabetes with HbA1C of 7.0 to 
10 % (both inclusive) while being on any combination of the following anti-diabetic therapies: 
Group A: Concomitant Anti-diabetic therapy + Placebo, n=20 
Wk 8* 
Wk 20 
Follow up 
Phone + In Person Screening 
Group B: Concomitant Anti-diabetic therapy + Cana 100mg, n=20 
Wk-0* 
Randomization 
Wk 16* 
Wk -2 


 
 
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metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-IV inhibitor, or sulfonylureas. As per 
ADA22, Metformin is the 1st line of care along with life-style modification. While Metformin on 
its own may affect inflammatory biomarkers, the effect is minimal at best, particularly in presence 
of endothelial dysfunction. Also both the placebo and Canagliflozin group will be on any 
combination of the following anti-diabetic therapies: metformin (1-2 grams), insulin, GLP-1 
agonists, a DPP-IV inhibitor, or sulfonylureas. Our study population will include equal numbers of 
adult male and female Type 2 diabetes (T2DM) patients aged 30-70 years, enrolled in both arms 
of the study. 
 
Patients will be treated with 100 mg of Cana or placebo for 16 weeks.  This time interval has 
been previously shown to be adequate to observe changes to Endothelial Progenitor Cells 
(EPCs)13,23. 
 
STUDY POPULATION 
Inclusion Criteria: 
 
1) Signed written informed consent 
 Before any study procedures are performed, subjects will have the details of the 
study described to them, and they will be given a written informed consent 
document to read. Then, if subjects consent to participate in the study, they will 
indicate that consent by signing and dating the informed consent document in the 
presence of study personnel. 
2) Target Population 
 Subjects with a diagnosis of Type 2 diabetes mellitus using criteria of the American 
Diabetes Association. 
 Currently treated with any combination of the following anti-diabetic therapies: 
metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-IV inhibitor, or 
sulfonylureas 
 HbA1C between 7.0% and 10% (both inclusive)   
 BMI 25-39.9 kg/m2 both inclusive. 
One of the prime outcome measure is estimation of renal function and accurate eGFR 
staging:Estimated Glomerular Filtration Rate Serum Cr should be used to estimate 
glomerular filtration rate (GFR). Estimated GFR (eGFR) is commonly reported by 
laboratories or can be estimated using formulae such as the Modification of Diet in Renal 
Disease (MDRD) study equation or the Chronic Kidney Disease Epidemiology Collaboration 
(CKD-EPI) equation. The latter is the preferred GFR estimating equation. GFR calculators 
are available at http:// www.nkdep.nih.gov.  
Abnormal urinary albumin excretion and eGFR may be used to stage chronic kidney disease 
(CKD). The National Kidney Foundation classification (Table below) is based on both 
kidney damage (UACR at or above 30 mg/g Cr) and eGFR. 
Normal UACR is defined as less than 30 mg/g Cr, and increased urinary albumin excretion is 
defined as at or above 30 mg/g Cr. Because of variability in urinary albumin excretion, 
ideally two (at least one) specimens of UACR should have been collected within a 3- to 6-
month period before considering a patient to have albuminuria. 
 
Stage -- Description  -- GFR (mL/min/1.73 m2 )  


 
 
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Stage 1 Kidney damage* with normal or increased GFR >=90  
          2 Kidney damage* with mildly decreased eGFR 60–89  
          3A Moderately decreased eGFR 45–59   
    3B Moderately decreased eGFR 30–45  
          4 Severely decreased eGFR 15–29  
          5 Kidney failure ,15 or dialysis  
*Kidney damage is defined as abnormalities on pathological, urine, blood, or imaging tests.24 
  
3) Age and Reproductive Status 
 Men and women, 30 to 70 years of age.  
 Enrolled women must not be pregnant, should not be breast-feeding and should 
not be taking estrogen hormone replacement therapy (HRT) during the study. 
 
The following is a definition of Women of Child Bearing Potential (WOCBP): 
 Women using the following methods to prevent pregnancy: Oral contraceptives, other 
hormonal contraceptives (vaginal products, skin patches, or implanted or injectable 
products), or mechanical products such as intrauterine devices or barrier methods 
(diaphragm, condoms, spermicides). 
 Women who are practicing abstinence. 
 Women who have a partner who is sterile (e.g., due to vasectomy).WOCBP must be using 
an acceptable method of contraception to avoid pregnancy throughout the study and for up 
to 4 weeks after the last dose of study drug in such a manner that the risk of pregnancy is 
minimized.  
 WOCBP must have a negative serum or urine pregnancy test result within 0 to 72 hours 
before the first dose of study drug.  
 Women must not be breast-feeding. 
Post-menopause is defined as: 
 Women who have had amenorrhea for  12 consecutive months and/or who have a 
documented serum follicle-stimulating hormone (FSH) level > 35 mIU/mL. 
 Women who have irregular menstrual periods and a documented serum FSH level 
> 35 mIU/mL. 
 Women who are taking hormone replacement therapy (HRT). 
 
Exclusion Criteria: 
 
 Type 1 diabetes mellitus 
 History of hyperosmolar non-ketotic coma 
 Abnormal CBC that is judged by physician to be unsafe to enroll or low hematocrit (<28 
UNITS).   
 History of pancreatitis 
 History of diabetic ketoacidosis in the last 3 months 
 History of cancer (except basal cell carcinoma and cancer that is cured or not active or 
being treated in the past 5 years) 
 Heart attack or stroke within 6 months of screening  


 
 
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 Clinically significant coronary and/or peripheral vascular disease that would be unsafe to 
enroll in the study. * 
 Statin use started or dose change in the last 3 months. * 
 CKD Stages 3B,4 and 5 (see definition above) 
 Use of oral or injectable anti-diabetic medication other than any combination of the 
following anti-diabetic therapies: metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-
IV inhibitor, or sulfonylureas currently, or in the past 1 month. * 
 Use of consistent long-term steroid medication (oral, inhaled, injected) within the last 3 
months. * 
 Uncontrolled inflammatory disease, or current chronic use of anti-inflammatory drugs 
within the last 3 months. * 
 Implanted devices (e.g., pacemakers) that may interact with Body Composition scale 
 Untreated Systolic Blood Pressure > 150 mmHg and diastolic Blood Pressure > 90 
mmHg  
 Active wounds or recent surgery within 3 months. * 
 Untreated hyper/hypothyroidism 
*These criteria may be allowed, judged on a case by case basis by the PI, with appropriate 
rationale.  
 
