Impact of Semaglutide (Long Acting GLP1 Agonist) on Peripheral Blood Derived CD34+ Endothelial Cells (EPCs) and Subcutaneous Fat Derived Mesenchymal Stromal Cells ( MSCs) in Type 2 Diabetes Subjects
Phase: PHASE4
Conditions: Diabetes Mellitus, Type 2
Sponsor: Sabyasachi Sen

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IMPACT OF SEMAGLUTIDE (LONG-ACTING GLP1 
AGONIST) ON PERIPHERAL BLOOD DERIVED CD34+ 
ENDOTHELIAL PROGENITOR CELLS (EPCS) AND 
SUBCUTANEOUS FAT DERIVED MESENCHYMAL 
STROMAL CELLS (MSCS) IN TYPE 2 DIABETES 
SUBJECTS
INVESTIGATOR-SPONSORED STUDY PROPOSAL
UNIVERSAL TRIAL NUMBER (UTN)
U1111-1209-6967
Department of Medicine, Endocrinology
The GW Medical Faculty Associates
2300 M Street, NW
Washington, D.C.
20037
Correspondence to:
Sabyasachi Sen, MD, PhD
Associate Professor of Medicine
ssen1@email.gwu.edu
Phone: 202-994-8560
Fax: 202-741-2490
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Table of Content
Page
1. Background and Significance 
3 
2. Specific Objectives
5
a. Primary Objectives
b. Secondary Objectives
c. Tertiary Objectives 
3. Research Design and Methods 
5
a. Study Hypothesis
b. Study Endpoints/Objectives
c. Study Type and Population
d. Inclusion and Exclusion Criteria
e. Withdrawal Criteria
4. Study Design 
10
5. Statistical Consideration 
15
6. Data handling and Record Keeping
16
7. Ethics
17
8. Study Schedule
18
9. Study Drugs and Materials
19
10.Concomitant Illness and Medications 
22
11.Adverse Events
22
12.Liability and Subject Insurance
26
13.Evaluability of Subjects
27
14.Premature Termination
27
15.Publication Plan
27
16.Appendix A: CD34+ Cell Analysis
27
17.Appendix B: Subcutaneous Fat Biopsy Procedure
28
18.Appendix C: Subcutaneous Fat Analysis 
28
19.Appendix D: Event Chart
29
20.References
30
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1. BACKGROUND AND SIGNIFICANCE:
Diabetes affects more than 9% 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 disease and blindness [2,4]. Endothelium and its progenitors, 
meaning endothelial progenitor cells (EPCs), are an established surrogate of cardiovascular risk 
outcome measures [4, 6]. EPCs have been defined as CD34+ cells [6] thereby identifying a 
defined homogenous population from a heterogeneous peripheral blood derived mononuclear 
cells.
We and others, have previously shown that EPCs can act as a cellular biomarker that is more 
reliable than serum based markers for CVD risk estimation [6, 10]. We demonstrated that gene 
expression in EPCs change within two weeks of an intervention such as aerobic exercise [10]. 
On the other-hand serum biomarkers usually take much longer time to change secondary to an 
intervention [4, 16, 17]. Also the paracrine effect of damaged endothelium is secondary to gene 
expression changes that have been altered in the progenitor cells several months ahead of 
discernable changes in serum based biomarkers such as endothelium based inflammatory 
markers. When serum inflammatory markers are elevated that may mean that the endothelium is 
already damaged/ inflamed and possibly irreversibly. [16, 17]. 
EPC are the future endothelium, therefore studying EPCs may help us to predict the effect of an 
intervention (such as a medication or exercise) on the future of endothelium and endothelial 
function. [5] In normal course of events, the 
EPCs transition to mature endothelium and 
replace endothelial cells after normal cell 
death 
cycle 
or 
programmed 
apoptosis. 
However, unfortunately, type 2 diabetes being 
a pro-inflammatory, high ROS disease [4] 
process, 
chronically 
depletes 
the 
EPC 
population 
by 
up-regulating 
apoptotic 
pathways mediated by p53 [4]. As an apoptotic 
condition, hyperglycemia even mild (such as 
prediabetes) affects immature EPCs more so 
than the mature endothelium. Hence, the 
damaged and inflamed mature endothelium, 
with time, is not replaced by EPCs [10] as the 
progenitor pool has been depleted. This maybe 
one of the reasons why vascular damage takes 
4-5 years to develop following onset of 
hyperglycemia. [2-4]
It is known that GLP1 agonist has positive effect on oxidative stress, and endothelial function, 
therefore semaglutide can be hypothesized to have a positive effect on EPC and endothelium and 
possibly reduce fat inflammation. [8,9]. It may also reduce transformation of multipotent 
mesenchymal stem cells (MSCs) towards more fat formation (prevent adipogenesis) which may 
explain weight reducing capability seen in semaglutide studies (SUSTAIN trials). The use of 
Endothelial Progenitor Cells (EPCs, defined as CD34+ cells) 
replace injured/ apoptotic mature endothelial cells [5]
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CD34+ cells and MSCs as a biomarker is novel. One can obtain CD34+ cells from a simple 
peripheral blood draw (without doing an invasive procedure). The blood is then sorted for a 
homogenous progenitor/stem cell population [6]. Role of CD34+ve EPCs in vascular biology, 
heart regeneration and collateral vessel formation as an endothelial progenitor cell is well 
established. It’s role as a biomarker is also being developed [7,10,12]. CD34+ cells are the most 
studied cardiovascular progenitor cells and its efficacy has been established in chronic diseases 
such as diabetes by Werner et al in 2005. [6,17]  
Similarly, one can obtain fat derived MSC from fat biopsies, particularly from overweight and 
obese individuals. Diabetes is not only a state of endothelial dysfunction, it is also a state of fat 
hyperplasia, insulin resistance at the level of muscle and fat and is associated with high ROS. 
Improvement of endothelial health is most likely paired with healthier fat. A state of healthier fat 
will be associated with healthy adipocytes, pre-adipocytes and healthy MSCs [3,13-15].
The weight reducing data from SUSTAIN 6 trial [16] using semaglutide at 0.5mg and 1.0mg, is 
encouraging. It has also shown significant improvement in blood pressure and HbA1C within 8 
weeks and definitely by 16 weeks even at a lower FDA approved dose of 0.5mg once a week.
These finding prompted us to use MSC as a fat surrogate and EPCs as an endothelial surrogate to 
establish a cellular mechanism behind the clinical trial findings. It may also shed light on cross-
talk between these two important insulin responsive tissues that contribute towards 
cardiovascular health.
We believe EPC is the ideal cellular 
vascular outcome biomarker while 
MSC is the ideal adipocyte health bio-
marker. 
Based 
on 
our 
recently 
published data on saxagliptin’s effect 
on EPC of subjects with Type 2 
Diabetes, we are confident that EPC is 
a robust endothelial marker with quick 
changes in number, function and gene 
expression, 
after 
appropriate 
intervention. [18].
The purpose of the present 
study is to study the effect of a long-
acting GLP-1 agonist, over a period of 
24 weeks and understand how it 
influences two different yet related 
cell types such as endothelium and 
adipocyte, both of which are key 
players in insulin resistance/sensitivity 
in the body [14,17].
Mesenchymal Stem Cells (MSCs) differentiate into osteoblasts, 
adipocytes and chondrocytes. MSCs is a multipotent stem cell that can 
be obtained from bone marrow, fat and any mesenchymal tissue [17] 
and should be able to differentiate in these 3 lineages in 
normoglycemic environment. Hyperglycemia and certain 
environments, such as PPARG agonists, presence of xeno-estrogens, 
accentuates adipogenic lineage thereby prevents osteogenesis and 
chondrogenesis. We would like to investigate how GLP1 influences 
this differentiation pathway
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2. SPECIFIC OBJECTIVES:
Primary Objective: semaglutide modification of fat derived mesenchymal stem cells and 
CD34+ cell number, function, and gene expression.
This will help identify genes and pathways that are responsive to semaglutide in the two 
different stem cells and identify associated proteins and pathways that may be quickly 
responsive and important in two different cell lineages
Secondary Objective: Investigate mitochondrial metabolism of undifferentiated cells such as 
MSCs derived from subcutaneous fat of subjects treated with or without semaglutide. 
This will Evaluate whether semaglutide prevents adipogenic differentiation of fat derived 
stromal cells which will explain clinical observation of weight loss (SUSTAIN- trial).
Tertiary Objective: To determine whether semaglutide improves vascular reactivity (arterial 
stiffness), change body habitus parameters, serum endothelial inflammatory markers and levels 
of appetite controlling hormones.
This will investigate whether semaglutide improves endothelial dysfunction.
3. RESEARCH DESIGN AND METHODS
Study Hypotheses: 
We hypothesize that GLP1 agonists, like semaglutide, have a positive effect on the EPC number, 
function, targeted gene expression, arterial stiffness and endothelium specific inflammatory 
markers.
Additionally, we hypothesize that semaglutide therapy will reduce adipogenesis and 
increase bone and cartilage formation by increasing cellular metabolism, as evidenced by 
increased mitochondrial biogenesis and increased cellular oxygen consumption rate (OCR, 
measured by SeaHorse). 
Study Endpoints: 
Primary Objective (Peripheral blood based Endothelium directed outcome measures): 
semaglutide modification of fat derived mesenchymal stem cells and CD34+ cell number, 
function, and gene expression.
1. CD34+ cell number, function, and gene expression. The CD34+ cell number will be 
measured as % of total mononuclear cell (MNC) population. The function will be 
assessed as migratory potential of the CD34+cells in response to SDF1a.  We will 
examine the cellular expression of genes that control endothelial function and cell 
survival and apoptosis genes such as SOD1-3, catalase, peroxidase, eNOS, vWF, hsCRP, 
IL-6, IL1β, TNF-alpha, COX2, endothelin 1, p53, p21, and caspases. This endpoint will 
be obtained by a venous blood draw. (Week 0, 8, 24).
Details: Upto 95 ml of peripheral blood will be obtained by venipuncture of which 65-70 mL 
of whole blood will be used to obtain CD34+ cells from the mononuclear cell (MNC) 
population. 20-25ml of whole blood will be used for biochemistry and serum ELISA assays.
MNC will be obtained from whole blood prior to CD34 column sorting following protocols 
described before and currently used in our laboratory for clinical studies [18]. A portion of the 
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MNCs will be plated to obtain and count colony formation units (CFU) at day 5 and an 
additional portion will be analyzed to determine increased or decreased presence of stem like 
cells (marked by CD34+, CD133+) by Fluorescence-activated cell sorting (FACS) The 
remaining MNCs will be sorted and separated by magnetic bead column method to obtain 
CD34+ cells (Miltenyi Biotec). 
Post-CD34 column sort we will undertake migration studies and mRNA isolation: 
First, we will count the number of viable CD34+ve cells obtained post column separation and 
divide that number by the total MNCs obtained to note the percentage of CD34+ve cells.
Next we will set up the migration assays using a Boyden’s chamber using SDF1 alpha at 0, 
10ng/ml and 100ng/ml concentration. The migration will be assessed at 24 hours using 
standard published methods [18].
We will undertake targeted gene expression by RT-PCR on CD34 positive cells from mRNA:  
a. Anti-oxidant genes (SOD 1-3, catalase and peroxidases), glucose transporters [4,13]
b. Endothelial function assay genes: endothelial NOS (eNOS), von-Willebrand’s factor (vWF), 
VEGF and KDR [17]
c. Inflammatory genes such as IL6, IL1β, TNFα, COX2 and endothelin-1 [4,17]
d. Apoptosis pathway genes such p53, p21, Bax-Bcl2 and caspase-3 & 9 [4,17]
Secondary Objective (Fat and fat derived MSC based outcome measures): 
1. Investigate targeted gene expression of subject’s subcutaneous fat.
SEMAGLUTIDE’S EFFECT ON FAT METABOLISM. THIS ENDPOINT WILL BE 
OBTAINED BY A SUBCUTANEOUS FAT BIOPSY OBTAINED FROM THE 
SUBJECT’S LEFT ABDOMEN. (WEEK 0 AND 24). We will evaluate mRNA gene 
expression and western blot protein estimation for mature fat and fat related transcription 
factors: leptin, perilipin, adiponectin, PPARG, CREBP-α and β, inflammatory markers: TNFα, 
IL-6, antioxidants: SOD-1 to 3 and Catalase in antioxidant MSC groups. We will estimate 
PGC1α, UCP-1 and PRDM-16 gene expression in the fat depots in order to verify browning or 
beiging of fat Genes, that will be tested are categorized as follows: 
Genes associated with 
Inflammation: IL-6 (interleukin 6), IL-10, IL1β, TNFα (tumor necrosis factor- alpha), 
Cellular Apoptosis p53, p21, caspase-3 and 9, 
Cellular Anti-oxidants: SOD1-3 (superoxide dismutases), CAT (catalase), GPX-1 & 3 
(glutathione peroxidase), 
Genes associated with mitochondrial complex subunits such as NDUF1 and SDHB [15,31], 
Genes associated with lipid formation and accumulation: CEBPα and CEBPβ (cholesterol 
ester binding protein α and β), PPARG, FABP4, Leptin, FGF21, Adiponectin and Perilipin;
Genes associated with mitochondrial activity such as COX2, COX4, TFAM, ATP5B, NRF-1, 
NRF-2, UCP1, PGC1A, PRDM-16, Sirtuin-1 and Sirtuin-3 
Genes associated with glucose transport and taste receptors: GLUT1, GLUT4, TAS1R3, 
TAS2R3.
2. Fat derived mesenchymal stromal cells (MSCs). We will investigate changes (up or 
downregulation) in bone, muscle, cartilage and adipocytes associated gene expressions in 
adipose tissue- derived MSCs. This endpoint will be obtained by a subcutaneous fat 
biopsy obtained from the subject’s left abdomen. (Week -8, 16)
Along with the genes noted above we will look at MSC differentiation [21-26] 
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Adipogenic Genes: CEBPΑ AND CEBPΒ (CHOLESTEROL ESTER BINDING PROTEIN 
Α AND Β), PPARG, FABP4, PLIN. 
Myogenic Genes: MYOD (Myogenin D)
Osteogenic Genes: RUNX2, BGLAP (osteocalcin), ALPL (alkaline phosphatase), BMP2, 
BMP6, SMADS3, TGF-B. 
Chondrogenic: COL1A, COL2A COLLAGEN TYPE II (CO-II), COLLAGEN TYPE XI 
(CO-XI), ACID PHOSPHATASE (ACP), CARTILAGE OLIGOMERIC MATRIX 
PROTEIN (COMP) AND ELASTIN
Tertiary Objective: To determine whether semaglutide improves vascular reactivity (arterial 
stiffness), change body habitus parameters, serum endothelial inflammatory markers and 
levels of appetite controlling hormones. 
 
