Clinical trial NCT02024477
Conditions: Type 2 Diabetes
Keywords: endothelial cells cellular biomarker endothelial dysfunction
Sponsor: George Washington University
Start date: 2013-11-01; Completion date: 2017-12-01
Enrollment: 42 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Type 2 diabetes is a national epidemic. Diabetes has undesirable effects on blood vessels which may contribute to heart disease. Endothelial Progenitor Cells(EPCs) are found in the blood . Research has shown that improving the survival of these special blood cells may decrease the harmful effects of diabetes on blood vessels and reduce or reverse heart disease. Saxagliptin is an FDA(Food and Drug Administration) approved prescription medicine used along with diet and exercise to lower blood sugar in people with Type 2 diabetes. It is in a class of diabetes medication called DPP-4 inhibitors. DPP-4 inhibitors have been shown to increase EPCs in patients with Type 2 diabetes.

Hypothesis: We believe poor viability and function of EPCs in early diabetes ultimately affects the repair and regeneration of the endothelium and that prompt intervention using saxagliptin with another oral hypoglycemic agent, Metformin, may reduce or reverse cardiovascular risk by improving EPC survival and function above and beyond adequate glucose metabolism control.

Detailed description:
Type 2 diabetes is a national epidemic 1,2 with significant macro and microvascular complications. Insulin resistance in prediabetes and early and late diabetes are associated with endothelial dysfunction.

A few studies indicate that EPCs can act as a suitable bio-marker for monitoring cardiovascular morbidity. In this proposal we suggest that EPCs or CD34 positive cells can act as a suitable cellular biomarker for estimating and following endothelial dysfunction in early type 2 diabetes patients.

EPCs have been used as a regenerative tool in ischemic myocardium and diabetic wound healing. Endothelial dysfunction with associated inflammation may be a consequence of excess super-oxide presence in a setting of diabetes which is a pro-oxidative stress condition causing EPC dysfunction and senescence. Therefore monitoring EPC number, function and gene expression may serve as a very useful cellular bio-marker for cardiovascular complications in early type 2 diabetes.

Though lifestyle modification has been proposed as a main stay for prevention and treatment of early type 2 diabetes, several new therapies for diabetes have been developed in recent years. Incretins and incretin mimetics appear to hold promise. Oral DPP-4 inhibitors have been shown to increase EPCs in patients with type 2 diabetes reportedly via SDF-1 alpha up-regulation. Interestingly, up-regulation of SDF-1 alpha and vascular endothelial growth factor (VEGF), both chemotactic factors increase mobilization and recruitment of EPCs in the face of acute ischemic injury for repair and regeneration.

Several studies have shown positive effect of incretins (Glucagon like peptide, GLP-1) and incretin receptor agonists (GLP-1 receptor agonists) on cardiovascular risk factors in type 2 diabetes patients and even in patients with chronic heart failure and left ventricular dysfunction who do not have diabetes.

DPP-4 Inhibitors may have cardio-protective effects of their own, as they increase bio-availability of endogenous GLP-1. They improve blood flow and nitric oxide production in endothelium. These are unique properties not demonstrated by other oral diabetes medications. The mechanism underlying these effects may be mediated by increased nitric oxide bioavailability but is not completely known. It is possible that Saxagliptin, a member of DPP-4 inhibitor group of drugs may be able to improve number and function of CD34+ endothelial progenitor cells by up-regulating chemotactic agent SDF1 alpha (DPP-4 degrades SDF-1) and its receptor CXCR47, 20, 21, 30, 31.

Poor viability and function of EPCs in early diabetes may ultimately affect the repair and regeneration of the endothelium and prompt intervention may reduce or reverse cardiovascular risk by improving EPC survival and function above and beyond adequate glucose metabolism control.

Therefore we would like to explore the effect of saxagliptin in addition to lifestyle intervention, on number and function and gene expression of EPC and impact on endothelial dysfunction in type 2 diabetes.

Arms: arm 1: Matching placebo 1 pill daily for 12 weeks arm 2: Saxagliptin 5mg once daily for 12 weeks
Interventions: intervention 1: Saxagliptin intervention 2: Placebo
Intervention descriptions: intervention 1: 5 mg tablet once daily for 12 weeks intervention 2: 1 tablet daily for 12 weeks

Protocol documents:
Prot_SAP_000.pdf: 
1 
Saxagliptin-Sen ISR CV181-305 
Protocol Version: 5.3 
Date: 12/21/2015 
TITLE PAGE 
 
Effect of Saxagliptin (DPP-4 Inhibitor) on Endothelial Progenitor Cells (EPCs) as 
a cellular biomarker for evaluating Endothelial Dysfunction in Early Type 2 
Diabetes patients 
 
 
 
BMS PROTOCOL NUMBER: CV181-305 
 
 
Sabyasachi Sen, MD 
Clinical Research Division  
George Washington University Medical Faculty Associates 
2150 Pennsylvania Ave NW  
Washington, D.C.  20037 
Phone - (202 741-2798 
Fax - (202) 659-8627 
 
 
Protocol Version/Date: Version 5.3  / December 2015 
 
 
 
 
 


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TABLE OF CONTENTS 
 
TITLE PAGE ..................................................................................... 1 
TABLE OF CONTENTS .................................................................... 2 
PROTOCOL SYNOPSIS ................................................................... 6 
1 
INTRODUCTION ............................................................... 11 
1.1 
Summary of Results of Investigational Program .......... 12 
1.1.1 
Pharmacology of Saxagliptin ......................................................... 12 
1.1.2 
Preclinical Toxicology of Saxagliptin ............................................ 13 
1.1.2.1 
Repeat-Dose Studies....................................................................... 13 
1.1.2.2 
Mutagenicity and Carcinogenicity ................................................. 14 
1.1.2.3 
Reproductive and Developmental Toxicity ................................... 14 
1.1.3 
Human Pharmacokinetics of Saxagliptin ...................................... 15 
1.1.3.1 
Absorption ....................................................................................... 15 
1.1.3.2 
Distribution ...................................................................................... 15 
1.1.3.3 
Elimination ....................................................................................... 15 
1.1.3.4 
Pharmacokinetics of Active Metabolites ....................................... 16 
1.1.3.5 
Dose and Time-dependencies ........................................................ 16 
1.1.3.6 
Special Populations ........................................................................ 16 
1.1.3.7 
Drug-drug interactions.................................................................... 17 
1.1.4 
Clinical Safety with Saxagliptin ...................................................... 17 
1.1.4.1 
Drug-Related Adverse Events ........................................................ 17 
1.1.4.2 
Drug-Related Serious Adverse Events .......................................... 18 
1.1.4.3 
Other Significant Drug-Related Adverse Events .......................... 18 
1.1.4.4 
Drug-Related Deaths ....................................................................... 20 
1.1.5 
Clinical Efficacy of Saxagliptin ...................................................... 20 
1.2 
Overall Risk/Benefit Assessment .................................. 21 
1.3 
Research Hypothesis ...................................................... 22 


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1.4 
Study Rationale ............................................................... 22 
2 
STUDY OBJECTIVES ...................................................... 22 
2.1 
Primary Objective ............................................................ 22 
2.2 
Secondary Objectives ..................................................... 22 
3 
ETHICAL CONSIDERATIONS ......................................... 23 
3.1 
Good Clinical Practice .................................................... 23 
3.2 
Institutional Review Board/Independent Ethics 
Committee ....................................................................... 23 
3.3 
Informed Consent ........................................................... 24 
4 
INVESTIGATIONAL PLAN ............................................... 25 
4.1 
Study Design and Duration ............................................ 25 
4.2 
Study Population ............................................................. 26 
4.2.1 
Inclusion Criteria ............................................................. 26 
4.2.2 
Exclusion Criteria ............................................................ 27 
4.2.3 
Discontinuation of Subjects from Treatment ................... 29 
5 
TREATMENTS .................................................................. 30 
5.1 
Study Treatment: Saxagliptin ......................................... 30 
5.1.1 
Identification .................................................................... 30 
5.1.2 
Packaging and Labeling ................................................. 30 
5.1.3 
Handling and Dispensing ............................................... 30 
5.2 
Drug Ordering and Accountability ................................. 31 
5.2.1 
Initial Orders .................................................................... 31 
5.2.2 
Re-Supply ...................................................................................... 31 
5.3 
Method of Assigning Subjects to a Treatment .............. 31 
5.4 
Selection and Timing of Dose for Each Subject ........... 31 
5.4.1 
Dose Modifications ......................................................... 31 
5.5 
Blinding/Unblinding ........................................................ 32 


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5.6 
Concomitant Treatments ................................................ 32 
5.6.1 
Prohibited and/or Restricted Treatments ...................... 32 
5.6.2 
Other Restrictions and Precautions .............................. 32 
5.7 
Treatment Compliance .................................................... 32 
6 
STUDY ASSESSMENTS AND PROCEDURES ............... 33 
6.1 
Study Schematic .................... Error! Bookmark not defined. 
6.2 
Time and Events Schedule .... Error! Bookmark not defined. 
6.3 
Study Materials ................................................................ 40 
6.4 
Safety Assessments ....................................................... 40 
7 
ADVERSE EVENT REPORTING ...................................... 40 
7.1 
Adverse Events ............................................................... 40 
7.1.1 
Serious Adverse Events ................................................. 41 
7.1.2 
Nonserious Adverse Events ........................................... 42 
7.1.3 
Adverse Events of Special Interest ................................ 42 
7.2 
Collection and Reporting ................................................ 43 
7.2.1 
Serious Adverse Event Collecting and Reporting ........ 43 
7.2.2 
Non-Serious Adverse Events (NSAEs) Collecting 
and Reporting .............................................................................. 44 
7.3 
Laboratory Test Abnormalities ...................................... 45 
7.4 
Overdose ......................................................................... 45 
7.5 
Pregnancy ........................................................................ 45 
7.6 
Potential Drug-Induced Liver Injury (DILI) ..................... 46 
7.7 
Other Safety Considerations .......................................... 46 
8 
DATA MONITORING COMMITTEE .................................. 46 
9 
STATISTICAL CONSIDERATIONS .................................. 47 
10 
STUDY MANAGEMENT ................................................... 49 