Physical and Laboratory Test Findings 
 Pre-existing  liver disease and/or ALT and AST >2.5X’s UNL 
 Serum creatinine levels ≥2.0  
 Estimated CrCl < 45 mL/min (measured by eGFR value) 
 Triglycerides >450 mg/dL 
Allergies and Adverse Drug Reactions 
 Subjects with a history of any serious hypersensitivity reaction to Cana or another 
SGLT2 inhibitor. 
Sex and Reproductive Status 
 Women in reproductive age group will be included in the study but encouraged to use 
contraceptive method to avoid pregnancy within 16 weeks of study duration. 
 Women who are pregnant or breast-feeding will be excluded. 
Other Exclusion Criteria 
 Prisoners or subjects who are involuntarily incarcerated. 
 Subjects who are compulsorily detained for treatment of either a psychiatric or physical 
(e.g., infectious disease) illness.  
 Patients who are active smokers 
 Patients who are pregnant 
 Nursing women 
 Post-menopausal women who are on estrogen hormone replacement therapy will be 
excluded.   
 Patients on low dose oral contraceptives will be allowed to participate as these 
formulations contain very low amounts of estrogens. 


 
 
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 Eligibility criteria for this study have been carefully considered to ensure the safety of the 
study subjects and to ensure that the results of the study can be used. It is imperative that 
subjects fully meet all eligibility criteria. 
 
DISCONTINUATION OF SUBJECTS FROM TREATMENT 
Subjects MUST discontinue from the investigational product (and non-investigational product at 
the discretion of the investigator) for any of the following reasons: 
 Withdrawal of informed consent (subject’s decision to withdraw for any reason) 
 Any clinical adverse event, laboratory abnormality, or inter-current illness which, in the 
opinion of the investigator, indicates that continued participation in the study is not in the 
best interest of the subject.  
 Patients who progress to stages 3 and beyond, post screening will be excluded 
 Pregnancy 
o Pregnant patients will be advised to contact the investigator or study staff 
immediately if they suspect they might be pregnant (e.g. missed or late menstrual 
period) at any time during study participation.  
 Loss of ability to freely provide consent through imprisonment or involuntary 
incarceration for treatment of either a psychiatric or physical illness (e.g. infectious 
disease).  
 If hyperglycemia and ketoacidosis is suspected, promptly discontinue treatment with 
Canagliflozin 
 All subjects who discontinue should comply with protocol-specified follow-up 
procedures outlined before. The only exception to this requirement is when a subject 
withdraws consent for all study procedures or loses the ability to consent freely (i.e., is 
imprisoned or involuntarily incarcerated for the treatment of either a psychiatric or 
physical illness). If a subject withdraws before completing the study, the reason for 
withdrawal must be documented appropriately. 
 
 
4 
TREATMENTS 
 
STUDY TREATMENT: CANAGLIFLOZIN 
Definition of Investigational Product: A pharmaceutical form of an active substance or placebo 
being tested or used as a reference in a clinical study, including products already with a 
marketing authorization but used or assembled (formulated or packaged) in a way different from 
the authorized form, or used for an unauthorized indication, or when used to gain further 
information about the authorized form. In this protocol, the investigational product is 
Canagliflozin. 
 
Definition of Non-Investigational Product: Other medications used in the study as support or 
escape medication for preventative, diagnostic, or therapeutic reasons as components of a given 
standard of care. In this protocol, the non-investigational products are Metformin, Insulin, GLP-1 
agonists, sulfonylureas, and DPP-IV inhibitors.  Patients will continue on any combination of the 
following anti-diabetic therapies: metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-IV 
inhibitor, or sulfonylureas, as per their study entry dose added to Cana or placebo. As mentioned 


 
 
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before, patients on oral hypoglycemic other than any combination of the following anti-diabetic 
therapies: metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-IV inhibitor, or sulfonylureas 
will not be invited to join the study.  
This study is being conducted under FDA mandated guideline for use in type 2 diabetes. 
Though in this particular study Cana is referred to as the investigational drug, it is not a novel 
drug and we should not need a separate IND for execution of this study. 
 
METHOD OF ASSIGNING SUBJECTS TO A TREATMENT 
Subjects, as per selection criteria below will be randomized to treatments using a permuted block 
design, developed by the Epidemiology & Biostatistics Research Core. This approach ensures 
groups will be approximately balanced at any time during the study and at study completion. 
Randomization will be performed by the MFA IDS Pharmacy staff who never come in contact 
with subjects.  
 
SELECTION AND TIMING OF DOSE FOR EACH SUBJECT 
The recommended dose of Cana is 100 mg PO will be given once daily. Cana can be taken with 
or without food. Cana would preferably be taken in the morning prior to the first meal. 
 
DOSE MODIFICATIONS 
No dose modification is necessary prior to CKD stage 3B (eGFR less than 45) 
 
BLINDING/ UNBLINDING 
Blinding is critical to the integrity of this clinical study. However, in the event of a medical 
emergency or pregnancy in a subject, in which knowledge of the investigational product is 
critical to the subject's management, the blind for that subject may be broken.  
Before breaking the blind of an individual subject’s treatment, the investigator should 
have determined that the information is necessary, i.e., that it will alter the subject’s immediate 
management. In many cases, particularly when the emergency is not investigational product-
related, the problem may be properly managed by assuming that the subject is receiving active 
product without the need for un-blinding.  
 
END OF STUDY UNBLINDING PROTOCOL 
Upon completion of the research study, we will undergo the following protocol in order to 
unblind the research data. 
 
Unblinding is the process by which the allocation code is broken so that the investigator, clinical 
staff and the trial statistician becomes aware of which intervention each subject enrolled in the 
research study was taking.  
Unblinding at the end of the study is required in order to make unmasked analysis in accordance 
with the study analysis plan. It is also conducted in order to inform the participants of which 
investigational product they were assigned to.  
 
Time to unblind: 
Unblinding shall be conducted when all subjects enrolled in the research study have finished 
treatment, and all follow up visits. There must be no plan to recruit any more subjects in the 
research study. Additionally, all data points and outcome measures for each research subject 


 
 
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must have been collected, and ideally compiled. Prior to unblinding there will be a data lock on 
clinical outcome measures and basic side outcome measures and associated  research data 
collected for the study.  
 
Procedure to unblind 
Once data (from both the clinical and the basic science side) and has been compiled and is data 
locked, the investigator can choose to unblind. The Principal Investigator must contact the study 
sponsor, and receive permission to un-blind. If, for unforeseen reasons, at the pre-determined 
date for full study unblinding the data analysis on the cellular or basic aspects of the study is 
lagging behind the clinical data outcome measures (though the data has been acquired) the 
Principal Investigator in consultation with the study sponsor may choose to un-blind the clinical 
outcome measures before the basic side data has been analyzed but compiled. Upon confirmation 
from the study sponsor, the principle investigator must make a written request to the designated 
party to unblind, hereto referred at the “unblinder”. The unblinder is the bio-statistician of the 
MFA, Dr. Richard Amdur. Upon receipt of an instruction to unblind, the unblinder will sign 
the request form, indicating their agreement to unblind. This form will then be taken to the 
pharmacy, MFA’s IDS, where a member of the IDS staff will take the form, and will give the 
unblinding study binder to the unblinder. The pharmacy will sign to indicate their release of the 
binder, and the unblinder will sign to indicate receipt of the binder. At this point in time the chain 
of custody of the pharmacy unblended binder has been transferred to the designated party to 
unblind. The form with all of the signatures will be provided to the study coordinator to be kept 
in the regulatory binder.  
 