Arterial Stiffness: We will acquire Pulse Wave Analysis and Vascular Flow using 
SphygmoCor CP system from ATCOR [19] as a measure of central arterial pressure and 
arterial stiffness. Vessel health will be assessed by degree of arterial stiffness, using 
arterial tonometry. 

Body Composition Scale: Height and weight will be measured and the body mass index 
(BMI=kg/m2) used as an indicator of relative weight. The body composition scale 
calculates body fat%, total body water%, fat free mass, etc., in addition to BMI.

Hip & Waist Measurements

Blood biochemistries and Urine microalbumin ratio: Patient blood biochemistries will 
be collected as a part of standard of care, and to monitor the overall health of the subjects 
throughout the protocol. We will be measure HbA1C, Lipid Panel, CRP, comprehensive 
metabolic panel (including liver panel), leptin, adiponectin, glucose, insulin, IL-1B, IL-6, 
IL-10, TNFα, leptin, ApoB, Apo A1 and urine micro-albumin /creatinine ratio.

Appetite controlling hormones: We plan to investigate changes in GLP1 (via LabCorp) 
and ghrelin (via ELISA). 
Study Type:
This is a single center, double-blind, placebo-controlled, parallel group, randomized phase IV 
clinical trial. There will be two treatment arms (Placebo and semaglutide), each with 20 patients. 
Subjects will be undergoing treatment for 16 weeks. 
    