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10.1 
Compliance with the Protocol ........................................ 49 
10.1.1 
Compliance with the Protocol and Protocol 
Revisions ......................................................................... 49 
10.2 
Records Retention .......................................................... 50 
10.2.1 
Records Retention .......................................................... 50 
10.2.2 
Study Drug Records........................................................ 50 
10.3 
Destruction of Investigational Product .... Error! Bookmark 
not defined. 
11 
GLOSSARY OF TERMS ................................................... 52 
12 
LIST OF ABBREVIATIONS .............................................. 53 
13 
REFERENCES .................................................................. 54 
 
 


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PROTOCOL SYNOPSIS 
Protocol Title: 
Effect of Saxagliptin (DPP-4 Inhibitor) on Endothelial 
Progenitor Cells (EPCs) as a cellular biomarker for 
evaluating Endothelial Dysfunction in Early Type 2 Diabetes 
patients 
Site Numbers & 
Names: 
GWU Medical Faculty Associates 
Research 
Hypothesis: 
We believe poor viability and function of EPCs in early 
diabetes ultimately affects the repair and regeneration of the 
endothelium and that prompts intervention using Saxagliptin 
with another oral hypoglycemic agent, Metformin, may 
reduce or reverse cardiovascular risk by improving EPC 
survival and function above and beyond adequate glucose 
metabolism control.   
Study Schema: 
Drugs / Doses / 
Length of Treatment) 
We propose a 2-arm randomized, parallel group, longitudinal 
study of 12-week intervention duration. The 12-week time 
interval has been previously shown adequate to observe 
changes in biochemistry, EPCs and, importantly, pulse wave 
velocity (PWV) changes. Patients will be randomized to 2 
groups: Control, (n=20), Metformin + Lifestyle + Placebo. 
Treatment, (n=20) Metformin+ Lifestyle+ Saxagliptin 
5mg.  
 


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Study Objectives: 
 Primary: 
 Secondary: 
The primary objective is to ascertain if addition of Saxagliptin 
improves endothelial dysfunction in early type 2 diabetes 
patients. 
The secondary objective is to establish CD34+ cell number, 
function and gene expression as important bio-markers to 
monitor endothelial function.  
 
Study Outcome Measures 
 
Primary:  
To investigate the effect of Saxagliptin and lifestyle 
modification (diet and aerobic exercise intervention) 
separately 
on 
Endothelial 
function 
in 
patients 
inadequately controlled (HbA1C ≥6% to ≤9%) with 
Metformin ≥1.0 to ≤2.0 grams daily.  
 Cellular markers. We will study pre and post 
Saxagliptin treatment changes in number, function and 
gene expression of patients’ peripheral blood-derived 
CD34+ cells.  
Secondary:  
 To investigate the effect of Saxagliptin treatment on 
serum endothelial inflammatory markers including high 
sensitivity C-reactive protein (hs-CRP), IL-6, TNF-
alpha, and fasting lipid profile.  
 Glycemic control will be evaluated by measuring 
fasting blood glucose, insulin, and HbA1c levels and 
assessing insulin resistance using HOMA-IR 
 Adiposity will be measured using the Tanita Body 
Composition Analyzer scale, measured as percentage 
body fat  
 Vessel health will be assessed by systolic and 
diastolic blood pressure and Arterial stiffness 
assessed using Vascular Flow and wave 
measurement equipment, SphygmoCor CP system 
from ATCOR. The sphygmocor system also allows us 
to estimate central aortic blood pressure.Resting 
Metabolic Rate (RMR, similar to Resting Energy 
expenditure measurement) at baseline, midway point 
(week 6) and post therapy. 
Study Design: 
Prospective, double-masked, randomized placebo-controlled 
trial  


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Accrual Goal: 
(Total number of 
subjects) 
N=42 
Accrual Rate: 
(Number of subjects 
expected per month) 
Recruit 42 patients in approximately 36 months. 
 
Estimated: 
FPFV: 
LPFV:  
Follow Up: 
(dd-mm-yy) 
FPFV: 01-10-12.    
LPFV: 01-06-15 
Correlative Studies:  
(PK/PD, etc.) 
N/A 
Inclusion Criteria: 
1. Adults aged 40-70 years.  
2. Diagnosis of type 2 diabetes within the previous 8 
years using criteria of the American Diabetes 
Association 
3. Currently treated with no hypoglycemic agents other 
than a stable dose (>3 months) of metformin (≥1.0 to 
≤2 grams daily).  
4. HbA1C between 6 to 9% (both inclusive)  
5. BMI 25 to 39.9 kg/m2 (both inclusive)  


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Exclusion Criteria: 
Patients with: 
1. Contraindications for moderate exercise 
2.  Implanted devices (e.g., pacemakers) that may 
interact with Tanita scale 
3. Previous coronary or cerebrovascular event within 6 
months of screening or active or clinically significant 
coronary and/or peripheral vascular disease 
4. Low hematocrit (< 28 UNITS).  
5. Pre-existing  liver disease and/or ALT and AST 
>2.5X’s UNL 
6. Kidney disease at levels defined by: serum creatinine 
levels ≥1.5 mg/dL for men, ≥1.4 mg/dL for women, 
estimated CrCl ≤50 mL/min 
7. History of pancreatitis, or cancer (except basal cell 
carcinoma)  
8. Statin use started (or dose change) in the last 3 
months,  
9. Use of oral or injectable anti-diabetic medication other 
than Metformin  
10. Daily use of a consistent long-term steroid medication 
(oral, inhaled, injected) within the last 3 months 
11. Systolic BP> 140 mmHg and Diastolic BP> 90 mmHg 
at screening visit.  
12. Active wounds or recent surgery within 3 months.  
13. Inflammatory disease, or chronic current use of anti-
inflammatory drugs within the last 3 months.  
14. triglycerides >400 mg/dL 
15. untreated hyper/hypothyroidism 
Additionally, patients who are active smokers, patients who 
are pregnant, nursing women, and post-menopausal women 
who are on hormone replacement therapy will be excluded. 
Patients on low dose oral contraceptives will be allowed to 
participate as these formulations contain lesser amount of 
estrogens. 


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Criteria for 
Evaluation:  
(Efficacy, safety,  
stopping rules, etc.) 
 
Subjects will be screened at week -4, then evaluated for end 
points at weeks 0, 6 and 12.  
Study participation will be stopped for adverse effects at the 
discretion of the study investigator. Subjects may stop study 
participation at any time and for any reason. 
Statistics: 
The total sample size requested, accounting for attrition over 
the 12-week period, is 20 subjects per group or 40 subjects 
total. Sample size estimates were based on the effects of 
exercise on CD34+/KDR+ cells as described in the literature.  
The effect of a single session, as well as extended training, 
on healthy subjects or those with existing cardiovascular 
conditions appears to increase the CD34+/KDR+ cells.    
This will provide 73% power (see statistics section).  A p-
value of less than 0.05 will be considered statistically 
significant. 
 


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1 
INTRODUCTION 
Type 2 diabetes is a national epidemic 1,2 with significant macro and microvascular 
complications3. Insulin resistance in prediabetes and early and late diabetes are associated 
with endothelial dysfunction4.  
A few studies indicate that EPCs can act as a suitable bio-marker 5-7 for monitoring 
cardiovascular morbidity. In this proposal we suggest that EPCs or CD34 positive cells can 
act as a suitable cellular biomarker for estimating and following endothelial dysfunction in 
early type 2 diabetes patients.  
EPCs have been used as a regenerative tool in ischemic myocardium and diabetic wound 
healing 8-13. Endothelial dysfunction with associated inflammation may be a consequence 
of excess super-oxide presence in a setting of diabetes which is a pro-oxidative stress 
condition causing EPC dysfunction and senescence14. Therefore monitoring EPC number, 
function and gene expression may serve as a very useful cellular bio-marker for 
cardiovascular complications in early type 2 diabetes. 
Though lifestyle modification has been proposed as a main stay for prevention and 
treatment of early type 2 diabetes, 2, 15-19 several new therapies for diabetes have been 
developed in recent years2. Incretins and incretin mimetics appear to hold promise. Oral 
DPP-4 inhibitors have been shown to increase EPCs in patients with type 2 diabetes 20 
reportedly via SDF-1 alpha up-regulation. Interestingly, up-regulation of SDF-1 alpha and 
vascular endothelial growth factor (VEGF), both chemotactic factors increase mobilization 
and recruitment of EPCs in the face of acute ischemic injury for repair and regeneration.21-
24.  
Several studies have shown positive effect of incretins (Glucagon like peptide, GLP-1) and 
incretin receptor agonists (GLP-1 receptor agonists) on cardiovascular risk factors in type 
2 diabetes patients20,25 and even in patients with chronic heart failure and left ventricular 
dysfunction who do not have diabetes26,27.  
DPP-4 Inhibitors may have cardio-protective effects of their own, as they increase bio-
availability of endogenous GLP-1. They improve blood flow and nitric oxide production 
in endothelium28, 29. These are unique properties not demonstrated by other oral diabetes 
medications 28. The mechanism underlying these effects may be mediated by increased 
nitric oxide bioavailability but is not completely known. It is possible that Saxagliptin, a 
member of DPP-4 inhibitor group of drugs may be able to improve number and function 