5 
ADVERSE EVENTS: DEFINITIONS, REPORTING, GUIDANCE 
I. 
Management of Safety Data 
This Study has been designated as an interventional study.  Janssen requirements for IIS 
interventional studies are all adverse events regardless of causality and special situations 
excluding those from subjects not exposed to a Janssen Medicinal Product and product quality 
complaints with or without an adverse event will be reported, once the subject has signed and 
dated an Informed Consent Form is obtained until the subject has completed participation in the 
study and for 30 days after the last dose of study drug. 
 
II. 
Definitions 
Adverse Event (AE) 
An adverse event is any untoward medical occurrence in a clinical study subject administered a 
medicinal (investigational or non-investigational) product. An adverse event does not necessarily 
have a causal relationship with the treatment. An adverse event can therefore be any unfavorable 
and unintended sign (including an abnormal finding), symptom, or disease temporally associated 
with the use of a medicinal (investigational or non- investigational) product, whether or not 
related to that medicinal (investigational or non-investigational) product. (Definition per 
International Conference on Harmonisation [ICH]). 
This includes any occurrence that is new in onset or aggravated in severity or frequency from the 
baseline condition, or abnormal results of diagnostic procedures, including laboratory test 
abnormalities. 
 
Adverse Events of Special Interest  


 
 
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Events that Janssen Scientific Affairs is actively monitoring as a result of a previously identified 
signal (even if non-serious).  These adverse events are: 
 
 Diabetic ketoacidosis (DKA) 
 Genital mycotic infection 
 Urinary tract infection 
 Osmotic diuresis-related AEs 
 Volume related AEs 
 
Individual Case Safety Report (ICSR) 
A valid ICSR must contain the four minimum criteria required to meet regulatory reporting 
requirements. 
 an identifiable subject (not disclosing the subject’s name and address) 
 an identifiable reporter (investigational site) 
 a Janssen medicinal product 
 an adverse event, outcome, or certain special situation 
 
The minimum information required is: 
 suspected Janssen product (doses, indication) 
 date of therapy (start and end date, if available) 
 batch or lot number, if available 
 subject details (subject ID and country) 
 gender 
 age at AE onset 
 reporter ID 
 adverse event detail (AE verbatim in English), onset date, relatedness, causality, action 
taken, outcome, (if available) 
 Janssen protocol ID 
 
Product Quality Complaint (PQC) 
A product quality compliant is related to a potential quality issue during manufacturing, 
packaging, release testing, stability monitoring, dose preparation, storage or distribution of the 
product, or delivery system.  Not all PQCs involve a subject.  Lot and batch numbers are of high 
significance and need to be collected whenever available. 
 
Examples of PQC include but not limited to: 
 Functional Problem: e.g., altered delivery rate in a controlled release product 
 Physical Defect:  e.g. abnormal odor, broken or crushed tablets/capsules 
 Potential Dosing Device Malfunction: e.g.,  autoinjector button not working, needle 
detaching from syringe 
 Suspected Contamination 
 Suspected Counterfeit. 
 
Serious Adverse Event (SAE) 


 
 
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A serious adverse event based on ICH and EU Guidelines on Pharmacovigilance for Medicinal 
Products for Human Use is any untoward medical occurrence that at any dose: 
 
 Results in death 
 Is life-threatening 
 (The subject was at risk of death at the time of the event. It does not refer to an event that 
hypothetically might have caused death if it were more severe.) 
 Requires inpatient hospitalization or prolongation of existing hospitalization 
 Results in persistent or significant disability/incapacity 
 Is a congenital anomaly/birth defect 
 Is a suspected transmission of any infectious agent via a medicinal product 
 Is medically important* 
 
*Medical and scientific judgment should be exercised in deciding whether expedited reporting is 
also appropriate in other situations, such as important medical events that may not be 
immediately life threatening or result in death or hospitalization but may jeopardize the subject 
or may require intervention to prevent one of the other outcomes listed in the definition above. 
These should usually be considered serious. 
 
NOTE: Death for ANY reason should be reported as a SERIOUS ADVERSE EVENT.  
 
For reports of hospitalization, it is the sign, symptom or diagnosis which led to the 
hospitalization that is the serious event for which details must be provided. 
 
Any event requiring in-patient hospital admission (or the prolongation of hospitalization) must 
be reported as an SAE. Events that do not meet the criteria for SAE reporting are: 
 Reasons described in the Protocol, e.g. drug administration, Protocol-required testing 
 Social reasons, e.g. overnight stay because of distance between home and hospital 
 Surgery or procedure planned and documented prior to entry into the Study. 
 
Any event requiring hospitalization or prolongation of hospitalization that occurs during the 
study must be reported as a serious adverse event, except hospitalizations for the following: 
 Hospitalizations not intended to treat an acute illness or adverse event (e.g., social 
reasons such as pending placement in long-term care facility) 
 Surgery or procedure planned before entry into the study. [Note: Hospitalizations that 
were planned before the start of data collection and where the underlying condition for 
which the hospitalization was planned has not worsened will not be considered serious 
adverse events. Any adverse event that results in a prolongation of the originally planned 
hospitalization is to be reported as a new serious adverse event.] 
 
The cause of death of a subject in a study within 30 days of the last dose of study drug, whether 
or not the event is expected or associated with the study drug, is considered a serious adverse 
event. 
 
Unlisted (Unexpected) Adverse Event/Reference Safety Information 


 
 
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An adverse event is considered unlisted if the nature or severity is not consistent with the 
applicable product reference safety information. For a medicinal product(s) with a marketing 
authorization, the expectedness of an adverse event will be determined by whether or not it is 
listed in the applicable product information. 
 
III. 
Special Situations  
Safety events of interest for a Janssen medicinal product that require expediting reporting and/or 
safety evaluation include, but are not limited to: 
 Drug exposure during pregnancy (maternal and paternal) 
 Overdose of a Janssen medicinal product  
 Exposure to a Janssen medicinal product from breastfeeding  
 Suspected abuse/misuse of a Janssen medicinal product 
 Inadvertent or accidental exposure to a Janssen medicinal product  
 Any failure of expected pharmacological action (i.e., lack of effect) of a Janssen 
medicinal product 
 Medication error involving a Janssen medicinal product (with or without patient exposure 
to the Janssen medicinal product, e.g., name confusion) 
 Suspected transmission of any infectious agent via a medicinal  
 Unexpected therapeutic or clinical benefit from use of a Janssen medicinal product 
 
These safety events may not meet the definition of an adverse event; however, they are treated in 
the same manner as adverse events. Special situations should be recorded on the Adverse Event 
page of the CRF. 
 