Study population:
We will randomize 40 subjects to start on study medications, with approximately 20% drop out 
rate over two years in a hope to retain approximately 32 individuals (16/group). We plan to 
screen approximately 60 subjects. This is a single site study, with the sole site being the GW 
Medical Faculty Associates in Washington, D.C.
We will attempt to recruit patients from across all races and ethnicities as much as possible to 
minimize ethnicity factors affecting results. Subjects only on metformin as diabetes medication 
will be included. Subject cohort with at risk for CVD being diabetic and obese but subjects with 
known history of myocardial infarction and cerebrovascular accident will be excluded. We want 
to study semaglutide without the confounding effect of any other anti-diabetic agents, other than 
metformin, in a patient population at risk of CVD, so that there is room for endothelial function 
improvement in this cohort.
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CVD risk in addition to type 2 diabetes will be an added as an inclusion criteria as determined by 
the investigator such as microalbuminuria or proteinuria (as defined by ADA), hypertension 
(labile and uncontrolled hypertension) and left ventricular hypertrophy, left ventricular systolic 
or diastolic dysfunction, or an ankle–brachial index [the ratio of the systolic blood pressure at the 
ankle to the systolic blood pressure in the arm] of less than 0.9, low HDL with 
hypertriglyceridemia (as defined by NCEP ATP III) , strong family history of CHD (as defined 
by NCEP ATP III) [27, 28] 
The primary outcome measures are based on endothelium, secondary outcome markers on fat/ fat 
derived MSCs and tertiary outcome markers are based on body composition, arterial stiffness, 
serum and urine biochemistries. All three outcome measures as elaborated above, are crucial in 
order to corroborate our cellular findings with currently accepted [17] clinical efficacy outcome 
measures such as arterial stiffness [19] and serum biochemistry. This design is similar to our 
recently published manuscript on Saxagliptin and cellular outcome measures [18].
Inclusion Criteria:
1. Age 30-70
2. Diagnosed with Type 2 diabetes mellitus 
3. Body Mass Index (BMI) between 25.0-45.0 (both inclusive)
4.  eGFR ≥ 30 mL/min/1.73 m2 by MDRD
5. HbA1C 7.0 – 10.0 %
6. Subjects on a stable dose of Metformin (1-2 grams), only, for 3 months prior to 
screening.
7. Ability to provide informed consent (and document informed consent by signature) 
before any trial-related activities are conducted. 
8. Additional CVD risk factor such microalbuminuria or proteinuria (as defined by ADA, 
UACR > 30 mg/g), hypertension (labile and uncontrolled hypertension) and left 
ventricular hypertrophy, left ventricular systolic or diastolic dysfunction, or an ankle–
brachial index [the ratio of the systolic blood pressure at the ankle to the systolic blood 
pressure in the arm] of less than 0.9, low HDL with hypertriglyceridemia (as defined by 
NCEP ATP III) , strong family history of CHD (as defined by NCEP ATP III and ATP 
IV).
9. Retinal examination within last 18 months of enrollment, showing no proliferative 
retinopathy 
Exclusion Criteria:
1. Uncontrolled hyperglycemia with fasting glucose >240 mg/dL (>13.3 mmol/L)
2. Liver disease with ALT, AST or ALP ≥ x3 ULN
3. Planned CV surgery or angioplasty in the past 1 month
4. History of established CVD
5. Known personal history of cerebral stroke or heart attack ( myocardial infarction)
6. All other diabetes medications other than metformin  
7. Personal or family history of medullary thyroid cancer (MTC)
8. Personal or family history of Multiple Endocrine Neoplasia Syndrome Type 2 (MEN 2)
9. GFR <30 mL/min/1.73 m2 by MDRD
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10. Prior surgery with chronic malabsorption (eg, bariatric) in prior 1 year
11. Clinically significant RBC disorders such as hemoglobinopathies
12. Diagnosis of Type 1 diabetes mellitus or history of GAD antibody positive status 
13. Chronic use of anti-inflammatory drugs for the last 3 months 
14. Beginning statin medications or change in statin dose in the past 1 month
15. Use of consistent long-term steroid medication (oral, inhaled, injected) within the last 1 
month
16. History of pancreatitis
17. Known or suspected allergy to GLP-1 agonists, excipients, or related products.
18. Active smokers
19. Active wounds (i.e. diabetic ulcers) or recent surgery within 1 month
20. Untreated hyper/hypothyroidism
21. Implanted devices (eg. Pacemaker) that may interact with Tanita scale
22. Any other clinical condition that would jeopardize patients safety while participating in 
this clinical trial
23. Women of child bearing potential who are not willing to use a contraceptive method to 
avoid pregnancy for the 16 weeks of study duration plus 2 months post treatment (for 
semaglutide washout).
24. Women who are pregnant or breastfeeding
25. Chronic or persistent alcohol or drug abuse
26. Prisoners or subjects who are involuntarily incarcerated
27. Subjects who are compulsorily detained for treatment of either a psychiatric or physical 
(eg. infectious disease) illness
28. Participation in another trial with an investigational drug within 30 days prior to informed 
consent.
29. Untreated or active hemorrhagic proliferative diabetic retinopathy
Exclusionary Laboratory Findings
30. Chronic Kidney Disease (CKD) stages 4 and 5 (estimated CrCl less than 30 mL/min)
31. Serum creatinine levels ≥1.8 with estimated CrCl < 60 mL/min
32. Triglycerides > 500 mg/dL
33. Low hematocrit (<28 Units)
Withdrawal Criteria:
This research study is entirely voluntary, and whether or not a subject takes part is entirely up to 
them. Subjects can withdraw at any time. If subjects withdraw after being randomized, all their 
data will be kept up to that date, unless a letter is written to the sponsor-investigator explicitly 
indicated against this. If subjects withdraw due to health reasons, they may be followed for 
safety reasons. 
Any subject with active or bleeding hemorrhagic proliferative diabetic retinopathy as a side 
effect, will be taken off the Investigational product but they can continue with the study till 
completion. 
Subjects who become pregnant during the study or have the intention of becoming 
pregnant will be removed from the study for safety reasons. There may be other reasons, up to 
the investigator’s discretion, for early removal of a research subject. 
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All medications and treatments not permitted during the trial are outlined in the inclusion 
/ exclusion criteria. 
Study Drug Interruption
Subject who interrupt their study drug dosage will be allowed to stay in the study if they resume 
their dosage within two weeks. 
The target for this trial is to keep the patient on maximum dosage of semaglutide, but if the 
patient can’t tolerate the maximum dosage of 1.0 mg /week, we will allow the patient to stay on 
the trial on the maximum tolerable dosage.  
Subject Replacement:
If a subject withdraws or is removed from the research study, they will not be replaced as we are 
already accounting for attrition with 40 subjects being randomized.
Rationale for Study Population:
This is a high impact clinical and translational project looking at the mechanism of how 
semaglutide along with metformin can improve number and function of endothelial progenitor 
cells (as a cellular marker of endothelial function) and fat derived mesenchymal stem cells (as a 
surrogate of fat metabolism and differentiation). The study population has been carefully selected 
to match the overall patient population that would most likely be receiving treatment with 
semaglutide. Patients have type 2 diabetes (HbA1C 7–10%), above normal BMI, and are at risk 
for CVD. We choose a population that at risk of CVD, rather than known history of CVD, as we 
believe this is a population that can have the greatest benefit from this medication, with a certain 
degree of positive chance in reversing endothelial dysfunction and deranged fat metabolism. We 
hope to corroborate our cellular findings with patient blood biochemistries gathered as used for 
regular standard of care for diabetes. We also hope to establish that monitoring function and gene 
expression of endothelial progenitors and fat derived MSCs will allow us to quantify CVD risk, 
monitor obesity at a stem and progenitor cell level, post GLP-1 treatment in patients with type 2 
DM. Effect on stem/progenitor cells will help us to predict how semaglutide would affect 
cardiovascular health in the near future in a specific subject/subjects. The exclusion criteria have 
been carefully selected to align with the prescriber information for semaglutide and ensure that 
this drug would be safe for study participants to take.  
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4. STUDY DESIGN
Group A: Metformin + Placebo, n=16
At Week 8*
Visit 2- 2nd 
samples obtained. 
Start of 1mg 
weekly dose
Week 28 Phone
Follow- up
          Screening
Week: -2: initial screening visit.       
Week:0: randomization and first 
samples obtained*. semaglutide starts 
to increase in dose: week 0 to wk 4 at 
0.25mg/week, followed by telephone 
call. semaglutide at 0.5mg/week from 
wk4 to wk 8. 
Week 24*
Visit 3, 3rd 
and final 
samples 
obtained
Group B: metformin + semaglutide (0.25mg/week for week 0 
– 4 , then increasing to 0.5mg/week for weeks 4 - 8, then 1 
mg/week for week 8 - 24 weeks) taken subcutaneously 
n=16 **
*Assessed at week: 0, 8 and 24: Biochemical and cellular markers to be obtained.
Blood for EPCs will be harvested at weeks 0, 8 and 24
MSCs from fat biopsies will be harvested at weeks 0 and 24.
Week 28 - A telephone call to subjects will be made 4 weeks after last dose of study medication 
to determine if there have been any adverse events. 
Time line:
Week -2: initial screening
Week 0: randomization, start on 0.25mg/week, sc, or placebo, Baseline-Studies: blood, fat biopsy and 
arterial stiffness.
Week 4: follow up phone-call to ascertain compliance
Week 4: start on 0.5mg/week, sc, or placebo, following phone call
Week 8: start on 1mg/ week, sc, or placebo, Studies: blood, and arterial stiffness.
Week 16: follow up phone-call to ascertain compliance at the mid-point of the study
Week 24: end of study: on 1mg/ week, sc, or placebo, Studies: blood, fat biopsy and arterial stiffness
Week 28: follow-up phone call for patient safety and satisfaction
Initial Screening: week -2
** 2 weeks of screening period will be used to help subjects adjust to the protocol and suggested lifestyle 
changes. (week -2 to week 0, two weeks)
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At the start of week 0, subjects will be randomized to treatment or placebo group. Treatment group will 
receive 0.25 mg /weekly semaglutide or placebo taken subcutaneously (week 0 to end of week 4, four 
weeks). Week 0 will be visit 1 
Patients randomized on semaglutide arm, will proceed to be started at 0.5g/week at the beginning of           
week: 4 and then increased to 1.0mg/week at the beginning of week 8. A matching placebo for either 
0.25mg or 0.5mg or 1.0mg will be administered to the subjects in the placebo arm.
At week 8 will be visit 2, when all parameters similar to visit 1 will be tested except fat biopsies.
At week 24, will be visit 3, when all parameters similar to visit 1 will be tested including fat biopsies.
Visit Procedures:
The study will consist of pre-screening, four visits to The GW Medical Faculty Associates, and a 
follow-up phone call. All procedures will take place at the GW MFA. 
Pre-Screening (Within 3 months of Screening Visit), 20 Minutes: Subjects who are interested in 
the research study will first be asked a series of questions either over the phone, or in-person in 
the MFA clinics to determine initial eligibility for the research study. See attached “Pre-
Screening Form”. This is called pre-screening and will be performed either over the phone, or 
when interested subjects are identified in the MFA clinic. If, when going through the questions, a 
subject is found ineligible due to the criteria, then the pre-screening will be stopped then. The 
responses to this study will only be recorded if the subjects are found eligible and are invited to 
an in-person screening visit, where they will sign the ICF. 
Screening Visit (week -2) 90 Minutes: During the screening visit, a trained member of the 
research staff will explain the study procedures and the participant will have the opportunity to 
ask questions. See “Consenting Procedures” below. After providing written consent, a copy of 
the signed ICF will be provided to that patient. Then the following screening procedures will be 
done: 

Medical history & medications reviewed and physical exam

Vitals & Anthropometric measures: heart rate, blood pressure, temperature, weight and 
height 

Biochemistry: Urine will be collected to perform a microalbumin/creatinine ratio, and 
pregnancy test (if applicable). A blood draw will be performed by trained staff to obtain 
approximately 22ml of blood (1.5 tablespoons). This blood will be used to check the 
standard of care labs:
o
Complete Blood Count (CBC)
o
Sedimentation Rate (Infection, anemia, and others)
o
Hemoglobin A1C (HbA1C, Blood sugar), 
o
Chemistry panel: ALT/AST (Liver function), Blood Urea Nitrogen (BUN) 
(Kidney function), Electrolytes (Kidney function), Fasting Glucose (Blood sugar)
o
Lipid Panel (Cholesterol, Triglycerides)
o
Thyroid Stimulating Hormone (TSH, Thyroid Function)

Dietary advice: Participants will be instructed not to change their eating habits, including 
alcohol intake and to maintain their weight and unsupervised activity level steady in a log 
throughout the study.
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Randomization Visit (week 0), Visit 1: 4hrs and 20 minutes: If the results of the screening tests 
show that the subjects are eligible for the study, then they will return in 2 weeks for their study 
randomization visit.

Inclusion / Exclusion criteria will be re-reviewed

Concomitant medications and recent medical changes will be reviewed

Assessment for adverse events

Randomize Subject

Dispense either 0.25mg / Week for week 0 to 4 of semaglutide or Placebo

Dispense either 0.5mg / Week for week 4 to 8 of semaglutide or Placebo

Dispense either 1.0mg / Week for week 8 to 24 of semaglutide or Placebo

Educate patients on drug + delivery by using the direction for use (DFU).
Visit 1 continued (week 0), 

Vitals: heart rate, blood pressure, temperature

Waist circumference, hip circumference 

Weight and body fat will be measured with bare feet on the body composition scale. This 
scale sends a mild electrical current through one’s body when they step on it. The 
electrical current is so small that it is not felt. This is a research related procedure only 
and is not usually done as part of a physical exam. This is NOT an investigational device.