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Saxagliptin-Sen ISR CV181-305 
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Date: 12/21/2015 
of CD34+  endothelial progenitor cells by up-regulating chemotactic agent SDF1 alpha 
(DPP-4 degrades SDF-1) and its receptor CXCR47, 20, 21, 30, 31.  
Poor viability and function of EPCs in early diabetes may ultimately affect the repair and 
regeneration of the endothelium and prompt intervention may reduce or reverse 
cardiovascular risk by improving EPC survival and function above and beyond adequate 
glucose metabolism control.  
Therefore we would like to explore the effect of Saxagliptin in addition to lifestyle 
intervention, on number and function and gene expression of EPC and impact on 
endothelial dysfunction in type 2 diabetes. 
This proposed study will recruit patients with type 2 diabetes of less than 8 years duration, 
who are already on Metformin (≥1.0 to ≤2 gm/d, stable dose for at least 3 months) but still 
remain poorly controlled with HbA1C between 6% and 9% (both values inclusive),  but 
not overtly out of control (> 9% of HbA1C). In these patients we seek to compare the effect 
of saxagliptin and lifestyle modification versus lifestyle modification and placebo on 
endothelial function, particularly the cellular end-point. Initially life-style intervention with 
diet and regular aerobic activity (as per Standards of Diabetes Care: Diabetes Care 
Supplement: Jan 2014) will be undertaken for 4 weeks. Subjects will begin taking 5.0 mg 
of Saxagliptin or placebo after initial 4 weeks of stabilization of life style modification and 
will be withdrawn from the study if the medication or placebo is not tolerated. We will use 
the maximum approved dose of saxagliptin in order to discern a definitive effect. 
1.1 
Summary of Results of Investigational Program 
1.1.1 
Pharmacology of Saxagliptin 
Saxagliptin has been shown to be a highly potent, reversible, competitive inhibitor of 
human DPP-4,65 with a Ki value of 1.3 ± 0.3 nM (0.41ng/mL) at 37°C. The 5-
hydroxyadamantyl metabolite of saxagliptin (5-hydroxysaxagliptin, BMS-510849), the 
major metabolite of saxagliptin both in vitro and in vivo in animals and humans, was also 
a competitive inhibitor of DPP-4 at 37°C, with a Ki value of 2.6 ± 1.0 nM (0.86 ng/mL. 2-
fold less potent than saxagliptin). 
The clinical pharmacology program consisted of 39 studies in 1090 subjects, 1037 of whom 
were dosed with saxagliptin. The Phase 2b and Phase 3 worldwide clinical development 
program in T2DM included 8 core studies in which 4607 subjects were randomized and 
treated. Of these, 3356 subjects received double-blind saxagliptin; an additional 66 subjects 


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Protocol Version: 5.3 
Date: 12/21/2015 
received open-label saxagliptin 10 mg in CV181011. Over 1000 subjects have received 
saxagliptin for more than 76 weeks, including 260 who received saxagliptin 10 mg, and 
over 450 subjects have received saxagliptin for more than 102 weeks. In studies CV181011 
and CV181014, 244 subjects have received saxagliptin for > 204 weeks. 66,67 
1.1.2 
Preclinical Toxicology of Saxagliptin 
The rat and dog were chosen as the standard species for in vivo safety studies based on 
comparative metabolism patterns and oral bioavailability. The cynomolgus monkey was 
added initially to model lymphocyte changes observed in humans (which could not be 
reproduced in the monkey) and subsequently at the request of the United States Food and 
Drug Administration (US FDA) for all sponsors developing DPP-4 inhibitors. Toxico-
kinetic data collected in the repeat-dose studies confirmed that adequate exposure was 
achieved.  
1.1.2.1 
Repeat-Dose Studies 
Saxagliptin was well tolerated in dogs at 1 mg/kg/day (AUC ≥ 286 ng•h/mL) for 12 months 
with effects at higher doses consisting primarily of GI (gastrointestinal) toxicity. A dose of 
25 mg/kg/day (AUC 50803 ng•h/mL) was overtly toxic and resulted in euthanasia of 1 
male after 9 doses due to saxagliptin-induced enteropathy with secondary minimal to mild 
decreases in serum protein and electrolytes related to intestinal dysfunction (diarrhea, 
emesis). Drug-related morphologic changes at 5 and 10 mg/kg/day were limited to minimal 
mixed hepatic infiltrates/inflammation (neutrophils, eosinophils, lymphocytes, and 
macrophages) around central veins and after 12 months, minimal to slight superficial 
erosions of the foot pad epidermis (corresponding to footpad cracking). All effects at 5 
mg/kg/day in the 3-month study were reversible; recovery was not assessed in the 12-
month study. The NOAEL was 1 mg/kg/day after 12 months of dosing, which resulted in 
respective AUC exposures for saxagliptin and metabolite of 4× and 2× the maximum 
recommended human dose (MRHD) of saxagliptin.  
The repeat-dose oral toxicity of saxagliptin in cynomolgus monkeys was characterized at 
doses of 0.03, 0.3, and 3 mg/kg/day for 3 months. No drug-related changes were observed 
at 0.03 or 0.3 mg/kg/day. At 3 mg/kg/day, multifocal skin lesions/scabbing was observed 
on the feet and/or tails of 4 of 14 monkeys. Drug-related effects at 3 mg/kg/day included 
skin lesions, minimal multi-tissue mononuclear-cell infiltrates (considered an exacerbation 
of a common background change), and minimal splenic, thymic, and/or bone marrow 
lymphoid hyperplasia. Healing of the skin lesions occurred during the dosing period, and 


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all findings were reversible after a 3-month recovery period. Respective systemic 
exposures (AUC) for saxagliptin and the metabolite at the NOAEL of 0.3 mg/kg/day were 
1 to 3× the MRHD.  
1.1.2.2 
Mutagenicity and Carcinogenicity  
The mutagenic and clastogenic potential of saxagliptin was tested at high concentrations 
and exposures in a battery of genetic toxicity studies including an in vitro Ames bacterial 
assay, an in vitro cytogenetics assay in primary human lymphocytes, an in vivo oral 
micronucleus assay in rats, an in vivo oral DNA repair study in rats, and an oral in vivo/in 
vitro cytogenetics study in rat peripheral blood lymphocytes.68 Saxagliptin was not 
mutagenic or clastogenic based on the combined outcomes of these studies. The major 
metabolite (BMS-510849) was not mutagenic in an in vitro Ames bacterial assay. Two-
year carcinogenicity studies were conducted in mice and rats at oral doses of 50, 250, and 
600 mg/kg/day and 25, 75, 150, and 300 mg/kg/day, respectively4.  Saxagliptin did not 
induce tumors in either mice or rats at the highest doses evaluated. The highest doses 
evaluated in mice were equivalent to approximately 900× (males) and 1210× (females) the 
human exposure at the MRHD. In rats, exposures were approximately 370× (males) and 
2300× (females) the exposures at the MRHD.  
1.1.2.3 
Reproductive and Developmental Toxicity  
In a rat fertility study, males were treated with oral gavage doses of 100, 200, and 400 
mg/kg/day for two weeks prior to mating, during mating, and up to scheduled termination 
(approximately 7 weeks total), and females were treated with oral gavage doses of 125, 
300, and 750 mg/kg/day for 2 weeks prior to mating through gestation Day 7.68 No adverse 
effects on fertility were observed at 200 mg/kg/day (males) or 125 mg/kg/day (females) 
resulting in respective exposures (AUC) of approximately 630× (males) and 805× 
(females) times human exposure at the MRHD. At higher, maternally toxic doses (300 and 
750 mg/kg/day), increased fetal resorptions were observed (approximately 2150× and 
6375× the MRHD). Additional effects on estrous cycling, fertility, ovulation, and 
implantation were observed at 750 mg/kg (approximately 6375× the MRHD). Saxagliptin 
was not teratogenic at any dose evaluated in rats or rabbits.68 At high doses in rats, 
saxagliptin caused a minor and reversible developmental delay in ossification of the fetal 
pelvis at ≥240 mg/kg/day (≥1560× the MRHD). Maternal toxicity and reduced fetal body 
weights were observed at 900 mg/kg/day (8290× the MRHD). In rabbits, the effects of 


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Date: 12/21/2015 
saxagliptin were limited to minor skeletal variations observed only at maternally toxic 
doses (200 mg/kg/day, exposures 1420× the MRHD).  
Saxagliptin administered to female rats from gestation Day 6 to lactation Day 20 resulted 
in decreased body weights in male and female offspring only at maternally toxic doses 
(≥250 mg/kg/day, exposures ≥1690× the MRHD).68 No functional or behavioral toxicity 
was observed in offspring of rats administered saxagliptin at any dose.  
1.1.3 
Human Pharmacokinetics of Saxagliptin 
Following a 5 mg single oral dose of saxagliptin tablet, the mean plasma AUC(INF) values 
for saxagliptin and BMS-510849 were 78 and 214 ng•h/mL, respectively. The 
corresponding maximum plasma concentration (Cmax) values were 24 and 47 ng/mL, 
respectively.  
1.1.3.1 
Absorption  
Saxagliptin was rapidly absorbed after oral administration, with Cmax usually attained 
within 2 hours after administration in the fasted state. In general, the Cmax and AUC values 
increased approximately equal to the increment in the saxagliptin dose (CV181001). 
Following a single oral dose of 5 mg saxagliptin in the fasted state, the mean terminal half-
life (T1/2) value for saxagliptin was 2.5 h, and no appreciable accumulation was observed 
upon repeated once-daily dosing at any dose.  
1.1.3.2 
Distribution  
The in vitro protein binding of saxagliptin and its major metabolite in human serum is 
below measurable levels.69 Thus, changes in blood protein levels in various disease states 
(e.g., renal or hepatic impairment) are not expected to alter the disposition of saxagliptin.  
1.1.3.3 
Elimination  
Renal excretion is a major elimination pathway for systemic saxagliptin and BMS-510849. 
Following a single 50 mg dose of 14C-saxagliptin, 24%, 36%, and 75% (on average) of 
the dose was excreted in the urine as saxagliptin, BMS-510849, and total radioactivity, 
respectively (CV181004). These data suggest that saxagliptin was extensively absorbed 
from the gastrointestinal tract and that BMS-510849 is a major metabolite of saxagliptin in 
humans. The average renal clearance of saxagliptin (∼200 L/min) was greater than the 
average estimated glomerular filtration rate (∼120 mL/min), suggesting some active renal 
excretion. For BMS-510849, renal clearance values were comparable to estimated 