Any special situation that meets the criteria of a serious adverse event should be recorded on a 
Serious Adverse Event Report Form and be reported to Janssen Scientific Affairs within 24 
hours of becoming aware of the event. 
 
IV. 
Pregnancy  
All initial reports of pregnancy must be reported to Janssen Scientific Affairs by the Sponsor 
Investigator within 24 hours of their awareness of the event using the Serious Adverse Event 
Form. Abnormal pregnancy outcomes (e.g. spontaneous abortion, stillbirth, and congenital 
anomaly) are considered serious adverse events and must be reported using the Serious Adverse 
Event Form.  
 
Any subject who becomes pregnant during the study must be promptly withdrawn from the study 
and discontinue further study treatment. 
 
Because the effect of the Janssen medicinal product on sperm is unknown, pregnancies in 
partners of male subjects exposed to a Janssen medicinal product will be reported by the Sponsor 
Investigator within 24 hours of their awareness of the event using the Serious Adverse Event 
Form.  Depending on local legislation this may require prior consent of the partner. 
 
Follow-up information regarding the outcome of the pregnancy and any postnatal sequelae in the 
infant will be required. 
 


 
 
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V. 
Reporting Procedures for Adverse Events and Pregnancies [and/or Pregnancies in 
Partners] 
All adverse events, whether serious or non-serious, related or not related, special situations, 
pregnancy exposures and/or pregnancies in partners following exposure to a Janssen medicinal 
product are to be documented by the investigator and recorded in the CRF and in the subject’s 
source records. Investigators must record in the CRF their opinion concerning the relationship of 
the adverse event to a Janssen medicinal product.  
All (serious and non-serious) adverse events reported for a Janssen medicinal product should be 
followed-up in accordance with clinical practice. 
 
All serious adverse events that have not resolved by the end of the study, or that have not 
resolved upon discontinuation of the subject's participation in the study, must be followed until 
any of the following occurs: 
 The event resolves 
 The event stabilizes 
 The event returns to baseline, if a baseline value/status is available 
 The event can be attributed to agents other than the study drug or to factors unrelated to 
study conduct 
 It becomes unlikely that any additional information can be obtained (subject or health 
care practitioner refusal to provide additional information, lost to follow-up after 
demonstration of due diligence with follow-up efforts) 
 
All serious adverse events, pregnancy exposures and/or pregnancies in partners for Janssen 
medicinal products under study should be reported directly by the Sponsor Investigator, within 
24 hours of becoming aware, to Janssen Scientific Affairs using the Janssen Scientific Affairs 
Serious Adverse Event Report Form. In the event the study is blinded, the Sponsor Investigator 
will submit an unblinded SAE or pregnancy exposure report to Janssen Scientific Affairs. 
 
All follow-up information for serious adverse events that are not resolved at the end of the study 
or by the time of patient withdrawal must be reported directly by the Sponsor Investigator, 
within 24 hours becoming aware, to Janssen Scientific Affairs using the Janssen Scientific 
Affair’s Serious Adverse Event Report Form. 
 
VI. 
Product Quality Complaints for Janssen Medicinal Products 
A PQC may have an impact on the safety and efficacy of the product. Timely, accurate, and 
complete reporting and analysis of PQC information from studies are crucial for the protection of 
patients, investigators, and Janssen Scientific Affairs, and are mandated by regulatory agencies 
worldwide. Janssen Scientific Affairs has established procedures in conformity with regulatory 
requirements worldwide to ensure appropriate reporting of PQC information.  Lot and/or Batch 
#s shall be collected or any reports of failure of expected pharmacological action (i.e., lack of 
effect). 
 
All initial PQCs involving a Janssen product under study must be reported to Janssen Scientific 
Affairs by the Sponsor Investigator within 24 hours after being made aware of the event. 
 


 
 
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If the defect for a Janssen product under study is combined with either a serious adverse event or 
non-serious adverse event, the Sponsor Investigator must report the PQC to Janssen Scientific 
Affairs according to the serious adverse event reporting timelines.  A sample of the suspected 
product should be maintained for further investigation if requested by Janssen Scientific Affairs. 
 
VII. 
Maintenance of Safety Information 
All safety data should be maintained in a clinical database in a retrievable format. The Institution 
and Sponsor Investigator shall provide all adverse events, both serious and non-serious, in report 
format. However, in certain circumstances more frequent provision of safety data may be 
necessary, e.g. to fulfill a regulatory request, and as such the data shall be made available within 
a reasonable timeframe at Janssen Scientific Affair’s request. 
 
VIII. 
Transmission Methods: 
The following methods are acceptable for transmission of safety information to Janssen 
Scientific Affairs: 
 Electronically via Janssen SECURE Email service (preferred) 
 For business continuity purposes, if SECURE Email is non-functional: 
o Facsimile (fax), receipt of which is evidenced in a successful fax transmission 
report 
 Telephone (if fax is non-functional). 
 
Please use the contact information and process information provided by Janssen Scientific 
Affairs. 
 
IX. 
Procedures for Reporting Adverse Events (AE), Serious Adverse Events (SAE), 
Pregnancy, and Product Quality Complaints (PQC) to Janssen Scientific Affairs 
A. AEs, SAEs, Special Situations and Pregnancy Reporting. 
The Institution and the Sponsor Investigator will transmit SAEs and Special Situations in a form 
provided by Janssen Scientific Affairs in accordance with Section VIII Transmission methods, in 
English within 24-hours of becoming aware of the event(s). 
 
All available clinical information relevant to the evaluation of a related SAE or Special Situation 
is required. 
 The Institution and/or Sponsor Investigator are responsible for ensuring that these cases 
are complete and if not are promptly followed-up. A safety report is not considered 
complete until all clinical details needed to interpret the case are received. Reporting of 
follow-up information should follow the same timeline as initial reports. 
 Copies of any and all relevant correspondences with regulatory authorities and ethics 
committees regarding any and all serious adverse events, irrespective of association with 
the Janssen Product under study, are to be provided to Janssen Scientific Affairs using a 
transmission method in Section VIII within 24 hours of such report or correspondence 
being sent to applicable health authorities. 
 
B. PQC Reporting 


 
 
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The Institution and the Sponsor Investigator will report any suspected PQC to the Janssen 
contact within 24 hours of becoming aware of the complaint.  The product should be quarantined 
immediately and if possible, take a picture. 
 