Urine sample for microalbumin/creatinine ratio, urine pregnancy test (if applicable), and 
exosome analysis

A 95ml blood draw for laboratory EPC analysis (Primary outcome, See Appendix A) and 
for the following standard of care labs:
o
Chemistry Panel (ALT/AST, BUN, Electrolytes, fasting glucose)
o
Hemoglobin A1C(HbA1C) (Blood sugar)
o
Lipid Panel (Cholesterol)
o
C-Reactive Protein (inflammation)
o
Adiponectin (measure of endothelial paracrine function)
o
Fasting Insulin levels (measure of insulin resistance)
o
Interleukin-6 (inflammation)

Subcutaneous Fat Biopsy (See Appendix B&C for details)

Arterial Stiffness (vessel health): Measurements of the blood vessels (pulse) in the wrist 
(radial), neck (carotid), and groin (femoral) area. This is a research related procedure, not 
routinely done. This is performed with a SphygmoCor Atcor machine, which is NOT an 
investigational device.

Flow Mediated Dilation: Endothelium-dependent process facilitating the relaxation of an 
artery in response to increased shear stress. It is a direct marker of nitric oxide bio-
availability. This is a research related procedure, not routinely done.
Week 4: phone call by coordinator to ascertain subject compliance and establish willingness of 
the subject to move from 0.25mg to 0.5 mg dose 
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
Week 8: Visit 2 ( start of 1mg dose), 2.5 hours: 

All procedures performed in visit 1 will be repeated in the exact same manner, except 
there will be no fat biopsy. 

Subjects will be dispensed either 1.0 mg/week of semaglutide or the matching placebo, 
based on their assigned randomization. Trial product is the same product and strength as 
V1, only the dose varies and thus the number of pen-injectors to be dispensed will be 
increased. 
Subjects will return all their used, partly used or unused study pens, and their exercise journal 
so that compliance can be measured.
Week 16: Mid-point of 1mg dose: phone call by coordinator to ascertain subject compliance. 
Week 24: Visit 3 (end of 16 weeks of 1mg dose), 4 hours: All procedures performed in visit 1 will 
be repeated in the exact same manner. There will be a second subcutaneous fat biopsy 
performed at this visit (6 months from the initial one). Subjects will return their old study 
pens, and their exercise journal so that compliance will be measured.
Follow-Up Phone Call (week 28), 10 minutes: Subjects will be called 4 weeks after completing 
their final visit in order to assess any AEs and check general subject health.
X-Chart:
Please See Appendix D
Consenting Process: 
Consenting will take place in a HIPAA Compliant, private room in the MFA. The protection of 
human subjects will be carefully managed in this study and subjects will be fully informed of 
their rights as research participants. Subjects will have the opportunity to discuss the study with 
their family and friends or think about it prior to agreeing to participate.Extensive discussion of 
risks and possible benefits of participation in this study will be provided to the subjects by 
trained study staff. An IRB approved consent form will describe the study procedures and risks 
in detail. In order to voluntarily participate in the study, informed consent will be obtained at the 
start of the screening study visit, before any screening or study procedures are conducted except 
from pre-screening. 
Written documentation of informed consent is required for all participants prior to 
undergoing any screening procedures or enrolling in the study. The subjects may withdraw 
consent at any time throughout the course of the study. A copy of the signed informed consent 
document will be given to the subjects for their records. The rights and welfare of the subjects 
will be protected by emphasizing to them that the quality of their medical care or their academic 
standing will not be adversely affected if they decline to participate in this study.
Screen Failures:
Upon completion of the screening visit, all of the data collected will be reviewed with the study 
staff. Ultimately, the investigator will made the decision on whether to enroll a subject who 
meets all inclusion / exclusion criteria, upon reviewing their PMH, Concomitant medications, 
laboratory results, and physical exam. Subjects who meet the eligibility criteria may not 
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necessarily be enrolled, based on the investigator’s discretion. If it is decided not to enroll a 
subject, they are classified as a screen failure. 
Subjects who screen fail can re-screen up to 2 times, with a minimum of a month 
between screenings. 
Subject Withdrawal Process: 
If a subject, wishes to withdraw from the research study, they must formally inform the research 
staff. Ideally, the research staff will get this in writing, and document the reason for the 
withdrawal. If subjects withdraw after being randomized, all their data will be kept up to that 
date, unless a letter is written to the sponsor-investigator explicitly indicated against this. If 
subjects withdraw due to health reasons, they may be followed for safety reasons. 
Data Handling:
For each enrolled patient, the medical record number, and study ID providing the link will be 
kept in a password protected file on the secure MFA network. This will be deleted once the study 
has closed. All study files that contain subjects' de-identified data, as well as files that contain the 
subject's consent forms will be kept in hard-copy files in the study coordinator's locked MFA 
office. These will be destroyed after study closure at the appropriately designated time (6 years).
All study data will be recorded on source documents, which will be kept in the subject’s 
study folders. These will be kept locked in the study coordinator’s office, and will only be 
accessible to study staff. All de-identified study data will also be uploaded to RedCap, a 
password protected database, for data compilation. No PHI will be placed on RedCap. 
Data analysis will be completed at either the MFA or Ross Hall. In order to maintain data 
security, the research staff will ensure that all data is completely de-identified prior to analysis 
beginning.  
Assessments for Safety
At each visit, the subject’s laboratory results will be assessed by the investigators. All laboratory 
samples will be collected during the screening and regular visits (1-3). They will be drawn by a 
trained member of the study staff, and processed and stored according to the guidelines for the 
specific tests being requested, and while adhering to OSHA guidelines. All laboratory results 
will be acquired from LabCorp, and results will be reported to the study coordinator. 
Laboratory results will be compared to the pre-determined “normal ranges”, and will be 
indicated if any value falls above or below this normal range. The investigator will then 
determine for all laboratory results, and especially for all values outside of the normal range, if it 
is clinically significant. 
Vitals and laboratory results will be monitored at every visit to see if any value classifies as an 
adverse event, in which case it will be reported via the appropriate manner (see AE Reporting 
Section)
Other Assessments
All samples being collected will be identified with the subjects’ specific study ID. Samples being 
sent to LabCorp will also include the subject’s DOB. Samples will be stored in the appropriate 
manner for the respective test being run. Samples that are planned to be sent to LabCorp will be 
sent same day via LabCorp courier. Samples being brought to GW Ross Hall for basic science 
lab processing will be transferred between clinical and basic science research staff at the GW 
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MFA. After transfer of care, samples will be maintained in the investigator’s lab, and only be 
accessible by members of the study staff. Samples will be destroyed upon completion of the 
study, and after a “data lock” has occurred. Some samples may be stored in the -70*C freezer of 
the MFA for later processing. 
Subject Compliance
Subject compliance will be measured by subjects returning the semaglutide pens at visits 2 and 
visit 3. A compliance calculation will then be done. If subjects have taken 70% of the dosage 
they were supposed to, they will be considered compliant with the study medication.
5. STATISTICAL CONSIDERATIONS:
Sample Size Calculation
Sandri et al. (2005, Study A, patients with peripheral arterial occlusive disease) [11] 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), [10] 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, these two quoted studies are different from our proposed study 
on patients’ with diabetes. It is well known that diabetes can affect CD34+ number and function, 
therefore in our proposed study number of CD34+ cell availability may be a limiting factor. 
Our recently concluded study also confirms that our power calculations are satisfactory 
[18]. Therefore, we propose to use double the number suggested by power calculation and use 
8x2=16 patients per group.
Data Analysis Methods
We will use generalized estimating equations (GEE) with robust standard error estimates to 
model within-subject and between-group differences over time. First we will investigate whether 
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an unstructured correlation matrix is best fitting for the error term based on examination of the 
working correlation matrix output. If an exchangeable structure fits the data, we will consider 
using a random-effects mixed model with restricted maximum likelihood, due to its weaker 
assumption for missing data on the response variable. Using either approach, of primary interest 
will be the group (drug treatment) by time interaction, which will tell us whether the change in 
the dependent variable over time differs between groups. We will include as covariates any pre-
treatment variables (e.g., demographics, baseline levels of the dependent variable, comorbidities, 
laboratory test values) that differ between groups even at a trend level of significance (p<.10).
Interim Analysis
No interim analysis will be performed, in order to keep all members of the research staff blinded, 
and to not introduce any bias. 
6. DATA HANDLING AND RECORD KEEPING:
For each enrolled patient, the medical record number, and study ID providing the link will be 
kept in a password protected file on the secure MFA network. This will be deleted once the study 
has closed. All study files that contain subjects' de-identified data, as well as files that contain the 
subject's consent forms will be kept in hard-copy files in the study coordinator's locked MFA 
office. These will be destroyed after study closure at the appropriately designated time (6 years).
All study data will be recorded on source documents, which will be kept in the subject’s study 
folders. All contact and communication with subjects will be recorded in progress notes, and 
maintained in the pateint folders. These will be kept locked in the study coordinator’s office, and 
will only be accessible to study staff. All de-identified study data will also be uploaded to 
RedCap, a password protected database, for data compilation. No PHI will be placed on RedCap. 
Data analysis will be completed at either the MFA or Ross Hall. In order to maintain data 
security, the research staff will ensure that all data is completely de-identified prior to analysis 
beginning.  
7. ETHICS:
This study will be reviewed and approved by the GW MFA and the GW IRB Full Board prior to 
beginning any recruitment or study procedures. A copy of the IRB approval letter and all IRB 
approved documents will be provided to the sponsor, upon approval. This study will be 
conducted in accordance with the Declaration of Helsinki, the ICH GCP Guidelines, and all other 
regulatory and legal requirements. Informed consent will be provided to subjects prior to 
conducting any study procedures. 
Study Risks
Blood Draw: This may result in discomfort at the site of the needle entry or bruising at the site. 
There is also a remote risk of fainting or local infection associated with drawing blood. To 
address this, only skilled and well-trained phlebotomists will carry out these procedures. In 
addition, peripheral blood draws (venipuncture) performed during this study for research will not 
exceed 95 mL per visit in visits 1-3. The total amount of blood drawn for the study, including 
22 mL from screening, is 307 mL.
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Urine collection: Participants will be asked at some visits to collect a urine sample. Some people 
do not like the idea of providing a urine sample and may experience difficultly with the 
collection as a result.
Medical Information: Participants will be asked to provide a medical history. Some participants 
may find this uncomfortable or embarrassing. We have minimized this risk by de-identifying the 
data obtained to ensure that this information is not linked to the subject’s identity. In addition, we 
will ensure that interviewers are properly trained to obtain this information in a comfortable and 
non-judgmental manner. There is, however, always a risk of loss of confidentiality. While all 
efforts will be dutifully undergone to maintain study confidentiality, it is a consideration. 
       