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glomerular filtration rate. A total of 22% (on average) of the administered radioactivity 
was recovered in feces representing the fraction of the saxagliptin dose excreted in bile 
and/or unabsorbed drug from the gastrointestinal tract.  
1.1.3.4 
Pharmacokinetics of Active Metabolites  
Cmax and AUC values for BMS-510849 increased approximately equal to the increment 
in the saxagliptin dose. Following single oral doses of 5 to 100 mg saxagliptin in the fed 
or fasted states, the mean BMS-510849 AUC values were between 2- and 7-times higher 
than the parent saxagliptin exposures on a molar basis.70 Following a single oral dose of 5 
mg saxagliptin in the fasted state (CV181037), the mean terminal half-life (T1/2) value for 
BMS-510849 was 3.1 h, and no appreciable accumulation was observed upon repeated 
once-daily dosing at any dose (CV181002 and CV181010).  
1.1.3.5 
Dose and Time-dependencies  
The single-dose pharmacokinetics of saxagliptin and BMS-510849 were predictive of their 
multiple-dose pharmacokinetics. No dose- or time-dependency was observed in any 
relevant pharmacokinetic parameter over 14 days of once-daily dosing with saxagliptin 
with doses ranging from 2.5 to 400 mg once daily (QD; CV181002 and CV181010). A 
linear pharmacokinetic model adequately described the plasma concentration time profile 
of saxagliptin and BMS-510849, suggesting the pharmacokinetics of saxagliptin and BMS-
510849 were linear to dose within the tested dose range of 1-100 mg. The estimated 
pharmacokinetic parameters were time-invariant. These data suggest that saxagliptin does 
not inhibit or induce its own metabolism or active transport.  
1.1.3.6 
Special Populations  
Saxagliptin may be administered without regard to meals. No dosage adjustment is 
considered necessary in elderly subjects on the basis of age. Body mass index (BMI), 
T2DM, gender, age, race, hepatic impairment, and mild renal insufficiency had no 
clinically meaningful effects on the pharmacokinetics of saxagliptin or BMS-510849. In 
subjects with moderate to severe renal impairment and end stage renal disease (i.e., 
creatinine clearance <50 mL/min), area under curve (AUC) values for saxagliptin and/or 
BMS-510849 were generally greater than 2-fold higher than the AUC values in healthy 
subjects.  


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1.1.3.7 
Drug-drug interactions 
Co-administration of saxagliptin with the following drugs had no meaningful effect on the 
pharmacokinetics of saxagliptin, BMS-510849, or the other drug tested: metformin, 
glyburide, pioglitazone, digoxin, simvastatin, rifampin, and combined oral contraceptives. 
Co-administration of saxagliptin and ketoconazole dosed to steady-state resulted in up to 
1.6- and 2.5-times increase in saxagliptin maximum observed concentration (Cmax) and 
area under curve extrapolated to infinity [AUC(INF)] values, respectively, and 20- to 
8-times decrease in the exposure to BMS-510849. However, the systemic exposures to the 
total active moieties of saxagliptin (saxagliptin + BMS-510849 corrected for relative 
differences in potency of DPP-4 inhibition) were not meaningfully altered by ketoconazole. 
Saxagliptin Cmax and AUC(INF) were also increased by 63% and 2.1-fold, respectively, 
when saxagliptin was co-administered with steady-state diltiazem. Higher geometric mean 
Cmax and AUV(TAU) for diltiazem were also found when co-administered with 
saxagliptin. The differences met statistical significance but were not considered clinically 
meaningful. Co-administration of saxagliptin with gastric acid controllers aluminum and 
magnesium hydroxides + simethicone, famotidine or omeprazole did not meaningfully 
alter the pharmacokinetics of saxagliptin or BMS-510849. 
1.1.4 
Clinical Safety with Saxagliptin 
Once-daily, orally administered saxagliptin was safe and well-tolerated at doses of up to 
400 mg QD for 2 weeks, 100 mg QD for 6 weeks, 40 mg QD for 12 weeks, and at doses 
of 2.5, 5, and 10 mg QD for up to 102 weeks.  
1.1.4.1 
Drug-Related Adverse Events 
In an extensive Phase 2b/3 program, the majority of reported adverse events were of mild 
intensity and did not require treatment discontinuation. The safety profile was generally 
consistent with placebo or comparator when saxagliptin was given as monotherapy, as add-
on combination treatment to metformin, SU, or TZD, and as initial therapy in combination 
with metformin. Although the rate of certain AEs was higher in subjects who received 
saxagliptin 10 mg compared with those who received 2.5 and 5 mg, saxagliptin 10 mg was 
also safe and well tolerated, providing a safety margin for the saxagliptin 5 mg dose. 
A pre-specified pooled analysis (the 5-study pooled analysis) of the 2 monotherapy 
studies, (CV181011 and CV181038) the add-on to metformin study (CV181014), the 
add-on to TZD study (CV181013) and the add-on to glyburide study (CV181040) 


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demonstrated that the overall incidence of AEs in patients treated with saxagliptin 5 mg 
was similar to placebo (72.2% vs. 70.6%). The majority of reported AEs were of mild 
intensity and did not require treatment discontinuation. There was no discernible 
difference in the clinical AE profile between the saxagliptin 2.5 mg and 5 mg doses. 
The most frequently reported AEs (incidence ≥ 5%) in active vs. placebo group were 
upper respiratory tract infection (7.7% vs. 7.6%), headache (6.5% vs. 5.9%), and 
urinary tract infection (6.8% vs. 6.1%). In the combination study with metformin 
(CV181-039), headache (7.5% in saxagliptin, 5.2% in metformin) and Nasopharyngitis 
(6.9% in saxagliptin, 4.0% in metformin) were the most frequently reported AEs 
(incidence ≥ 5%). In September 2015, the FDA reported a new AE of severe and 
disabling joint pain (less than 5% incidence.)  
 
The frequency of AEs leading to discontinuation from study therapy was generally low for 
all treatment groups. In the placebo-controlled pooled safety analysis, the overall frequency 
of AEs leading to discontinuation of study medication was comparable between the 
saxagliptin 2.5 mg and placebo groups (2.2% and 1.8%, respectively) but was greater for 
patients in the saxagliptin 5 group (3.3%). The most frequently reported AEs (> 1 subject) 
leading to study discontinuation, which were numerically higher in patients who received 
saxagliptin compared with placebo, were: lymphopenia, increased blood creatine kinase 
(CK), increased blood creatinine, nausea, and eye pain. Rates of discontinuation for rash 
were similar in patients who received saxagliptin and placebo, whereas the rate of 
discontinuation for AEs of increased weight, depression, and angina pectoris were higher 
in patients who received placebo compared with saxagliptin. 
1.1.4.2 
Drug-Related Serious Adverse Events 
In the placebo-controlled pooled safety analysis, the frequency of SAEs was identical 
(3.4%) for the saxagliptin 5 mg and placebo groups. The frequencies of SAEs were 
comparable for the saxagliptin 5 mg plus metformin and metformin monotherapy groups 
(2.5% and 2.4%, respectively) in the combination with metformin study (CV181039). 
1.1.4.3 
Other Significant Drug-Related Adverse Events 
Hypoglycemia: Treatment with saxagliptin led to rates of hypoglycemia that were 
generally similar compared with placebo. This is consistent with the mechanism of action 
of DPP-4 inhibitors, which exert their insulinotropic effects on the β-cell in a glucose-


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dependent manner. In the add-on to sulphonylurea (SU) study, the rate of hypoglycemia 
was numerically higher in patients who received 2.5 or 5 mg of saxagliptin added on to an 
intermediate dose of glyburide compared with up-titration of glyburide monotherapy plus 
placebo. 
Skin related AEs: The frequency of skin-related AEs was generally comparable between 
subjects who received saxagliptin 5 mg and placebo. Overall, evaluation of the Phase 3 
clinical data has not revealed any signals that correlate to the skin findings during 
non-clinical toxicology studies in the monkey, including reversible erosive and/or 
ulcerative skin lesions with scab formation. Overall, evaluation of the Phase 3 clinical data 
with exposures up to 206 weeks did not reveal any signals that correlate to the skin findings 
in the cynomolgus monkey. 
Local edema: The proportion of patients with AEs of localized edema, an event of special 
interest given reports of symptomatic edema of the hands and feet in patients who received 
another member of the DPP-4-inhibitor class, was generally similar in patients who 
received saxagliptin and placebo with the exception of the add-on to TZD study, where 
there was a higher rate of events constituting localized edema compared to placebo in 
patients treated with saxagliptin 5 mg. The majority of these events in the saxagliptin 5 mg 
plus TZD group were for pedal edema with no imbalance seen for events of hand edema. 
Across the clinical program, the majority of events of localized edema were of mild to 
moderate intensity and did not lead to study discontinuation. 
Hypersensitivity Reactions 
 A 24-week analysis of a grouping of hypersensitivity-related events in a pool of 5 placebo-
controlled Phase 3 studies showed an incidence of 1.5% and 0.4% in patients who received 
saxagliptin 5 mg and placebo, respectively. None of these events in patients who received 
saxagliptin required hospitalization or were reported to be life-threatening by the 
investigators. 
Lymphopenia: The frequency of investigator reported AEs of lymphopenia was similar for 
subjects who received saxagliptin and placebo. Mean lymphocyte counts remained stable 
and within normal limits with daily dosing up to 102 weeks. Overall, a small decrease in 
mean absolute lymphocyte count was observed at a dose of saxagliptin 5 mg and above. At 
the 5 mg dose, the mean decrease was approximately 100 cells/μL relative to placebo (from 
baseline absolute lymphocyte count of approximately 2200 cell/μL) based on a pooled 
analysis of the 5 placebo-controlled clinical studies including saxagliptin as monotherapy, 