X. 
Reconciliation of SAEs 
At a minimum, on a quarterly basis and at the end of the Study, Janssen Scientific Affairs will 
provide to the Institution and/or Sponsor Investigator, a listing of all SAEs reported to Janssen 
Scientific Affairs.  The Sponsor Investigator will review this listing and provide any 
discrepancies to Janssen Scientific Affairs. 
 
Upon request, Institution and/or Sponsor Investigator shall provide Janssen Scientific Affairs 
with a summary list of all SAEs, and AEs of Special Interest and Special Reporting Situation 
reports to date, for reconciliation purposes. 
 
XI. 
Dissemination of Safety Information from Janssen Scientific Affairs to 
Institution/Sponsor Investigator  
Janssen Scientific Affairs agrees to provide to the Sponsor Investigator SUSAR reports for the 
Study Product as they become available until all subjects in the Protocol have completed their 
last Study visit according to the Protocol (i.e. Last Subject Last Visit has occurred).  
 
 


 
 
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6 
STUDY SCHEMATIC: TIME AND EVENTS SCHEDULE 
 
Procedure 
Phone 
Screening 
(Before 
week -2) 
Screening 
Visit 
(Week -2) 
Baseline/ 
Randomization 
Visit 1 
(Week 0) 
During 
Treatment 
Visit 2 
(Week 8) 
End-of-
Treatment 
Visit 3 
(Week 
16) 
Follow 
up 
phone 
call 
(Week 
20) 
Eligibility Assessments  
 
 
 
 
 
Informed Consent 
 
 
X 
 
 
 
 
Inclusion / Exclusion 
Criteria 
X 
X 
 
 
 
 
Medical History 
 
X 
 
 
 
 
Safety Assessments 
 
 
 
 
 
 
Physical Examination 
 
X 
 
 
X 
 
Targeted Physical 
Examination (as 
needed) 
 
 
X 
X 
X 
 
Vital Signs 
 
X 
X 
X 
X 
 
Assessment of Signs 
and Symptoms 
 
X 
X 
X 
X 
 
Adverse Events 
Assessment 
 
 
X 
X 
X 
X 
Laboratory Tests 
(Biochemical) 
 
Screening 
X 
X 
X 
 
Urine Pregnancy Test 
(As needed) 
 
X 
X 
X 
X 
 
Spot Urine Sample 
 
 
X 
X 
X 
 
Waist/Hip 
Measurements 
 
 
X 
X 
X 
 
Body Composition 
Scale 
 
 
X 
X 
X 
 
Efficacy Assessments 
 
 
 
 
 
 
Peripheral blood draw 
for CD34+cell harvest 
 
 
X 
X 
X 
 
Pulse wave analysis 
and Pulse wave 
velocity assessment 
 
 
X 
X 
X 
 
RMR 
 
 
X 
X 
X 
 
Clinical Drug Supplies 
 
 
 
 
 
 
Randomize 
 
 
X 
 
 
 
Dispense Study 
Treatment 
 
 
X 
X 
 
 
 
RESEARCH STUDY DESIGN AND METHODS 
Patient Definition and Selection:  


 
 
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Adults aged 30-70 years will be recruited mainly from the GWU MFA research patient database 
provided by MFA Information Technology, and then contacted via email and phone, from the 
endocrinology and other provider clinics at GWU and through referrals from the Veterans Affairs 
(VA) clinics. Suitable advertisements for the study patient enrollment will be posted. Patients will 
be included in the study if they have been diagnosed with type 2 diabetes within the previous 15 
years using criteria of the American Diabetes Association22, and are currently treated with any 
combination of the following anti-diabetic therapies: metformin (1-2 grams), insulin, GLP-1 
agonists, a DPP-IV inhibitor, or sulfonylureas. We wanted to capture a population where the 
subjects will be classified as having early stage of diabetes where endothelial dysfunction can be 
reversed. Late stages of diabetes may be associated with irreversible vascular damage. We plan 
to enroll subjects with HbA1C between 7.0% and 10% (both inclusive) and with BMI 25-39.9 
kg/m2 (both inclusive). Post enrollment, patients will be randomized to Cana 100 mg or placebo 
as detailed in Study Design for direct comparison between the two regimens.  
Before any study procedures are performed, subjects will have the details of the study 
described to them, and they will be given a written informed consent document to read. Then, if 
subjects consent to participate in the study, they will indicate that consent by signing and dating 
the informed consent document in the presence of study personnel. 
 
Recruitment:  
Subjects will be recruited from the physician clinics at GWU, Medical Faculty Associates, where 
Dr. Sen is a clinician, as well as through referral from the VA. All patients will receive 
counseling regarding the study aims and methods.  
 
Study Design Overview:  
We propose a 2-arm randomized, double-masked, placebo-controlled, parallel group, 
longitudinal study of 16-weeks duration. (See study design below), recruited over 2 years.  The 
16 week time interval has been previously shown adequate to observe changes in endothelial 
progenitor stem cells, biochemistry and importantly, PWV changes25,26. Patients will be 
randomized to 2 groups: 
Control, (n=20), metformin, insulin or Combo + Placebo 
Treatment, (n=20) metformin, insulin or Combo + Cana 100mg 
 
This proposed study is based on patients with type 2 diabetes, who are on any combination of the 
following anti-diabetic therapies: metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-IV 
inhibitor, or sulfonylureas but still remain poorly controlled with HbA1C between 7.0% and 10% 
(both inclusive), but not overtly out of control (> 10% of HbA1C). 
Though our study is over 16 weeks we do not anticipate significant drop in HbA1C in 
between the two groups in this relatively short time period to act as a confounding factor on 
CD34+ cell number, function and gene expression.  
Subjects will be taking 100 mg of Cana or placebo. Dose titration of metformin, will not 
be allowed.  Dose titration of insulin will be allowed to avoid hypoglycemia while maintaining 
HbA1C under 10%.  
 
Most clinical studies looking at HbA1C reduction were of a much longer duration of 6 months or 
above and most DPP-4 inhibitor studies are with animal EPCs where comparison with human 
CD34+ population is difficult to make. In one study27 where saxagliptin was used for 12 weeks 


 
 
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in drug-naïve type 2 diabetes patients there was placebo-subtracted HbA1c reduction noted of 
0.45-0.63%. It showed mean placebo-subtracted reductions in fasting serum glucose of 20 
mg/dL. From our laboratory studies of in vitro human EPC studies we do not think glucose level 
change of 20 mg/dL is significant enough to account for changes in EPC number, function and 
gene expression. However we will take the possibility of glycemia change/ reduction in 
experimental arm into account during statistical analysis of the results between the Canagliflozin 
and placebo groups. 
 
Visits with outcome measurements will be scheduled for the morning while overnight fasting 
(except water) to reduce diurnal variability, and clinical parameters will be obtained at scheduled 
office visits as depicted in the study schema and will be measured at each outcome visit as 
follows: 
 
Week -2: Subjects will be phone screened to determine initial eligibility. If the phone-screening 
indicates possible eligibility, then the subjects will come to an in-person screening visit that same 
week (If within two weeks that is acceptable). 
In-person screening (Week -2): Subjects will be screened and eligibility will be verified based on 
physical and laboratory tests. Equal numbers of male and female patients will be recruited.  
 