Tanita Body Composition Scale may cause electrical devices inside the body to malfunction. It 
should not be used by people with pacemakers or other similar devices.  
Subcutaneous fat biopsies: Collection of subcutaneous adipose tissue biopsies may result in pain, 
bruising, hematoma, infection, and scarring. The procedure will be performed under sterile 
technique to minimize the chances of infection. Local anesthetic will be used to minimize pain. 
Ice will be applied to the site immediately after the procedure to limit bruising, swelling and 
tenderness. After each biopsy, patients will be monitored by the study physician. Post-biopsy the 
incision site will be cleaned and closed with adhesive wound closures and covered with gauze 
and translucent dressing tape. Study participants will be instructed to report to the study 
phyisician any changes at the biopsy site including bleeding, secretion, erythema, pain, and signs 
and symptoms of infection. Study participants will be instructed to self-monitor after their study 
visit. An MFA phyisican who is both trained and has experience with performing fat biopsies 
will be performing  the procedure.
Study Medication: semaglutide. The following warnings and precautions are listed:

The most common adverse reactions, reported in ≥ 5% of patients treated with 
semaglutide, are: Nausea, Vomiting, Diarrhea, Abdominal pain and Constipation. 

Pancreatitis has been reported in clinical trials. If pancreatitis is suspected, study 
medication will be discontinued immediately. 

Diabetic Retinopathy Complications have been reported in a clinical trial. We will 
monitor patients with a history of diabetic retinopathy.

Hypoglycemia is a risk with concomitant use of insulin secretagogues or insulin. 
semaglutide itself does not cause hypoglycemia Blood sugars will be closely monitored, 
and insulin dose titrations will be made, if necessary. 

Thyroid C-cell tumors were found in rodents. It is unknown if semaglutide causes C-cell 
tumors in humans (including MTC). 

Acute Kidney Injury: There have been post-marketing reports of acute kidney injury 
and worsening of chronic renal failure, in patients treated with GLP-1 receptor agonists. 
Renal function will be monitored throughout the study, especially in patients reporting 
severe gastrointestinal reactions.
Steps Taken To Minimize Risks
The inclusion/exclusion criteria have been created to only allow a patient population that is safe 
to take semaglutide (within the clinical indication). Throughout the study subject's health will be 
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monitored at all study visits, and regular Adverse Event assessments will be performed. If a 
subject does have an adverse event during the study they are instructed to seek medical care, and 
then to notify the study staff. They can be seen in an interim visit if necessary to asses any 
adverse events. If patients cannot tolerate semaglutide they will be removed from the study.  
All study files will be kept in a locked office in the study coordinator's office. All study 
information will be transferred from the paper source documents to the secure website, RedCap. 
All other study information, for example the subject link, will be kept in a password protected 
excel on the MFA network, only accessible to study staff. Research personnel are trained 
regarding how to collect, enter, and code subject information and the information gathered in this 
study will not become part of the participants’ medical records. Any samples sent to outside 
laboratories for future analyses will be identified only with the participants’ study code number, 
and not their name or other identifying information.
Potential Benefits

Improvement in diabetes condition 

Improved cardiovascular health and possible decrease in body weight.

Continual monitoring of their diabetes progression and overall health every 8 weeks 
through standard of care labs.

Information gained from this research study may help other people with diabetes in the 
future.
8. STUDY SCHEDULE:
As the study involves both blood draw and fat biopsies recruitment may be slow.
The project will be performed over a 2.5 years period:  3 months start up, 24 months recruitment 
and intervention, 3 months data compilation, analysis and publication.  We estimate a total of 40 
subjects will be enrolled to achieve 32 completed subjects (16 in each group).
Start of Study: March 2019
FPFV: August 2019
LPFV: August 2021
LPLV: Dec 2021
Final Study Report: June 2022
Publication: Dec 2022
9. STUDY DRUGS AND MATERIALS:
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 
semaglutide.
This study is being conducted under FDA mandated guideline for use in type 2 diabetes. Though 
in this particular study semaglutide 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.
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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.  Patients will 
continue on their anti-diabetic therapy of metformin as per their study entry dose. Doses may be 
altered if laboratory results indicate this is necessary.
Study medication(s) / devices(s)
semaglutide 0.25mg/week for 4 weeks followed by 0.5mg/week for 4 weeks, during initial 8 
week period, 
with Matching Placebo
semaglutide 1.0mg/week for week 9 to week 24,
With Matching Placebo
Novo Nordisk will supply the investigator with 0.25mg/week, 0.5mg/week and 1.0mg/week of 
semaglutide, and their respective matching placebo. Global clinical supply will provide active 
and placebo study drug, which may be packaged differently from commercial product
Packaging and Labelling of Study Medication(s)
Packaging for the medication will be in the following format:
A single trial product (Name: semaglutide, Strength: 1.34mg/ml) will be provided for all three 
doses along with its matching placebo, it will be in the form of 1.5 ml prefilled pen injector 
containing solution for sc injection. Subject will be supplied with sufficient trial product at 
dispensing visits to cover entire period between two visits. 
NovoFine® Plus needles (not provided by Clinical Supplies, requested separately in ISS 
website) 
The pens will be labelled by Novo Nordisk  as “Either semaglutide or Placebo”. Trial product are 
packed blinded from Novo Nordisk and will not be re-labelled locally.  On the label the 
following or similar will be stated "semaglutide 1.34 mg/ml or placebo" to maintain blinding of 
staff and subject. All packs are uniquely numbered and a total DUN (Dispensing Unit Number) 
list providing the information about which numbers are active and which are placebo will be 
provided to unblinded IDS staff. This list is to be used when dispensing trial product according 
to treatment allocation. 
Storage and Drug Accountability of Study Medication(s)
Prior to first use
After first use
Refrigerated 
36°F to 46°F 
(2°C to 8°C)
Room Temperature 
59°F to 86°F
(15°C to 30°C)
Refrigerated 
36°F to 46°F 
(2°C to 8°C)
Until expiration date of drug
For up to 56 days (8 weeks)
Additional Condition
DO NOT FREEZE and PROTECT FROM LIGHT
Storage of the study medication prior to dispensation will be done by IDS. Calibrations are 
performed continuously, and any temperature excursions are extensively recorded, with 
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accompanying alarms to the IDS Pharmacist. The investigator will ensure the availability of 
proper storage conditions and record and evaluate the temperature.
No trial medication will dispensed to any person not enrolled in the study. Used trial medication 
(study pens) will be returned to IDS for compliance purposes, but will be kept separately from 
the un-used, non-dispensed, drug. All returned drug will be promptly disposed of upon recording 
compliance, in the appropriate manner. 
Extensive logs will be kept by IDS pharmacy documenting subject randomization, and 
dispensation logs, as well as product return. 
Auxiliary Supply
The budget is inclusive of all other auxiliary supplies will be necessary. All supplies will be 
purchased by the investigator using the study budget funds, and do not need to be provided by 
the Novo Nordisk. 
Randomization and Blinding
This study is a double-blind trial. All study staff will remain blinded to the treatment allocation, 
in addition to the patients. The only individuals who are un-blinded are IDS staff, who never 
have any patient contact or interaction. Treatment allocation is assigned by a block 
randomization method performed by the Investigational Drug Services (IDS) Pharmacy Staff. 
Upon receiving a prescription from the investigator for a new subject to be enrolled, IDS 
computes what their randomization will be. They then prepare the appropriate study product, and 
re-label all items to be dispensed to ensure that it is blinded. 
Breaking of Blinded Codes
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. 
Code break will be provided by the study staff who was responsible for randomization in the first 
place. 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. When the a code is broken, the person breaking the 
code must record the time, date and reason as well as his/her initials in the source documents. All 
codes (whether broken or not) will be kept throughout the trial period. Accountability of all 
broken or unbroken codes (hard copy or electronic) will be performed at or after trial closure.
End of Study Un-Blinding Protocol
Upon completion of the research study, we will undergo the following protocol in order to 
unblind the research data.
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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 
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.
10. CONCOMITANT ILLNESSES AND MEDICATIONS:
Definitions:
Concomitant illness: any illness that is present at the start of the trial (i.e. at screening). 
Concomitant medication: any medication other than the trial product(s) that is taken during the 
trial, including the screening and run-in periods.
After signing the informed consent, all concomitant illness and medications are recorded in a 
study log. Throughout the trial, at every visit these two lists are reviewed with the subject, and 
any changes / updates are made on the log. If any change would alter eligibility for the study, the 
investigator will inform Novo Nordisk.
Information gathered regarding concomitant illness includes: Onset date, Diagnosis, Date of 
resolution / ongoing, concomitant medications taken for the illness
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Information gathered regarding concomitant medications includes: Medication name, Start date, 
dosage/frequency, indication, stop date (if applicable)
11. ADVERSE EVENTS:
Adverse Event Collecting and Reporting
The collection of adverse event (AEs) information will begin at initiation of signed informed 
consent. Adverse events can be spontaneously reported or elicited during open-ended 
questioning, examination, or evaluation of a subject. To prevent reporting bias, subjects should 
not be questioned regarding the specific occurrence of one or more adverse events. AEs will be 
followed to completion of the study, if considered not related, or until resolution or stabilization 
if considered related. They will be reported as SAEs if they become serious. The investigator 
will comply with all local legal, regulatory, and IRB requirements.
If known, the diagnosis of the underlying illness or disorder should be recorded, rather than its 
individual symptoms. The following information should be captured for all AEs: onset, duration, 
intensity, seriousness, relationship to investigational product, action taken, and treatment 
required. If treatment for the event was administered, it should be recorded in the medical record. 
The investigator will report these to the IRB and Novo Nordisk upon continuing review 
(annually). The investigator must supply Novo Nordisk and the IRB with any additional 
information requested.
Serious Adverse Event Collecting and Reporting
Following the subject’s written consent to participate in the study, all SAEs, whether related or 
not related to study drug, will be collected, including those thought to be associated with 
protocol-specified procedures. All SAEs will be collected that occur within 30 days of 
discontinuation of dosing or within 30 days of the last visit for screen failures. 
The investigator will report any SAE to the IRB, within 6 business days of knowledge of 
the event. An SAE report will be completed for any event where doubt exists regarding its status 
of seriousness. If the investigator believes that an SAE is not related to study drug but is 
potentially related to the conditions of the study (such as withdrawal of previous therapy, or a 
complication of a study procedure), the relationship will be specified in the narrative section of 
the SAE Report Form.
The sponsor-investigator will report to Novo Nordisk all SAEs, SUSARs, and SADRs at the 
same time such events are reported to regulatory authorities or within 15 days from the sponsor-
investigator becoming aware of such adverse events, whichever comes first.
Minimum Information to Report for any SAE, SUSAR, SADR
The sponsor-investigator will collect the following information at minimum for each of these 
events:
1. Study Name, and PI
2. Patient ID
3. Patient demographics (age, sex, race/ethnicity, etc.)
4. Event (diagnosis if possible)
5. Drug involved, and any actions take with the study drug
6. Reporter identification (e.g. Name, or initials)
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7. Investigator determined causality of event
8. Outcome of the event, if possible
Definitions
Adverse Event (AE):
An AE is any undesirable medical event occurring to a subject in a clinical trial, whether or not 
related to the trial product(s). This includes events reported from the first trial related activity 
after the subject has signed the informed consent and until post treatment follow-up period as 
defined in the protocol. The following should not be recorded as AEs, if recorded as medical 
history/concomitant illness on the CRF at screening:
• Pre-planned procedure, unless the condition for which the procedure was planned has worsened 
from the first trial related activity after the subject has signed the informed consent
• Pre-existing conditions found as a result of screening procedures
Clinical Laboratory Adverse Event:
All laboratory test results captured as part of the study will be recorded following institutional 
procedures. Test results that constitute SAEs should be documented and reported as such.
The following laboratory abnormalities should be documented and reported appropriately:

Clinical laboratory abnormality regarded as clinically significant i.e. an abnormality that 
suggests a disease and/or organ toxicity and is of a severity, which requires active 
management, (i.e. change of dose, discontinuation of trial product, more frequent follow-
up or diagnostic investigation).

Any laboratory abnormality that required the subject to have study drug discontinued or 
interrupted

Any laboratory abnormality that required the subject to receive specific corrective therapy.
Serious Adverse Event (SAE):
A serious AE is an experience that at any dose results in any of the following:
• Death
• A life-threatening* experience
• In-patient hospitalisation or prolongation of existing hospitalization
• A persistent or significant disability/incapacity
• A congenital anomaly/birth defect
• Important medical events that may not result in death, be life-threatening*, or require
  Hospitalisation may be considered an SAE when, based upon appropriate medical judgement, 
they may jeopardise the subject and may require medical or surgical intervention to prevent 
one of the outcomes listed in this definition
• Suspicion of transmission of infectious agents 
*The term life-threatening in the definition of SAE refers to an event in which the subject was at 
risk of death at the time of the event. It does not refer to an event which hypothetically might 
have caused death if it was more severe.
NOTE: The following hospitalizations are not considered SAEs:
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
A visit to the emergency room or other hospital department lasting less than 24 hours that 
does not result in admission (unless considered an “important medical event” or a life-
threatening event)

Elective surgery planned or anticipated before signing consent

admissions as per protocol for a planned medical/surgical procedure

Routine health assessment requiring admission for baseline/trending of health status (e.g., 
routine colonoscopy)

Medical/surgical admission for purpose other than remedying ill health state that was 
planned before study entry. Appropriate documentation is required in these cases.