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and as add-on therapy to metformin, TZD and SU. While the clinical significance of the 
decreases in lymphocyte count relative to placebo is not known, the decreases were not 
associated with clinically relevant AEs. 
Platelet counts: Results from the Phase 3 clinical studies demonstrated no clinically 
meaningful or consistent effect on platelet counts.  
In the five pooled, monotherapy and placebo-controlled combination studies, the 
frequencies of AEs in the SOC Infection and Infestations were comparable in the 
saxagliptin 2.5 mg, saxagliptin 5 mg, and placebo groups (36.4%, 35.9%, and 34.8% 
respectively); a higher frequency of AEs was observed in the saxagliptin 10 mg group 
(40.1%).  
There was no evidence for an association of saxagliptin treatment with an increased risk of 
elevated liver function tests or serum creatinine. 
Effect on QTc interval: In the clinical pharmacology program, results from a thorough QTc 
study at daily doses of saxagliptin up to 40 mg QD and analyses from other studies at daily 
doses up to 400 mg QD demonstrated an absence of an effect of saxagliptin and BMS-
510849 on QTc interval in humans.  
1.1.4.4 
Drug-Related Deaths 
Overall, death was an infrequent event and occurred with comparable frequencies in the 
saxagliptin and comparator groups. In the core Phase 3 studies, a total of 24 patients died 
during the short-term and long-term periods: 4 subjects in the saxagliptin 2.5 mg group, 3 
subjects in the 5 mg group, 5 subjects in the saxagliptin 10 mg group, 7 subjects in the 
placebo group, and 5 subjects in the metformin monotherapy group. 
1.1.5 
Clinical Efficacy of Saxagliptin 
Saxagliptin Phase 2b and Phase 3 worldwide clinical development program in T2DM 
included eight studies in which over 4600 subjects combined with the results from clinical 
pharmacology studies support the oral dose of saxagliptin 5 mg once daily as the usual 
clinical dose in a wide range of subjects with T2DM, as either monotherapy, add-on 
combination therapy with metformin, a thiazolidinedione (TZD), or a sulfonylurea (SU), 
or initial combination therapy with metformin. 
In the Phase 2b dose-ranging study, administration of saxagliptin 5 mg was associated with 
significant inhibition of plasma DPP-4 activity at the trough of the dosing interval as well 


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as clinically meaningful decreases in A1C, fasting plasma glucose (FPG), and postprandial 
serum glucose. The results from the short-term periods of the Phase 3 studies confirmed 
clinically meaningful benefits of saxagliptin 5 mg on A1C, as well as FPG, postprandial 
glucose, insulin, C-peptide, and glucagon levels. In the monotherapy studies (CV181011 
and CV181038), add-on combination therapy studies (CV181014, CV181013, CV181040 
with metformin, TZD, glyburide, respectively), and initial combination study with 
metformin (CV181039), treatment with saxagliptin produced statistically significant 
reductions in A1C relative to control. A greater percentage of subjects treated with 
saxagliptin achieved target glycemic goals including A1C levels < 7% compared with 
subjects treated with placebo or active comparator. The saxagliptin 5 mg groups achieved 
greater reductions from baseline in A1C than the saxagliptin 2.5 mg groups in five of the 
six core Phase 3 studies (one of the core studies, Study CV181039, did not have a 2.5 mg 
treatment group). There was no consistent evidence for an incremental efficacy benefit for 
10 mg beyond that seen for the 5 mg dose.  
Saxagliptin treatment consistently demonstrated a beneficial antihyperglycemic effect 
across subgroups of demographic and baseline diabetes characteristics. 
1.2 
Overall Risk/Benefit Assessment 
The efficacy of saxagliptin for the treatment of patients with T2DM was demonstrated in 
a single Phase 2b study and confirmed in 6 double-blind, placebo- or active-controlled 
clinical studies. In these studies, 4673 subjects (4607 subjects randomized and treated and 
66 patients treated in the open-label cohort of CV181011) received at least one dose of 
study medication, including 3422 receiving saxagliptin and 1251 receiving either placebo 
or metformin plus rescue medication if necessary. Treatment with saxagliptin 2.5, 5, and 
10 mg was demonstrated to be generally safe and well tolerated. The overall frequencies 
of AEs for saxagliptin 2.5 and 5 mg were comparable to placebo. Also, the initial 
combination study with metformin showed that the safety profile of saxagliptin 5 mg plus 
metformin was generally comparable to either agent used as monotherapy.  
Evaluation of the clinical safety and clinical efficacy data indicate an acceptable 
risk/benefit profile at the dose of saxagliptin planned for this study, 5 mg daily, as a 
monotherapy, and also in combination with metformin XR 1500 mg. Type 2 diabetes 
mellitus is a progressive disease, and a combination of 2 agents with complimentary 
mechanisms of action, such as metformin and saxagliptin, may offer considerable 
therapeutic advantage. 


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1.3 
Research Hypothesis 
We believe poor viability and function of EPCs in early diabetes ultimately affects the 
repair and regeneration of the endothelium and that prompt intervention using saxagliptin 
with another oral hypoglycemic agent, Metformin, may reduce or reverse cardiovascular 
risk by improving EPC survival and function above and beyond adequate glucose 
metabolism control.   
1.4 
Study Rationale 
Though certain DPP-4 inhibitors have been shown to augment vascular function and 
endothelial progenitor cells, no such data is available for Saxagliptin.  
2 
STUDY OBJECTIVES 
2.1 
Primary Objective 
To investigate the effect of saxagliptin and lifestyle modification (diet and aerobic exercise 
intervention) separately on Endothelial function in patients whose HbA1C is between 6 to 
9% (both inclusive) despite treatment with metformin (≥1.0 to ≤2 gm/d), stable dose for at 
least 3 months) 
Cellular markers5-7,30.We will use patient’s peripheral blood derived CD34+ cells looking 
at number, function and gene expression changes pre and post saxagliptin. 
2.2 
Secondary Objectives 
To investigate the effect of saxagliptin treatment on serum endothelium inflammatory 
markers including: C-reactive protein (hs-CRP), IL-6, TNF-alpha, Leptin, Adiponectin,  
and fasting lipid profile 6, 30  
 
Glycemic control will be evaluated by measuring fasting blood glucose, insulin levels and 
HbA1c. Fasting blood glucose and insulin will be used to assess insulin resistance with 
HOMA-IR 32  
 
Adiposity will be measured using the Tanita Body Composition Analyzer scale, measured 
as percentage body fat 33 
 
Vessel health will be assessed by systolic and diastolic blood pressure and Arterial 
stiffness will be assessed using Vascular Flow and wave measurement equipment, 
SphygmoCor CP system from ATCOR. The sphygmocor system also allows us to 
estimate central aortic blood pressure. 
 
 


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The biochemical or bio-inflammatory markers are an important marker of endothelial 
inflammation and dysfunction along with glycemic markers and markers of arterial 
stiffness. 
3 
ETHICAL CONSIDERATIONS 
3.1 
Good Clinical Practice 
This study will be conducted in accordance with Good Clinical Practice (GCP), as defined 
by the International Conference on Harmonisation (ICH) and in accordance with the ethical 
principles underlying European Union Directive 2001/20/EC and the United States Code 
of Federal Regulations, Title 21, Part 50 (21CFR50). 
The study will be conducted in compliance with the protocol. The protocol, any 
amendments, and the subject informed consent will receive Institutional Review 
Board/Independent Ethics Committee (IRB/IEC) approval/favorable opinion before 
initiation of the study. 
All potential serious breaches must be reported to Astra-Zeneca (AZ) immediately. A 
serious breach is a breach of the conditions and principles of GCP in connection with the 
study or the protocol, which is likely to affect, to a significant degree, the safety or physical 
or mental integrity of the subjects of the study or the scientific value of the study. 
Study personnel involved in conducting this study will be qualified by education, training, 
and experience to perform their respective tasks.  
This study will not use the services of study personnel where sanctions have been invoked 
or where there has been scientific misconduct or fraud (e.g., loss of medical licensure; 
debarment).  
3.2 
Institutional Review Board/Independent Ethics 
Committee 
Before study initiation, the investigator must have written and dated approval/favorable 
opinion from the IRB/IEC for the protocol, consent form, subject recruitment 
materials/process (e.g., advertisements), and any other written information to be provided 
to subjects. The investigator or sponsor should also provide the IRB/IEC with a copy of 
the Investigator Brochure or product labeling information to be provided to subjects, and 
any updates.  


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The investigator should provide the IRB/IEC with reports, updates, and other information 
(e.g., expedited safety reports, amendments, and administrative letters) according to 
regulatory requirements or institution procedures. 
3.3 
Informed Consent 
Investigators must ensure that subjects or, in those situations where consent cannot be 
given by subjects, their legally acceptable representative are clearly and fully informed 
about the purpose, potential risks, and other critical issues regarding clinical studies in 
which they volunteer to participate. 
Investigators must: 
1) Provide a copy of the consent form and written information about the study in the 
language in which the subject is most proficient prior to clinical study participation. 
The language must be non-technical and easily understood. 
2) Allow time necessary for subject or subject's legally acceptable representative to 
inquire about the details of the study. 
3) Obtain an informed consent signed and personally dated by the subject or the 
subject's legally acceptable representative and by the person who conducted the 
informed consent discussion.  
4) Obtain the IRB/IEC’s written approval/favorable opinion of the written informed 
consent form and any other information to be provided to the subjects, prior to the 
beginning of the study, and after any revisions are completed for new information. 
5) If informed consent is initially given by a subject’s legally acceptable representative 
or legal guardian, and the subject subsequently becomes capable of making and 
communicating their informed consent during the study, then consent must 
additionally be obtained from the subject. 
6) Revise the informed consent whenever important new information becomes 
available that is relevant to the subject's consent. The investigator, or a person 
designated by the investigator, should fully inform the subject or the subject's 
legally acceptable representative or legal guardian, of all pertinent aspects of the 
study and of any new information relevant to the subject's willingness to continue 
participation in the study. This communication should be documented.  
The rights, safety, and well-being of the study subjects are the most important 
considerations and should prevail over interests of science and society. 