Week 0: At this stage patients will be randomized into the study. Subjects will have the first set 
of measurements. We will make sure that patients still meet the inclusion criteria. They will be 
started on medication or placebo. 
Weeks 0, 8 and 16: Subjects will all have the same measurements and will continue on study 
medication or placebo. All primary and secondary measures as outlined before will be carried 
out. 
 
PRIMARY OBJECTIVE 
The cellular outcome measures are as follows: 
To determine whether 4 months of Canagliflozin modifies CD34+ cell number, gene 
expression and function. 
 
Protocol for obtaining CD34+ cells: We will obtain a total of approximately 95 mL of peripheral 
blood per visit. Of these 95 mL, 60-70 mL will be used to obtain CD34+ cells from mononuclear 
cell (MNC) population and 25-35 mL for biochemistry and plasma ELISA assays. No samples 
will be stored for future research. MNC will be obtained from whole blood similar to protocols 
described before13,14. MNCs will be put through CD34 magnetic bead column to obtain CD34+ 
cells (Miltenyi Biotec). Purity of CD34+ cells, post sort, usually is above 90%, to be verified by 
FACS analysis. 
A. From Mononuclear (MNC) population (pre CD34+ magnetic column sorting): MNC cells 
(prior to CD34 magnetic bead sorting) will be also used to assay CFU Hill Colony 
forming unit (Stem Cell Technologies- Cat#05900) media for Colony Forming unit 
(CFU) Assay. CFU will be counted on 5-7 days post plating (from initial plating).  
B. From CD 34+ cell population (Post Sorting): 
a. Migration Assay using SDF1Alpha concentrations of 0, 10, 100ng/ml and VEGF-
A concentrations of 20 and 50 ng/ml, using 150,000 cells in 300ul serum free cell 
suspension media per insert.  


 
 
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b. Gene expression studies. Genes to be assessed on sorted CD34 positive cells: 
i. Endothelial lineage cell surface markers: CD34, VEGFR2 (KDR), CD31, 
CD144 
ii. For Anti-oxidant gene expression: Superoxide dismutase (SOD) 1, 2 and 
3, catalase, glutathione- peroxidase. 
iii. Apoptosis pathway: p53, p21, Bcl2, caspase-3 
iv. Endothelial Function Assay Gene: endothelial Nitric Oxide Synthase, von-
Willebrand’s Factor 
v. Genes associated with progenitor cell chemotaxis: VEGF-A, SDF1 alpha, 
CXCR4. 
 
SECONDARY OBJECTIVE 
To determine whether use of Canagliflozin alters other cardio-metabolic health indicator 
parameters such as: arterial stiffness measures, resting metabolic rate, body composition 
measures, hs-CRP, IL-6, TNF-alpha (inflammatory markers), fasting lipid profile, levels of 
insulin, glucose appetite controlling hormones and urine levels of microalubumin 
(Microalbumin/Creatinine ratio).. 
 
We expect to corroborate our findings of Aim 1 (cellular outcome measures) with traditional 
markers of endothelial/ vascular function such as serum biochemistry and arterial stiffness. 
A. Effect of Cana on Serum Biochemistry & Urine:  
a. Looking at inflammation, apoptosis and anti-oxidant protein levels: Highly 
selective C-reactive protein (hs-CRP), IL-1, IL-6, TNF-alpha, MCP-1 , fasting 
glucose, comprehensive metabolic panel, insulin and lipid profile.  
b. Plasma SDF1 alpha (ELISA) and GLP-1 (ELISA) will be estimated to assess 
endothelial health and factors that may influence CD34+ cell chemotaxis 
c. Estimation of Creatinine Clearance and proteinuria: 24hr urinary protein estimation 
and creatinine clearance (obtained via micro-albumin/creatinine ratio from a spot 
urine sample). 
i. Optional sub-study to investigate urine exosomes. See page 31. 
d. The glomerular filtration rate (GFR) will be estimated using the CKD-EPI 
(Chronic Kidney Disease Epidemiology Collaboration) equation. The CKD-EPI 
equation, expressed as a single equation, is: 
GFR = 141 X min (Scr/κ,1)α X max(Scr/κ,1)-1.209 X 0.993Age X 1.018 [if 
female] X 1.159 [if black]; where Scr is serum creatinine (mg/dL), κ is 
0.7 for females and 0.9 for males, α is –0.329 for females and –0.411 for 
males, min indicates the minimum of Scr/κ or 1, and max indicates the 
maximum of Scr/κ or 117. 
 
B. Effect of Cana on Body Habitus (Determination of body composition and visceral 
fat) AND Resting Metabolic Rate.   
a. Height and weight will be measured and the body mass index (BMI=kg•m-2) 
used as an indicator of relative weight. The body composition scale calculates 
body fat% and total body water% in addition to BMI. 


 
 
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b. We also intend to follow Resting Metabolic Rate (RMR): at weeks 0, 8 and 16. 
Indirect calometric measurements of basal metabolic rate will be obtained using 
machine obtained from Korrs Medical. This is a noninvasive procedure. We plan 
to measure the oxygen that the body consumes.  It will help us calculate a 
patient’s Resting Energy Expenditure (REE), commonly referred to as a Resting 
Metabolic Rate (RMR).   As our protocol is directed to overweight and obese 
individuals using incretin therapy, it will be beneficial to note if the treatment 
modifies RMR. Description: The machine ReeVue by KORR directly measures 
the concentration of oxygen breathed out by each patient.  The patient breathes 
through a simple mouthpiece as all the exhaled air is collected and 
analyzed.  Because there is a direct correlation between oxygen consumed and 
calories burned (4.813 calories for every milliliter of oxygen consumed), an 
accurate measurement of oxygen consumption is an effective measurement of 
calorie consumption. The machine uses a one-way valve in the disposable 
(MetaBreather) mouthpiece.  This draws in fresh room air with each inspiratory 
breath and eliminates concerns about cross contamination.  The disposable 
mouthpiece is thrown away. It is un-necessary to clean the equipment or the 
tubings between each patient. This equipment is FDA approved and has been used 
in physician offices as an oxygen uptake test with associated CPT code #9469027. 
Other related trials have shown weight loss but effect on metabolic rate has not 
been studied18. 
C. Effect of Cana on Assays of Arterial Stiffness:  
a. We will acquire Pulse wave analysis and Vascular Flow using SphygmoCor CP 
system from ATCOR as a measure of central arterial pressure and arterial 
stiffness. Again SGLT2 inhibitors have been shown to reduce systolic blood 
pressure but effect on stiffness has not been looked at, except with 
Empagliflozin17,18. 
b. We also intend to acquire Pulse wave analysis and Vascular Flow using 
SphygmoCor CP system from ATCOR to measure central arterial pressure and 
arterial stiffnesss28,29,30,31,32. The secondary measures are indirect measures of 
endothelial inflammation in early type 2 diabetes patients11,12,19,33,34.  
D. Other Measures: 
a. Urine pregnancy testing for women of child bearing potential. 
b. Creatinine Clearance and proteinuria estimation (obtained via micro-
albumin/creatinine ratio from a spot urine sample).   
 