Admission encountered for another life circumstance that carries no bearing on health 
status and requires no medical/surgical intervention (e.g., lack of housing, economic 
inadequacy, caregiver respite, family circumstances, administrative).
Serious Adverse Drug Reaction (SADR): 
An adverse drug reaction (ADR) is an adverse event (AE) for which a causal relationship to the 
trial product is at least possible i.e. causal relationship is conceivable and cannot be dismissed. 
Serious adverse reaction (SAR): Adverse event which fulfils both the criteria for a Serious 
Adverse Event and the criteria for an Adverse Reaction.
Suspected Unexpected Serious Adverse Reaction (SUSAR): 
An SAE which is unexpected and regarded as possibly or probably related to the trial/study 
product by the investigator. 
Medical Events of Special Interest (MESI): A MESI is either
1. A medication error (e.g. wrong drug administration or wrong route of 
administration) o
2. A suspected transmission of an infectious agent via the product
Non-Serious Adverse Event:
A non-serious AE is any AE which does not fulfil the definition of an SAE.
Severity Assessment Definitions:
• Mild: Transient symptoms, no interference with the subject’s daily activities
• Moderate: Marked symptoms, moderate interference with the subject’s daily activities
• Severe: Considerable interference with the subject’s daily activities, unacceptable
Relationship to study medication Assessment Definitions:
• Probable: Good reasons and sufficient documentation to assume a causal relationship
• Possible: A causal relationship is conceivable and cannot be dismissed
• Unlikely: The event is most likely related to an etiology other than the trial product
The semaglutide Prescriber Information packet will be used to evaluate all unexpected events 
and adverse reactions.
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Outcome Categories and Definitions:
A. Recovered: Fully recovered or by medical or surgical treatment the condition has 
returned to the level observed at the first trial related activity after the subject signed the 
informed consent
B. Recovering: The condition is improving and the subject is expected to recover from the 
event. This term should only be used when the subject has completed the trial
C. Recovered with sequelae: As a result of the AE, the subject suffered persistent and     
significant disability/incapacity (e.g. became blind, deaf, paralysed). Any AE recovered 
with sequelae should be rated as an SAE
D. Not recovered
E. Fatal
F. Unknown
Collection, Recording and Reporting of Adverse Events
All events meeting the definition of an adverse event must be collected and reported from the 
first trial related activity after the subject has signed the informed consent and until the end of the 
posttreatment follow-up period as stated in the protocol. For specific timelines on reporting, 
please see above. 
Follow-up of Adverse Events
During and following a subject’s participation in the trial, the sponsor-investigator and institution 
will provide adequate medical care to the study subject for any study-related adverse events, 
including clinically significant laboratory values related to the study. However, the GW Medical 
Faculty Associates, GWU, GWU Hospital or the sponsor does not have a program to provide 
compensation or free medical treatment for research-related injury. This would be covered by the 
subjects’ insurance, or whatever route of payment they typically use for medical care.
All adverse events classified as serious or severe or possibly/probably related to the trial product 
must be followed until the subject has recovered and all queries have been resolved. For cases of 
chronic conditions follow-up until the outcome category is “recovered” is not required, as these 
cases can be closed with an outcome of “recovering” or “not recovered”.
All other adverse events must be followed until the outcome of the event is “recovering” (for 
chronic conditions), or “recovered” or until the end of the post-treatment follow-up stated in the 
protocol, whichever comes first, and until all queries related to these AEs have been resolved.
Pregnancy
If, following initiation of the investigational product, it is subsequently discovered that a study 
subject is pregnant or may have been pregnant at the time of investigational product exposure, 
including up to 2 months post product administration, the investigational product will be 
permanently discontinued in an appropriate manner (e.g., dose tapering if necessary for subject 
safety). Protocol-required procedures for study discontinuation and follow-up must be performed 
on the subject unless contraindicated by pregnancy (e.g., x-ray studies). Other appropriate 
pregnancy follow-up procedures should be considered if indicated. Pregnancy complications 
should be recorded as adverse event(s). If the infant has a congenital anomaly/birth defect this 
must be reported and followed up as a serious adverse event.
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The investigator or designee will immediately notify the Novo Nordisk of this event immediately 
(within 24 hours) in accordance with SAE reporting procedures. Follow-up information 
regarding the course of the pregnancy, including perinatal and neonatal outcome and, where 
applicable, offspring information will be reported.
Any pregnancy that occurs in a female partner of a male study participant should be reported to 
the sponsor. Information on this pregnancy will be collected on the Pregnancy Surveillance 
Form.
Precautions/Over-dosage
Correct dosing of the investigational product will be overseen by subject drug returns. If a 
subject does overdose, then this is considered an SAE. Seriousness can be assessed on case by 
case basis and based on the associated consequences. Appropriate SAE reporting guidelines will 
be followed. 
12. LIABILITY AND SUBJECT INSURANCE:
During and following a subject’s participation in the trial, the sponsor-investigator and institution 
will provide adequate medical care to the study subject for any study-related adverse events, 
including clinically significant laboratory values related to the study. However, the GW Medical 
Faculty Associates, GWU, GWU Hospital or the sponsor does not have a program to provide 
compensation or free medical treatment for research-related injury. This would be covered by the 
subjects’ insurance, or whatever route of payment they typically use for medical care.
The sponsor-investigator will be responsible for the conduct of the study and that the sponsor-
investigator agrees to defend, indemnify, and hold harmless Novo Nordisk, any of its parent 
companies, affiliates, or subsidiaries, and their respective officers, directors, employees, agents, 
representatives, distributors, salespersons, customers, licensees, and end-users from and against 
any claim, suit, demand, loss, damage, expense or liability imposed by any third party arising 
from or related to: (a) any breach of sponsor-investigator's obligations or representations; or (b) 
sponsor-investigator’s negligent or grossly negligent use or willful misuse of the study drug, the 
results, or services derived therefrom.  This indemnification shall not apply in the event and to 
the extent that a court of competent jurisdiction or a duly appointed arbiter determines that such 
losses or liability arose as a result of Novo Nordisk’s gross negligence, intentional misconduct, 
or material breach of its responsibilities. 
13. EVALUABILITY OF SUBJECTS:
Subjects who meet the eligibility criteria and are deemed suitable by the investigator will be 
included in the research study. The study statistician will made the determination, upon data 
analysis about whether to include certain data elements in the conglomerate data set, and outliers 
may be excluded. Prior to a subject’s data been removed, the reasons for their exclusion must be 
documented and signed by those responsible prior to database release. The documentation must 
be stored together with the remaining trial documentation.
14. PREMATURE TERMINATION OF STUDY:
If, it is deemed by the investigator, Novo Nordisk, the IRB, the FDA, or any other governing 
body that the study is unfit to continue due to safety, non-compliance, or any other reason, then 
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all study activity will cease immediately. A formal letter regarding the reason for premature 
study termination will be submitted to the IRB and Novo Nordisk. 
15. PUBLICATION PLAN:
The clinical trial will be registered with clinicaltrial.gov prior to the enrolment of the first 
subject. 
Upon completion of the study, the investigator plans to submit to a peer-reviewed scientific 
journal within 6 months of last patient, last visit. Results of the study will be reported on 
clinicaltrials.gov for public access. 
16. APPENDIX A: CD34+ CELL ANALYSIS
We will obtain of 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 20 mL for biochemistry and 
serum ELISA assays. MNC will be obtained from whole blood prior to CD34 column sorting 
following protocols described before and currently used in our laboratory for clinical studies. Some 
MNCs will be plated to obtain and count colony formation units (CFU) at day 14. Some MNCs 
will be analyzed to assay increased or decreased presence of stem like cells (marked by CD34+, 
CD133+) using a Fluorescence-activated cell sorting (FACS) analyzer (part of GW Core facilities). 
Rest of the MNCs will be put through column to obtain CD34+ cells (Miltenyi Biotec). 
Post-CD34 column sort we will undertake: 
1. CD34+ cell Migration assay using SDF1α conc.at 0, 10, 100ng/ml 
2. RT-PCR on CD34 positive cells for:  
a. Anti-oxidant genes (SOD 1-3, catalase and peroxidases), glucose transporters
b. Endothelial function assay genes: endothelial NOS (eNOS), von-Willebrand’s factor (vWF)
c. Inflammatory genes such as IL6, IL10, IL1β, TNFα, COX2 and endothelin-1 
d. Apoptosis pathway genes such p53, p21, Bax-Bcl2 and caspase-3 & 9
e. Mitochondrial genes: TFAM, NRF-1 &2, ATP5B, cytochrome oxidase 2 & 4, PGC1A, PRDM-
16, UCP1
17. APPENDIX B: SUBCUTANEOUS FAT BIOPSY PROCEDURE
Two to three grams of subcutaneous adipose tissue from the left abdomen will be collected. This 
will be done as follows: 
Patient will be fasting for 10 hours prior to the procedure. The patient will lie down supine on an 
exam table. The physican performing the biopsy will perform a minor abdominal exam, and will 
palpate the abdomen. They will identify the area, between the left iliac crest and the unbillicum 
where the procedure will be performed, and indciate to the trained assistant. The area will then 
be thoroughly cleaned with proper anesthetic procedures. A fenestrated drape will be placed over 
the abdomen, revealed the site of the procedure. Local anesthesia is applied to the area 
(Lidocaine with or without Biocarbonate). After a few minutes, the physician will palpate the 
anesthetized area with a scalpal blade to ensure the subejct is apporpriate anestehtized. A small 
incision of ≤ 1 inch is made with a scalpel. Once the layer of subcutenous fat has been reached, a 
sterilized small-diameter Bergstrom needle will be inserted into the abdomen, attached to a 
suction synringe. The physican will manipulate the Bergstrom needle against the fat, while the 
assistant applies suction. The needle will then be extracted from the abdomen, and the fat will be 
removed from the needle. A second, sterilized larger-diameter Bergstrom needle will now be 
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inserted and the technique will be repeated. The fat is deposited into a sterile container. The 
wound will be closed with any combination of: 1-2 stitches of the physicians choosing, 
dermabond, steristrips, and bandaids. This will be decided in the moment, based on physician 
judgement. Local pressure and an ice pack will be applied for 10 minutes. 
18. APPENDIX C: SUBCUTANEOUS FAT ANALYSIS
Subcutaneous adipose tissue biopsies: 2-3gms of subcutaneous adipose tissue from the abdomen 
will be collected. Samples will be stored at -80°C 
Transcriptomics (RNA-seq and RT-PCR): Adipose-derived RNA will be sent to a commercial 
laboratory for RNAseq sequencing at >50 bp SE. Results will be analyzed by Dr. Mazumder to 
identify changes in RNAseq, using group*intervention ANCOVA and Pearson correlation 
coefficients in Partek Genomics Suite software (Partek Inc., St Louis, Missouri). Resultant lists 
of dysregulated transcripts will be tested for representation in known biological pathways via 
Ingenuity Pathway Analysis Suite (QIAGEN, Valencia, CA). 
Adipose tissue MSC for gene expression and SeaHorse OCR Estimation: MSCs will be 
obtained from fat biopsies, and post culture, will be assessed for gene expression and cellular 
OCR. Targeted gene expression assays similar to CD34+ genes as described above. To study 
differentiation program of MSCs post exercise, mRNA gene expression looking at individual 
lineages as shown in Fig 2 will be interrogated. Other than mRNA analysis we plan to culture 