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4 
INVESTIGATIONAL PLAN 
4.1 
Study Design and Duration 
Study Schema, 42 Type 2 diabetic Subjects Aged 40-70 on stable dose of Metformin 
 
 
+/- 3 days  for visits Wk 0  Wk 6 and Wk 12, if the particular day is not feasible    
*Assessed at week 0, 6 and 12: Biochemical and cellular markers of endothelial function 
and Pulse wave analysis and velocity (PWA and PWV). 
                         
Available related studies and Expected Results: Studies measuring changes to 
EPCs in type 2 diabetes patients are very limited. To the best of our knowledge there are 
no studies examining the effect of saxagliptin in early diabetes on patient’s CD34/KDR+ve 
cells and comparing that to the effect of lifestyle + metformin on CD34+ve cell population.  
The regenerative potential of EPC is thought to be due to direct incorporation into vascular 
endothelium and/or the secretion of angio-supportive factors to enhance endothelial repair 
and function17, 18, 19.  Our studies and others’ 11,12,34, have shown that various disease states, 
including those seen in diabetes, can deplete and damage EPC, thereby diminishing their 
regenerative potential.  The inability to maintain or repair damaged endothelial tissue leads 
to cumulative vascular dysfunction and cardiovascular disease.  
 
We hypothesize that monitoring number, function and gene expression of endothelial 
progenitors will allow us to quantify cardiovascular disease risk at the onset and 
regenerative potential post intervention at a cellular level. It will also help us to identify 
and correlate the best endothelial function bio-chemical inflammatory marker as an 
early indicator of cardiovascular disease progression in early type 2 diabetes. 
   
Group A: Metformin + Lifestyle + Placebo, n=21 
Wk 6* 
Wk 12* 
          Screening 
Group B: Metformin + Lifestyle + Saxagliptin 5mg, n=21 
Wk-0* 
Randomization 
Wk -4 
 


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Therefore, studying the effects of Saxagliptin which can increase SDF1 alpha levels and 
possibly CXCR4 receptor expression as an intervention on the number and health of EPCs 
may not only provide insight on the biological role of these cells, but they can be used as 
a tool to study vascular biology and lastly can be a potential target for prevention of 
diabetes vascular complications. 
  
We expect to establish that saxagliptin improves endothelial function that is EPC number, 
function, gene expression and bio-inflammatory markers beyond its glycemic control 
effect in type 2 diabetes patients. 
 
Saxagliptin 5 mg or placebo will be added to patients who have type 2 diabetes diagnosed 
less than 8 years with HbA1C of 6 to 9 % (both inclusive) while being on lifestyle 
modification and Metformin (≥1.0 to ≤2 gm/d orally).  
Our study population will include equal numbers of adult male and female Type 2 
diabetes (T2DM) patients aged 40-70 years, enrolled in both arms of the study. 
Patients will be treated with 5 mg of Saxagliptin or placebo with lifestyle modification 
for 12 weeks.  This time interval has been previously shown to be adequate to observe 
changes to Endothelial Progenitor Cells (EPCs)5,6. 
 
4.2 
Study Population 
For entry into the study, the following criteria MUST be met. 
4.2.1 
Inclusion Criteria 
1) Signed Written Informed Consent 
 Before any study procedures are performed, subjects will have the details of the 
study described to them, and they will be given a written informed consent 
document to read. Then, if subjects consent to participate in the study, they will 
indicate that consent by signing and dating the informed consent document in 
the presence of study personnel. 
2) Target Population 
 Subjects with a diagnosis of Type 2 diabetes mellitus within the previous 8 
years using criteria of the American Diabetes Association. 
 Currently treated with no hypoglycemic agents other than a stable dose (>3 
months) of metformin (≥1.0 to ≤2 grams daily).  
 HbA1C between 6% and 9% (both inclusive)   
 BMI 25-39.9 kg/m2 both inclusive. 
 


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3) Age and Reproductive Status 
 Men and women, 40 to 70 years of age.  
 Reproductive Status: Definition of Women of Child-Bearing Potential 
(WOCBP). WOCBP comprises women who have experienced menarche and 
who have not undergone successful surgical sterilization (hysterectomy, 
bilateral tubal ligation, or bilateral oophorectomy) or who are not post-
menopausal (see definition below). 
Post-menopause is defined as:  
 Women who have had amenorrhea for  12 consecutive months (without 
another cause) and especially with a documented serum follicle-stimulating 
hormone (FSH) level > 35 mIU/mL. 
 Women who have irregular menstrual periods and a documented serum FSH 
level > 35 mIU/mL. 
 Women who are taking hormone replacement therapy (HRT). 
The following women are WOCBP: 
 Women using the following methods to prevent pregnancy: Oral 
contraceptives, other hormonal contraceptives (vaginal products, skin patches, 
or implanted or injectable products), or mechanical products such as 
intrauterine devices or barrier methods (diaphragm, condoms, spermicides). 
 Women who are practicing abstinence. 
 Women who have a partner who is sterile (e.g., due to vasectomy).WOCBP must 
be using an acceptable method of contraception to avoid pregnancy throughout the 
study and for up to 4 weeks after the last dose of study drug in such a manner that 
the risk of pregnancy is minimized.  
 WOCBP must have a negative serum or urine pregnancy test result (minimum 
sensitivity 25 IU/L or equivalent units of HCG) within 0 to 72 hours before the first 
dose of study drug.  
 Women must not be breast-feeding. 
 
4.2.2 
Exclusion Criteria 
 Type 1 diabetes mellitus 
 History of diabetic ketoacidosis or hyperosmolar nonketotic coma 
 Hemoglobinopathies with low hematocrit (hematocrit below normal limits that may 
impair exercise tolerance) or abnormal CBC  
 History of pancreatitis, or cancer (except basal cell carcinoma) 
 Previous coronary or cerebrovascular event within 6 months of screening or active 
or clinically significant coronary and/or peripheral vascular disease 


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 Statin use started (or dose change) in the last 3months,  
 Daily use of oral or injectable anti-diabetic medication other than Metformin  
 use of any form of steroid medication (oral, inhaled, injected or nasal) within the 
last 3 months 
 Systolic BP> 140 mmHg and Diastolic BP> 90mmHg at screening visit.  
 Active wounds or recent surgery within 3 months  
 Inflammatory disease, or current use of anti-inflammatory drugs  
 Untreated hyper/hypothyroidism 
 Contraindications for moderate exercise 
 Implanted devices (e.g., pacemakers) that may interact with Tanita scale 
 
Physical and Laboratory Test Findings 
 Pre-existing  liver disease and/or ALT and AST >2.5X’s UNL, 
 serum creatinine levels ≥1.5 mg/dL for men, ≥1.4 mg/dL for women, estimated 
CrCl ≤50 mL/min) 
 Triglycerides >400 mg/dL  
Allergies and Adverse Drug Reactions 
 Subjects with a history of any serious hypersensitivity reaction to saxagliptin or 
DPP-4 inhibitor.  
Sex and Reproductive Status 
 WOCBP who are unwilling or unable to use an acceptable method to avoid 
pregnancy for the entire study period.  
 Women who are pregnant or breastfeeding. 
See Section on WOCBP above (Section 4.2.1, item # 3.) 
Prohibited Treatments and/or Therapies 
 Treatment with systemic cytochrome P450 3A4 (CYP 3A4) inhibitors 
Other Exclusion Criteria 
 Prisoners or subjects who are involuntarily incarcerated. 
 Subjects who are compulsorily detained for treatment of either a psychiatric or 
physical (e.g., infectious disease) illness.  
 
Additionally, patients who are active smokers, patients who are pregnant, nursing 
women, and post menopausal women who are on hormone replacement therapy 
will be excluded.  Patients on low dose oral contraceptives will be allowed to 
participate as these formulations contain lesser amount of estrogens. 


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Eligibility criteria for this study have been carefully considered to ensure the safety of the 
study subjects and to ensure that the results of the study can be used. It is imperative that 
subjects fully meet all eligibility criteria. 
4.2.3 
Discontinuation of Subjects from Treatment 
Subjects MUST discontinue investigational product (and noninvestigational product at the 
discretion of the investigator) for any of the following reasons: 
 Withdrawal of informed consent (subject’s decision to withdraw for any reason). 
 Any clinical adverse event, laboratory abnormality, or intercurrent illness which, in 
the opinion of the investigator, indicates that continued participation in the study is 
not in the best interest of the subject. 
 Changes in estimated CrCL to ≤50 mL/min necessitating reduction in saxagliptin 
dose 
 Treatment with systemic cytochrome P450 3A4 (CYP 3A4) inhibitors necessitating 
reduction in saxagliptin dose 
 Pregnancy 
 Instruct WOCBP to contact the investigator or study staff immediately if they 
suspect they might be pregnant (e.g., missed or late menstrual period) at any 
time during study participation. Institutional policy and local regulations should 
determine the frequency of on-study pregnancy tests for WOCBP enrolled in 
the study. 
 The investigator must immediately notify BMS if a study subject becomes 
pregnant. 
 Loss of ability to freely provide consent through imprisonment or involuntary 
incarceration for treatment of either a psychiatric or physical (e.g., infectious 
disease) illness. 
All subjects who discontinue should comply with protocol-specified follow-up 
procedures outlined in Section 6. The only exception to this requirement is when a 
subject withdraws consent for all study procedures or loses the ability to consent freely 
(i.e., is imprisoned or involuntarily incarcerated for the treatment of either a psychiatric or 
physical illness). If a subject withdraws before completing the study, the reason for 
withdrawal must be documented appropriately. 