STUDY MATERIALS 
Janssen, manufacturers of Cana is expected to provide Cana at no cost for this study.  
 
SAFETY ASSESSMENTS 
Study drug toxicities will be assessed continuously. Adverse events will be evaluated on a 
continuous basis while the patient is on study and until 30 days after the last dose of study drug. 
Patients should be followed until all treatment-related adverse events have recovered to baseline 
or are deemed irreversible by the principal investigator.  
 
 


 
 
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7 
STATISTICAL CONSIDERATIONS 
 
STATISTICAL ANALYSIS 
Sandri et al. (2005, Study A, patients with peripheral arterial occlusive disease) found that in the 
control group, CD34+ cells increased from 372 cells per mL blood at baseline (SD 156) to 402 
(SD 183) at 4 weeks. In their exercise training group, the increase was from 458 (SD 252) at 
baseline to 2977 (SD 852) at 4-weeks.  We created a simulated data set with these parameters in 
order to calculate the percent of CD34+ cell variation that was explained by the group x time 
interaction. We tested two random effects mixed models, one without and the other with, a group 
x time interaction (group is treatment vs control; time is pre vs. 4-weeks post). The percent 
variance explained = (Vno-int – Vint)/Vno-int, where Vno-int is the residual covariance 
parameter estimate without a group x time interaction term in the model, and Vint is the residual 
covariance parameter estimate with a group x time interaction term in the model. We found that 
based on the baseline and 4-week mean and standard deviation of CD34+ cell concentrations 
reported by Sandri et al. (2005), the interaction explained 84.5% of the variance in CD34+ cell 
concentration (a very large effect size). The correlation among repeated measures was r=.62. 
Using these parameters in G-Power3 (version 3.1.3), with 2 groups measured at 2 time points, in 
order to achieve power >.95 would require a total sample size of 6 subjects (3 per group) each 
measured at 2 time points. Assuming 25% subject loss, we would need to start with 4 per group. 
If the group x time interaction explained only 50% of the variance in CD34+ cell count (still a 
large effect), we would need total sample size of 8 (4 per group) in order to achieve power >.95. 
In order to account for possible 25% drop-out, 6-8 per group should be randomized.  
In Sen et al. (2015), in a sample of patients with pre-diabetes (n=11), CD34+ cell number 
increased from 0.8 (SEM 0.1) before exercise, to 1.4 (SEM 0.2) after exercise, a pre-post effect 
size (Cohen’s d) of 1.81.  In order to detect an effect of this size using a 2-tailed paired t-test, 
with alpha=.05, for power >.80, >.90, and >.95, the number of subjects required would be 5, 6, or 
7, respectively.   
However the two quoted studies quoted above are very different from our proposed study on 
patients’ with established endothelial dysfunction entity such as diabetes. It is well known 
(Werner et al, Ref No. 11) that diabetes can affect CD34+ number and function, therefore in our 
proposed study, number of CD34+ cell availability may be a limiting factor. Therefore we 
propose to use double the number suggested by power calculation and use 8x2=16 patients per 
group so that CD34+ cell based end-point studies are not compromised.  
DATA ANALYSIS 
Digital data files will be stored in a protected limited-access folder on the MFA network. Only 
the Study Biostatistician and the PI will have direct access to the data once it is on this server. 
The data file will have random subject identifiers and will have no HIPAA identifiers.  
 
The distributional assumptions of all measures will be examined. Means and standard deviations 
will be computed for continuous measures and proportions for categorical variables. Graphical 
representations of the mean group slopes and individual slopes will be generated and inspected.  


 
 
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Study groups will be compared to determine whether any imbalance between the groups on 
patient characteristics remain after randomization.  If imbalance is evident, by inspection, all 
models will be adjusted for the unbalanced covariates.  Change in outcome over time will be 
examined using a multilevel approach with linear mixed models (LMM)35,36.  The longitudinal 
multilevel modeling approach will enable us to examine characteristics of within-person change, 
as well as between-group differences that may influence change.  LMMs handle missing data 
more efficiently than traditional ANOVA designs. In the linear mixed models, the patient is 
considered a random effect and the outcome measured at specific time points is nested within 
patient. Time (week 0, 8, 16) will be modeled as a continuous variable in order to assess linear 
effects from 0 to 8 to 16 weeks. In order to determine whether there are non-linear time effects 
(e.g., rapid increase from 0 to 8 weeks followed by little change from 8 to 16 weeks; or initial 
decrease at 8 weeks followed by increase at 16 weeks, etc.) we will also need to include models 
in which time is defined as a class variable (with 0 as the reference group). Subsequent analyses 
will add study group and other patient characteristics as covariates. We are particularly interested 
in whether there is a significant interaction between study group and time, adjusting for other 
patient level covariates.  A significant interaction would indicate that the slopes in outcome over 
time differ by study group.  If the global test of interaction is significant, subsequent significance 
testing of the pair wise comparisons of group slopes group will be adjusted for multiple 
comparisons.  The model also includes coefficients for the group-by-time interactions and 
random effects for patient and time. 
 
𝑌𝑖𝑗= 𝛽0 + 𝛽1𝑇𝑖𝑚𝑒𝑖𝑗+ 𝛽2𝐺𝑟𝑜𝑢𝑝𝑖+ 𝛽3(𝑇𝑖𝑚𝑒𝑖𝑗 𝑥 𝐺𝑟𝑜𝑢𝑝𝑖) + + 𝜀𝑖𝑗 
 
The interaction between GROUP and TIME will be modeled in a similar fashion using two 
additional indicator variables.   The above multivariable model enables us to adjust for time-
varying covariates, such as glucose.  Since glucose will be measured contemporaneously with 
the outcomes at each visit, however, causal interpretations of glucose and any outcome are 
suspect.  As such, we will consider glucose as a nuisance covariate, and consider any relationship 
between glucose and an outcome as merely associative.   
 