MSCs in different mesenchymal tissue (fat, bone, cartilage, muscle) specific culture media as per 
protocol, followed by staining to confirm differentiation [13].
We have presented similar investigations looking at fat derived MSC metabolism and 
differentiation obtained from prediabetes subjects, post aerobic exercise. [26].
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Appendix E: Optional modified study procedure during COVID19 
Pandemic
Due to the COVID19 pandemic and emergency orders in place we have adopted study procedures that 
will minimize patient and staff exposure to SARS-cov-2 during study procedure. We had many patients 
who are interested in our trials but does not feel safe to take public transportation to come to site for 
study procedures hence we have adopted a hybrid approach to keep our studies going. This study process 
will include virtual study visits, field phlebotomy staff and shipment of drugs via medical courier to 
subject’s home in the DMV area. During screening process subject will be offered both the in site study or 
the virtual option if they are not comfortable with their transportation options to make their way to Foggy 
Bottom. We will focus on the core set assessments, which are EPC cell counts, which are vital to the study 
and answers the primary research questions posed. Any assessment that cannot be done remotely will be 
considered optional in this format.  
Screening: Interview will be done by the study staff and PI virtually to assess eligibility. All blood work 
done in the past 6 months for standard of care will be considered valid for screening purpose. If the patient 
does not have the specific blood work to fulfil the inclusion criteria they will not be enrolled. A urine 
pregnancy test (if female subjects) along with the consent paperwork will be mailed to the patient 
beforehand with return label. The patient will perform the urine pregnancy test inform the study team of 
the result. After the informed consent process, the patient will sign it and mail it back with the included 
return mailer. Some patient may choose to receive the informed consent digitally, sign it and email it back. 
In those cases, the consent will be received back digitally. The study staff will sign it and file it. The urine 
pregnancy test will be mailed separately in that case. 
Enrollment V1:    After the enrollment criteria is fulfilled, Dr Sen will sign the prescription and send it to 
IDS with planned shipment of the study drugs on the planned enrollment date. IDS at GW MFA has 
contracted a medical courier service to ship study drugs to patients. Dr Sens lab at GW is currently working 
on a contract with a mobile phlebotomy company to go to patient’s place of residence to draw research 
blood samples.   A mobile phlebotomy order will be sent out for the planned enrollment date. We will be 
using a third party mobile phlebotomy company called “POW or Phlebotomy on Wheels”. They will receive 
an order form with patient name, address and phone number. After they do the draw they will drop off 
the blood and urine samples to our GW research staff at GW campus. The research staff will send the 
appropriate blood tubes to lab corp and basic science lab. 
During the zoom call the patient will be counselled on the use of the drug, they will be trained on the use 
of the injectable study medication. Patient will be instructed to contact study staff if there is any issue or 
go to the emergency room due to any medical emergency.  Secondary outcomes such as arterial stiffness 
and mesenchymal cell assessment ( fat biopsy) will be considered optional in this format. 
V2 and V3: Procedure from V1 will be followed.  
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19. APPENDIX E: X-CHART
Phone 
Screening
(Before week -
2)
Screening 
Visit
(Week -2)
Randomization 
Visit 1
(Week 0)
Follow up 
phone call 
(week 4)
Visit 2
(Week 8)
Follow up 
phone call 
(week 16)
Visit 3
End-of-
Treatment
 (Week 24)
Follow up 
phone call
(Week 28)
Written Informed Consent 
Obtained
X
Inclusion / Exclusion Criteria 
Reviewed
X
X
X
Medical History
X
X
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
X
X
X
Laboratory Tests (Biochemical)
X
X
X
Urine Pregnancy Test (As needed)
X
X
X
X
Spot Urine Sample
X
X
X
X
Waist/Hip Measurements
X
X
X
Body Composition Scale
X
X
X
Blood Draw for Primary Outcome 
Measures
X
X
X
Arterial Stiffness Measurements
X
X
X
Subcutaneous Fat Biopsy
X
X
Randomize
X
Dispense Study Drug
X
0.25 mg/Week 
or Placebo For 4 
weeks
AND
0.5 mg/Week 
or Placebo For 
another 4 weeks.
X
1.0 mg/Week 
or Placebo
Subject compensation
X
X
X
Handout Direction for use (DFU)  
Chart
X
Subject compliance check 
X
X
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20. REFERENCES:
1.
Department of Health and Human Services, NIH and National Center for Chronic Disease Prevention and 
Health Promotion, “National Diabetes Statistics Report 2017.” 
2.
American Diabetes Association, “Standards of medical care in diabetes--2014,” Diabetes Care, vol. 37    
Suppl 1, pp. S14–80, Jan. 2014. 
3.
J. García-Castro, C. Trigueros, J. Madrenas, J. A. Pérez-Simón, R. Rodriguez, and P. Menendez, 
“Mesenchymal stem cells and their use as cell replacement therapy and disease modelling tool,” J. Cell. 
Mol. Med., vol. 12, no. 6B, pp. 2552–2565, Dec. 2008. 
4.
Kundu N, Domingues CC, Chou C, Ahmadi N, Houston S, Jerry DJ, Sen S. Use of p53-Silenced 
Endothelial Progenitor Cells to Treat Ischemia in Diabetic Peripheral Vascular Disease. JAHA. J Am Heart 
Assoc. 2017;6(4): e005146. 
5.
Hristov M, Erl W, Weber PC. Endothelial progenitor cells: mobilization, differentiation, and homing. 
Arterioscler Thromb Vasc Biol. 2003 Jul 1;23 (7):1185-9.
6.
N. Werner, S. Kosiol, T. Schiegl, P. Ahlers, K. Walenta, A. Link, M. Böhm, and G. Nickenig, “Circulating 
endothelial progenitor cells and cardiovascular outcomes,” N. Engl. J. Med., vol. 353, no. 10, pp. 999–
1007, Sep. 2005. 
7.
D. W. Losordo, R. A. Schatz, C. J. White, J. E. Udelson, V. Veereshwarayya, M. Durgin, K. K. Poh, R. 
Weinstein, M. Kearney, M. Chaudhry, A. Burg, L. Eaton, L. Heyd, T. Thorne, L. Shturman, P. 
Hoffmeister, K. Story, V. Zak, D. Dowling, J. H. Traverse, R. E. Olson, J. Flanagan, D. Sodano, T. 
Murayama, A. Kawamoto, K. F. Kusano, J. Wollins, F. Welt, P. Shah, P. Soukas, T. Asahara, and T. D. 
Henry, “Intramyocardial transplantation of autologous CD34+ stem cells for intractable angina: a phase 
I/IIa double-blind, randomized controlled trial,” Circulation, vol. 115, no. 25, pp. 3165–3172, Jun. 2007. 
8.
Rizzo M, Abate N, Chandalia M, Rizvi AA, Giglio RV, Nikolic D, Marino Gammazza A, Barbagallo I, 
Isenovic ER, Banach M, Montalto G, Li Volti G. Liraglutide reduces oxidative stress and restores heme 
oxygenase-1 and ghrelin levels in patients with type 2 diabetes: a prospective pilot study. J Clin Endocrinol 
Metab. 2015 Feb;100(2):603-6. 
9.
Nandy D, Johnson C, Basu R, Joyner M, Brett J, Svendsen CB, Basu A. The effect of liraglutide on 
endothelial function in patients with type 2 diabetes. Diab Vasc Dis Res. 2014 Nov;11(6):419-30.
10. Sabyasachi Sen, Sarah Witkowski, Ann Lagoy, Ashequl M. Islam: A six-week home exercise program 
improves endothelial function and CD34+ circulating progenitor cells in patients with pre-diabetes. J 
Endocrinol Metab.2015; 5 (1-2):163-171, doi: http://dx.doi.org/10.14740/jem273w. 
11. M. Sandri, V. Adams, S. Gielen, A. Linke, K. Lenk, N. Kränkel, D. Lenz, S. Erbs, D. Scheinert, F. W. 
Mohr, G. Schuler, and R. Hambrecht, “Effects of exercise and ischemia on mobilization and functional 
activation of blood-derived progenitor cells in patients with ischemic syndromes: results of 3 randomized 
studies,” Circulation, vol. 111, no. 25, pp. 3391–3399, Jun. 2005. 
12. Sen S, Merchan J, Dean J, Ii M, Gavin M, Silver M, Tkebuchava T, Yoon YS, Rasko JE, Aikawa R. 
Autologous transplantation of endothelial progenitor cells genetically modified by adeno-associated viral 
vector delivering insulin-like growth factor-1 gene after myocardial infarction.  Hum Gene Ther. 2010 
Oct;21(10):1327-34. doi: 10.1089/hum.2010.006. 
13. Sabyasachi Sen, Cleyton Dominguez, Carol Rouphael, Cyril Chou, Chul Kim, Nagendra Yadava: Genetic 
modification of human mesenchymal stem cells helps to reduce adiposity and improve glucose tolerance in 
an obese diabetic mouse model: Stem Cell Research & Therapy.2015,6:242.DOI:10.1186/s13287-015-
0224-9. URL: http://stemcellres.com/content/6/1/242. 
14. Sen S, Strappe P, O'Brien T: Gene transfer in endothelial dysfunction and hypertension. Methods Mol Med. 
2005; 108:299-314. 
15. Zaghloul A, Al-Bukhari TA, Al-Pakistani HA, Shalaby M, Halawani SH, Bajuaifer N, Teama SH. Soluble 
endothelial protein C receptor and high sensitivity C reactive protein levels as markers of endothelial 
dysfunction in patients with type 1 and type 2 diabetes mellitus: their role in the prediction of vascular 
complications. Diabetes Res Clin Pract. 2014 Dec;106(3):597-604. 
16. Steven P. Marso, M.D., Stephen C. Bain, M.D., Agostino Consoli, M.D., Freddy G. Eliaschewitz, M.D., 
Esteban Jódar, M.D., Lawrence A. Leiter, M.D., Ildiko Lingvay, M.D., M.P.H., M.S.C.S., Julio 
Rosenstock, M.D., Jochen Seufert, M.D., Ph.D., Mark L. Warren, M.D., Vincent Woo, M.D., Oluf Hansen, 
M.Sc., Anders G. Holst, M.D., Ph.D., Jonas Pettersson, M.D., Ph.D., and Tina Vilsbøll, M.D., D.M.Sc., for 
the SUSTAIN-6 Investigators*     Semaglutide and Cardiovascular Outcomes in Patients with Type 2 
Diabetes. N Engl J Med 2016; 375:1834-1844. November 10, 2016DOI: 10.1056/NEJMoa1607141. 
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17. Cleyton C. Domingues, Nabanita Kundu, Fiona J. Dore and Sabyasachi Sen. Genetic Modification of Stem 
Cells in Diabetes and Obesity. Agricultural and Biological Sciences » "Genetic Engineering - An Insight 
into the Strategies and Applications". 2016. Intech Publications, Croatia.
18. Fiona J. Dore†, Cleyton C. Domingues†, Neeki Ahmadi, Nabanita Kundu, Yana Kropotova, Sara Houston, 
Carol Rouphael, Aytan Mammadova, Linda Witkin, Anamil Khiyami, Richard L. Amdur and 
Sabyasachi Sen. The synergistic effects of saxagliptin and metformin on CD34+ endothelial progenitor 
cells in early type 2 diabetes patients: a randomized clinical trial. Cardiovascular Diabetology 201817:65. 
19. Khiyami AM, Dore FJ, Mammadova A, Amdur RL, Sen S. The Correlation of Arterial Stiffness with 
Biophysical Parameters and Blood Biochemistry. Metab Syndr Relat Disord. 2017 May;15(4):178-182. 
doi: 10.1089/met.2016.0136. Epub 2017 Feb 22.
20. M. Sandri, V. Adams, S. Gielen, A. Linke, K. Lenk, N. Kränkel, D. Lenz, S. Erbs, D. Scheinert, F. W. 
Mohr, G. Schuler, and R. Hambrecht, “Effects of exercise and ischemia on mobilization and functional 
activation of blood-derived progenitor cells in patients with ischemic syndromes: results of 3 randomized 
studies,” Circulation, vol. 111, no. 25, pp. 3391–3399, Jun. 2005.
21. Hwang YC1, Jeong IK, Ahn KJ, Chung HY. Circulating osteocalcin level is associated with improved 
glucose tolerance, insulin secretion and sensitivity independent of the plasma adiponectin level. Osteoporos 
Int. 2012 Apr;23(4):1337-42. doi: 10.1007/s00198-011-1679-x. Epub 2011 Jun 9. 
22. Cho YD, Yoon WJ, Kim WJ, Woo KM, Baek JH, Lee G, Ku Y, van Wijnen AJ, Ryoo HM. Epigenetic 
modifications and canonical wingless/int-1 class (WNT) signaling enable trans-differentiation of 
nonosteogenic cells into osteoblasts. J Biol Chem. 2014 Jul 18;289(29):20120-8. doi: 
10.1074/jbc.M114.558064. Epub 2014 May 27.
23. Rawadi G, Vayssière B, Dunn F, Baron R, Roman-Roman S. BMP-2 controls alkaline phosphatase 
expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res. 2003 
Oct;18(10):1842-53. 
24. Montagnana M, Lippi G, Danese E, Guidi GC. The role of osteoprotegerin in cardiovascular disease. Ann 
Med. 2013 May;45(3):254-64. doi: 10.3109/07853890.2012.727019. Epub 2012 Oct 30. Review. 
25. Human polymorphonuclear neutrophils express RANK and are activated by its ligand, RANKL. Riegel A, 
Maurer T, Prior B, Stegmaier S, Heppert V, Wagner C, Hänsch GM. Eur J Immunol. 2012 Apr;42(4):975-
81. doi: 10.1002/eji.201141786. 
26. Nabanita Kundu, Cleyton C Domingues, Edina Paal, Peter Kokkinos, Eric S Nylen, Sabyasachi Sen.: Effect 
of Exercise Training on Fat-Derived Mesenchymal Stromal Cells (MSCs) in Prediabetes Subjects, 
moderated poster presentation at the American Diabetes Association's 77th Scientific Sessions, June 9-13, 
2017 in San Diego, California. Manuscript under review at Metabolic Syndrome and Related Disorders. 
27. Marso SP, Daniels GH, Brown-Frandsen K, Kristensen P, Mann JF, Nauck MA, Nissen SE, Pocock S, 
Poulter NR, Ravn LS, Steinberg WM, Stockner M, Zinman B, Bergenstal RM, Buse JB, LEADER Steering 
Committee, LEADER Trial Investigators. Liraglutide and cardiovascular outcomes in type 2 diabetes. N 
Engl J Med. 2016;375:311-322. 
28. “Overview of Clinical Guidelines in Lipid Management- National Lipid Association” 
https://www.lipid.org/sites/default/files/6-_lipid_u-_guidelines-_jones.pdf 
IRB NUMBER: NCR191206
IRB APPROVAL DATE: 08/25/2021