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5 
TREATMENTS 
5.1 
Study Treatment: Saxagliptin 
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 saxagliptin. 
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 product is 
Metformin.  Patients will continue on Metformin as per their study entry dose added to 
Saxagliptin or placebo.  Metformin is not an investigational drug and will not be provided 
by AstraZeneca Pharmaceuticals LP Bristol Myers Squibb.  Subject will obtain continue 
to obtain Metformin as they did prior to study entry. 
5.1.1 
Identification 
Saxagliptin 5 mg tablets are pink, biconvex, round, film-coated tablets with “5” printed on 
one side and “4215” printed on the reverse side, in blue ink. 
5.1.2 
Packaging and Labeling 
Saxagliptin film-coated tablets in strengths of 1 mg, 2.5 mg, 5 mg, and 10 mg (as the free 
base) have been developed. The tablets are manufactured from saxagliptin and inactive 
ingredients including lactose, microcrystalline cellulose, croscarmellose sodium, 
magnesium stearate, and film-coating materials (Opadry® II white and Opadry® II 
yellow). The tablets are supplied in high-density polyethylene bottles containing two 
desiccant and charcoal canisters, with cotton coil and tightly closed with a child resistant 
cap.  
5.1.3 
Handling and Dispensing 
The investigational product should be stored in a secure area according to local regulations. 
The investigator is responsible for ensuring that it is dispensed only to study subjects and 
only from official study sites by authorized personnel, as dictated by local regulations. 


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The investigator is responsible for ensuring that the investigational product is stored under 
the appropriate environmental conditions (temperature, light, and humidity), as described 
below: 
Saxagliptin tablets should be stored at temperatures between 15C and 20C (59F and 
77F) in tightly closed containers.  If concerns regarding the quality or appearance of the 
investigational product arise, do not dispense the investigational product, and contact BMS 
immediately. 
5.2 
Drug Ordering and Accountability 
5.2.1 
Initial Orders 
Contact the BMS protocol manager for information.  
5.2.2 
Re-Supply 
Contact the BMS protocol manager for information.  
5.3 
Method of Assigning Subjects to a Treatment 
Subjects, as per selection criteria below will be randomized to treatments using a permuted 
block design, developed by the Epidemiology & Biostatistics Research Core. This 
approach ensures groups will be approximately balanced at any time during the study and 
at study completion.  
5.4 
Selection and Timing of Dose for Each Subject 
The recommended dose of saxagliptin is 5 mg PO will be given once daily. Saxagliptin can 
be taken with or without food. 
5.4.1 
Dose Modifications 
A single dosage adjustment to 2.5 mg daily is recommended for patients with moderate or 
severe renal impairment, or with end-stage renal disease (CrCl ≤50 mL/min, approximately 
corresponding to serum creatinine levels of ≥1.7 mg/dL in men and ≥1.5 mg/dL in women). 
Assessment of renal function is recommended before initiation of saxagliptin, and 
periodically thereafter. Given that this study protocol includes only saxagliptin 5 mg dose, 
the participation of any study subject requiring dose adjustment while on study medication 
will be discontinued (see section 4.2.3.). 


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5.5 
Blinding/Unblinding 
Blinding is critical to the integrity of this clinical study. However, in the event of a medical 
emergency or pregnancy in a subject, in which knowledge of the investigational product 
is critical to the subject's management, the blind for that subject may be broken.  
Before breaking the blind of an individual subject’s treatment, the investigator should have 
determined that the information is necessary, i.e., that it will alter the subject’s immediate 
management. In many cases, particularly when the emergency is not investigational 
product-related, the problem may be properly managed by assuming that the subject is 
receiving active product without the need for unblinding.  
5.6 
Concomitant Treatments 
5.6.1 
Prohibited and/or Restricted Treatments 
Ketoconazole significantly increased saxagliptin exposure. Similar significant increases in 
plasma concentrations of saxagliptin are anticipated with other strong cytochrome P450 
3A4/5 (CYP3A4/5) inhibitors. The dose of ONGLYZA should be limited to 2.5 mg when 
coadministered with a strong CYP3A4/5 inhibitors (e.g., ketoconazole, atazanavir, 
clarithromycin, indinavir, itraconazole, nefazodone, nelfinavir, ritonavir, saquinavir, and 
telithromycin).  
Given that this study protocol includes only saxagliptin 5 mg dose the participation of any 
study subject requiring dose adjustment while on study medication will be discontinued 
(see section 4.2.3.). 
5.6.2 
Other Restrictions and Precautions 
Saxagliptin should not be used in patients with type 1 diabetes mellitus or for the treatment 
of diabetic ketoacidosis.   
5.7 
Treatment Compliance 
Subjects will be given study drug at each dispensing visit with enough study drug to 
last until the next visit.  The subjects will return all unused medication and 
compliance will be assessed.  Subjects will be in compliance if they have taken at 
least 75% of study drug.  
 
 


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6 
STUDY ASSESSMENTS AND PROCEDURES 
 
6.1 
STUDY SCHEMATIC 
 
6.2  TIME AND EVENTS SCHEDULE 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Time and Events Schedule for Protocol  
Procedure 
Screening 
Visit 
(Week -4) 
Baseline/ 
Randomization  
Visit 1 
(Week 0) 
During 
Treatment 
Visit 2 
(Week 6) 
End-of-
Treatment 
Visit 3 
(Week 12) 
 
Eligibility Assessments 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
Inclusion/Exclusion Criteria 
X 
 
 
 
 
Medical History 
X 
 
 
 
 
Safety Assessments 
 
 
 
 
 
Physical Examination 
X 
 
 
X 
 
Targeted Physical Examination 
 
X 
X 
X 
 


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Time and Events Schedule for Protocol  
Procedure 
Screening 
Visit 
(Week -4) 
Baseline/ 
Randomization  
Visit 1 
(Week 0) 
During 
Treatment 
Visit 2 
(Week 6) 
End-of-
Treatment 
Visit 3 
(Week 12) 
 
Vital Signs (BP, RR, Temp, HR) 
X 
X 
X 
X 
 
Assessment of Signs and 
Symptoms 
 
X 
X 
X 
 
Adverse Events Assessment 
X 
X 
X 
X 
 
Laboratory Tests (Biochemical) 
screening 
X 
X 
X 
 
Pregnancy Test (if applicable) 
X 
X 
X 
X 
 
Efficacy Assessments 
 
 
 
 
 
Peripheral blood draw for 
CD34+cell harvest 
 
X 
X 
X 
 
Pulse wave analysis  
Pulse wave velocity assessment 
 
X 
X 
X 
 
Hip / Waist Measurements 
 
X 
X 
X 
 
Tanita Scale 
 
X 
X 
X 
 
REE                                                    
Clinical Drug Supplies 
 
X 
 
X 
 
X 
 
 
Randomize 
 
X 
 
 
 
Dispense Study Treatment 
 
X 
X 
 
 
 
Screening visit= week-4; Visit 1= week 0, Visit 2= week 6 and Visit 3= week 12. 
+/- 3 day window for visits 1-3 


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Research Study Design and Methods: 
 
Patient Definition and Selection: Adults aged 40-70 years will be recruited mainly from 
the MFA research patient database provided by MFA Information Technology. The 
remaining patients will be recruited from the endocrinology clinic at GWU and referring 
primary care physicians. Suitable advertisements for the study patient enrollment will be 
posted. Patients will be included in the study if they have been diagnosed with type 2 
diabetes within the previous 8 years using criteria of the American Diabetes Association2, 
and are currently treated with no other hypoglycemic agents other than a stable dose (>3 
months) of metformin (≥1.0 to ≤2 grams daily). We plan to enroll subjects with HbA1C 
between 6% and 9% (both inclusive) and with BMI 25-34.9 kg/m2 (both inclusive). Post 
enrollment, patients will be randomized to saxagliptin 5 mg or placebo as detailed in Study 
Design for direct comparison between the two regimens.  
Before any study procedures are performed, subjects will have the details of the study 
described to them, and they will be given a written informed consent document to read. 
Then, if subjects consent to participate in the study, they will indicate that consent by 
signing and dating the informed consent document in the presence of study personnel. 
Recruitment: Subjects will be recruited from the local general physician patient 
population and General Endocrinology and Diabetes department at GWU, Medical Faculty 
Associates, where Dr. Sen is a clinician. All patients will receive counseling regarding the 
study aims and methods.  
 