Privacy Protection 
All data collected for the research study will be collected under a random subject ID.  
 All samples being sent to LabCorp for standard of care labs will be labeled with the 
subject ID, DOB, Date of visit, time of collection, and Visit number. These samples will 
not be stored as they will be sent down to LabCorp immediately.  
 The whole blood and plasma being used for ELISA and CD34+ analysis will be labeled 
with the subject ID, Date of visit, and Visit number. No additional data will be provided 
with the samples. These samples will be analyzed and stored at The George Washington 
University Ross Hall in Dr. Sen’s Laboratory,  
 The urine sample being used for the exosome sub-study will be labeled with the subject 
ID, Date of visit, time of collection, and Visit number. They will also be provided with 
subject gender, and age. These samples will be analyzed and stored (as per the protocol 
below) at the VA by Dr. Eric Nylen. 
All other study data will be both electronically stored on a secure server, and physically stored as 
patients files locked in the Department of Medicine. The only individuals who will have access 


 
 
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to the study data will be the members of the research staff team, as indicated in the GW IRB 
Form “Research Team Personnel Form (HRP-201)”. 
 
8. APPENDIX A: OPTIONAL URINE EXOSOME STUDY, 
Veterans Affairs, Dr. Eric Nylen 
A urine sample is already required as a part of Dr. Sen’s Canagliflozin protocol. This urine will 
be used for urine pregnancy tests (if applicable), and labcorp tests for microalbumin/creatinine 
ratio. There will be excess urine.  
 
 
Subjects will have the option to opt in or out of a research study being conducted by Dr. Eric 
Nylen of the VA, in which the urine will undergo a urine exosome analysis.  
 
Purpose of this Study: Exosomes are cell-derived vesicles that are present in many and perhaps 
all biological fluids, including blood, urine, and cultured medium of cell cultures. The reported 
diameter of exosomes is between 30 and 100 nm, which is larger than LDL, but much smaller 
than for example, red blood cells. Exosomes are either released from the cell when 
multivesicular bodies fuse with the plasma membrane or they are released directly from the 
plasma membrane.[3] Evidence is accumulating that exosomes have specialized functions and 
play a key role in, for example, coagulation, intercellular signaling, and waste 
management.[1] Consequently, there is a growing interest in the clinical applications of 
exosomes. Exosomes can potentially be used for prognosis, therapy, and biomarkers for health 
and disease. In this study we are particularly interested in exosomes released from podocytes as a 
marker of podocyte function and podocyte inflammation. Podocytes are often considered a 
modified endothelium and one of its more commonly used parameter is microalbuminuria. 
In this study we are using urine exosomes as an added parameter of kidney function other than 
microalbuminuria and GFR calculation. 
 
The procedure for this study is as follows: 
10-50 mL of the fresh spot urine is placed into a urine cup containing 1 tab of cOmplete Ultra - 
protease inhibitor cocktail (Roche) for exosome analysis performed at the Veterans Affairs (VA) 
Maryland Health Care System.  
The specimen can be refrigerated at 4ºC for ~ 1 week, frozen at -80C, or processed immediately. 
Transfer to the VA will be done following appropriate procedures outlined in the MFA-VA 
Materials Transfer Agreement (MTA). The urine will be processed for exosomes as follows:  
 
The supernatant may be subjected to ultracentrifugation, with final centrifugation at 200,000g for 
2 hr at 4ºC to obtain the urinary exosome pellet. The pellet would then be tested for exosomes 
markers, namely CD63, CD9, CD81, Hsp70, and WT-1, nephrin and podocalyxin, using one of 
two methods. Preferably mRNA will be extracted and then RT-PCR will be run. Alternative 
analysis will be done using gel electrophoresis followed by Western blot techniques.   
 
Method 1: Urine exosome mRNA extraction 
mRNA extraction from exosomes will be accomplished by using commercial kits (Norgen 
Biotek Corp., Catalog # 47200). Thereafter, a pre-amplification step will take place, this is a 


 
 
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necessary step to measure mRNA in exosomes and will be followed by quantification of specific 
endothelium and podocyte proteins such as CD9, WT-1, nephrin and podocalyxin by RT-PCR. 
 
Total urinary protein content, albumin and creatinine will be measured via Lapcorp for the 
same urine samples that have been collected.  
 
Method 2: Gel electrophoresis and Western blot  
Exosome pellet will be dissolved in Laemli buffer and proteins will be resolved by gel 
electrophoresis (4-12% polyacrylamide gel) under denaturing conditions. Subsequently, proteins 
will be transferred to a PVDF membrane by Western blotting using a dry blotting method (iBlot). 
The blot will be incubated with blocking solution for 1h, and subsequently exposed to a solution 
with the first antibody overnight, at 4ºC. After washing (5 x 5 min), the membrane will be 
exposed to a secondary antibody (horseradish peroxidase [HRP]-conjugated) for 1h at room 
temperature. Finally, the membrane will be incubated with an HRP substrate reagent to reveal 
the proteins bound to the antibodies tested. Quantitative analysis will be performed by 
densitometry readings of the positive signals corrected to creatinine concentration of the urine 
aliquot equivalent to each exosome sample.  
 
End-point: Exploratory but in conjunction with urine albumin/creatinine ratio will help to 
establish impact of Linagliptin on renal function in diabetes related CKD population. 
 
 


 
 
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3 M. Afkarian, M. C. Sachs, B. Kestenbaum, I. B. Hirsch, K. R. Tuttle, J. Himmelfarb, and I. H. de Boer, “Kidney 
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8 N. Werner, S. Kosiol, T. Schiegl, P. Ahlers, K. Walenta, A. Link, M. Böhm, and G. Nickenig, “Circulating 
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9 D. W. Losordo, R. A. Schatz, C. J. White, J. E. Udelson, V. Veereshwarayya, M. Durgin, K. K. Poh, R. Weinstein, 
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10 Zinman B, Wanner C, Lachin JM, Fitchett D, Bluhmki E, Hantel S, Mattheus M, Devins T, Johansen OE, Woerle 
HJ, Broedl UC, Inzucchi SE; EMPA-REG OUTCOME Investigators. Empagliflozin, Cardiovascular Outcomes, and 
Mortality in Type 2 Diabetes.  N Engl J Med. 2015 Sep 17.  
 
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Medicine. 2005;108:299-314 
 
12 Krenning G, Dankers PY, Drouven JW, Waanders F, Franssen CF, van Luyn MJ, Harmsen MC, Popa ER. 
Endothelial progenitor cell dysfunction in patients with progressive chronic kidney disease. Am J Physiol Renal 
Physiol. 2009 Jun;296(6):F1314-22 
 
13 Jialal I, Devaraj S, Singh U, Huet BA. Decreased number and impaired functionality of endothelial progenitor 
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14 Sen S, Merchan JA, Dean J, et al. Autologous transplantation of endothelial progenitor cells genetically modified 
by adeno-associated viral vector delivering IGF-1 gene following myocardial infarction. Human Gene Therapy Oct 
2010: 21: 1327-1334.  
 
                                                          
 


 
 
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15 Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis model assessment: Insulin resistance and beta-cell 
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