Study Design Overview: We propose a 2-arm randomized, double-masked, placebo-
controlled, parallel group, longitudinal study of 12-weeks duration. (See study design 
below), recruited over 2.5 years.  The 12 week time interval has been previously shown 
adequate to observe changes in biochemistry, EPCs and, importantly, PWV changes17-19,36. 
Patients will be randomized to 2 groups: 
Control, (n=20), Metformin + Lifestyle + Placebo 
Treatment, (n=20) Metformin+ Lifestyle+ Saxagliptin 5mg 
This proposed study is based on patients with type 2 diabetes of less than 8 years duration, 
who are already on Metformin (≥1.0 to ≤2 gm/d, stable dose for at least 3 months) but still 
remain poorly controlled with HbA1C between 6% and 9% (both inclusive),  but not 
overtly out of control (> 9% of HbA1C). Our study design is similar to that of Fadini et al 


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20where Sitagliptin was compared to placebo with endothelial progenitor cells as a outcome 
measure in patients with type 2 diabetes over 4 weeks. Though our study is over 12 weeks 
we do not anticipate significant drop in HbA1C in between the two groups in this relatively 
short time period to act as a confounding factor on CD34+ cell number, function and gene 
expression. Most clinical studies looking at HbA1C reduction were of a much longer 
duration of 6 months or above and most DPP-4 inhibitor studies are with animal EPCs 
where comparison with human CD34+ population is difficult to make. In one study58 where 
saxagliptin was used for 12 weeks in drug-naïve type 2 diabetes patients there was placebo-
subtracted HbA1c reduction noted of 0.45-0.63%. It showed mean placebo-subtracted 
reductions in fasting serum glucose of 20 mg/dL. From our laboratory studies of in vitro 
human EPC studies we do not think glucose level change of 20 mg/dL is significant enough 
to account for changes in EPC number, function and gene expression. However we will 
take this possibility into account during statistical analysis of the results between the 
saxagliptin and placebo groups. 
Lifestyle Intervention for Type 2 diabetes patients2:  
To keep uniformity within the cohort regarding lifestyle during the length of the study 
duration, all patients at entry will ideally receive dietary advice from the research 
coordinator, a diabetes educator, a registered dietitian (as per American Diabetes 
Association clinical guidelines2), specifically geared towards type 2 diabetes patients. They 
will be advised to adhere to 150 minutes of weekly aerobic exercise (50-70% of maximal 
heart rate) as best as possible. Patient’s activity will be monitored using accelerometers 
and will be downloaded at regular intervals by the research coordinator.  
Subjects will be taking 5.0 mg of Onglyza (saxagliptin) or placebo. We choose to use the 
maximum approved dose of saxagliptin in order to discern a definite effect. 
Visits with outcome measurements will be scheduled for morning following overnight 10 
hours fast to reduce diurnal variability, and clinical parameters will be obtained at 
scheduled office visits as depicted in the study schema and will be measured at each 
outcome visit as follows: 
Week -4: Subjects will be screened and eligibility will be verified based on physical and 
laboratory tests. We will aim to recruit equal numbers of male and female patients will be 
recruited. All patients will be educated on the recommended lifestyle changes of 
maintaining a level of aerobic activity of 150 minutes/ week (non-supervised)and ideally 
receive dietary advice from the research coordinator, a diabetes educator, or a registered 


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dietitian (as per American Diabetes Association clinical guidelines2) specifically geared 
towards type 2 diabetes patients.  We plan to monitor activity using accelerometers. We 
will allow 4 weeks for the patients to reach a steady state while implementing life style 
(diet and exercise) changes. 
Week 0: At this stage patients will be randomized into the study. Subjects will have the 
first set of measurements. We will make sure that patients still meet the inclusion criteria. 
They will be started on medication or placebo and will be encouraged to continue 150 
minutes/week of moderate physical exercise and dietary management for the remainder of 
the study. 
Weeks 0, 6 and 12: Subjects will all have the same measurements and will continue on 
study medication or placebo. All primary and secondary measures as outlined before will 
be carried out. 
 Primary Measures: CD34+ cell number, function and gene expression.  
 Mononuclear cell population (day 0 of harvest) will be analyzed by FACS to obtain 
cell counts of different endothelial precursor lineage cells. Combinations of 
following cell markers will be used, such as CD34, KDR(VEGFR2), CD31, CD144 
(VE-Cadherin), CD133 ( a progenitor cell marker of indeterminate lineage) and 
CXCR4 (SDF1α receptor) 5,6,8,10,20,21,59 
For: 
 CD34/KDR+  
 CD34/ CXCR4+ 
 CXCR4 + CD31 
 KDR + CD34 
 Some of the MNC population will be put through a human CD34 magnetic bead 
column using Miltenyi Biotech Column to obtain CD34+ and CD34- cells. The 
CD34+ cells will be used for 
 Colony Forming Unit Assay (CFU Assay) from mononuclear cell population (prior 
to magnetic bead sorting) 
 Migration assay in response to SDF-1 (0, 10, 100 ng/mL) and if sufficient cells are 
available we will note migration in response to SDF1 (0, 10, 100 ng/mL)  
 Gene expression assays: Genes analyzed will be eNOS, PECAM-1, IGF-1, VEGF-
alpha, VEGFR2, SDF-1, CXCR4 (receptor for SDF1 ), anti-oxidants MnSOD, 
Catalase, Cu-Zn SOD, Glutathione Peroxidase, extracellular SOD, pro-
inflammatory markers such as IL6, TNF-alpha and Endothelin-1 genes and  
apoptosis cascade genes such as p53, p21, Bcl-2, GPX, Caspase-3 will be analyzed.   
 
Secondary Measures:  


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 Highly selective C-reactive protein (hs-CRP), IL-6, TNF-alpha, Leptin ,18, 28, and 
fasting lipid profile  
 Serum SOD activity and SDF1 alpha (ELISA), VEGF-A (ELISA) and GLP-1 (ELISA) 
levels will also be assessed14,20,24,39. 
 Assessment of insulin sensitivity using the HOMA-IR32, calculated from individual 
serum measures (fasting glucose (mg/dl)* insulin (µU/mL)/405) 37.  
 Adiposity will be measured using the Tanita Body Composition Analyzer scale, 
measured as percentage body fat, BMI, waist-hip ratio5,55  
 Fasting lipid profile will be checked as a marker of insulin resistance and lipo-toxicity 
at weeks 0, 6, and 12 5.  
 We also intend to acquire Pulse wave analysis and Vascular Flow using SphygmoCor 
CP system from ATCOR to measure central arterial pressure and arterial stiffness38,42. 
The secondary measures are indirect measures of endothelial inflammation in early 
type 2 diabetes patients. 30, 31, 32, 55, 56, 57.  
 Urine pregnancy testing for women of child bearing potential  
We will obtain a total of85mL of peripheral blood per visit. Of these 85 mL, 60 mL 
will be used to obtain CD34+ cells from MNC population and 25 mL for biochemistry 
and serum ELISA assays.  
 
Methodology of Cellular Measure of Endothelial Function:  
This is the strongest and most direct endothelial functional parameter, yet the least 
explored.  
Here we plan to measure stem cell precursors of endothelium in subjects with type 2 
diabetes as a bio-marker of endothelial health.  
Day 0: FACS Analysis for CD34/CD31/KDR/CD133/CD144+, CXCR4 cells (note 
percentage) 
Sort MNC for CD34+ cells for Migration, Tube Formation Assay (Functional Assay) 
And gene expression assays.  


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Functional Assay (Cellular): At Day 0, post cell sorting for CD34 + cells we will note 
functional assays of these cells.  
We plan to look at:  
 Colony Forming Unit Assay 
 Migration assay in response to SDF-1 and VEGF-A. 
Colony Forming Unit Assay: MNC’s (prior to CD34 magnetic bead sorting) will be plated 
in Human Fibronectin Coated 6 well (5 million) or 12 well plates (2 million) from BD 
Biosciences or Millipore. Number of CFU will be determined at Day 5, post plating. 
Migration property of the patient’s CD34+ cells in response to SDF-1 will be assessed 
immediately post cell sorting on day 0 using modified Boyden chamber. Briefly, CD34+ 
cells (1-2x10e5) will be placed in the upper compartment, as shown below. Concentrations 
tested will be SADF-1 at concentrations of 0, 10, 100 ng/ml and if sufficient cells remain, 
VEGF-A at concentrations of 0, 20, 50 ng/mL12,34 dissolved in basal endothelial culture 
media. Other than this protocol one can also use Migration Assay kit using 3micron pore 
size from Cell BioLabs Inc. (catalog # CBA-103). 
 
 
These tests will evaluate the functional (migration) property of CD34+ endothelial 
progenitor cells 60. 
 
 
 
 
 
Chemotactic factor, 
SDF-1α or VEGF-A 
 CD34+ve cells 


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The CD34+ cells obtained at day 0 from peripheral blood derived mononuclear cell 
population will be analyzed for mRNA expression of various endothelial function related 
genes,  
 The gene expression changes should indicate changes in paracrine properties of 
CD34+ cells pre and post saxagliptin therapy,  
 Genes analyzed will be eNOS, and PECAM-1, VEGF, VEGFR2, SDF-1alpha, 
CXCR4, IGF-1 36,42,43. Intra-cellular antioxidant genes such as MnSOD, Catalase 
and GPX will also be noted22,23,44,45. eNOS is a critical gene regulating EPC’s nitric 
oxide production capability22. PECAM-1 is platelet endothelial cell adhesion 
molecule and indicates inflammatory potential of endothelial lineage cells. VEGF 
and IGF-1 are growth factors produced by healthy EPCs. MnSOD and Catalase are 
anti-oxidants which may be important endothelial anti-inflammatory agents and 
may promote healing in hyperglycemic conditions 9,12,31,46. Pro-inflammatory 
markers such as IL6, TNF-alpha and Endothelin-1 genes and apoptosis cascade 
genes such as p53, p21, Bcl-2, will be analyzed in adition to other genes previously 
mentioned.   
5.8 
Study Materials 
Astra-Zeneca (AZ) will provide saxagliptin and matching placebo at no cost for this study.  
5.9 
Safety Assessments 
Study drug toxicities will be assessed continuously. Adverse events will be evaluated on a 
continuous basis while the patient is on study and until 30 days after the last dose of study 
drug. Patients should be followed until all treatment-related adverse events have recovered 
to baseline or are deemed irreversible by the principal investigator. 
6 
ADVERSE EVENT REPORTING 
6.1 
Adverse Events 
An Adverse Event (AE) is defined as any new untoward medical occurrence or worsening 
of a pre-existing medical condition in a patient or clinical investigation subject 
administered an investigational (medicinal) product and that does not necessarily have a 
causal relationship with this treatment. An AE can therefore be any unfavorable and 
unintended sign (including an abnormal laboratory finding, for example), symptom, or 


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disease temporally associated with the use of investigational product, whether or not 
considered related to the investigational product. 
The causal relationship to study drug is determined by a physician and should be used to 
assess all adverse events (AE). The casual relationship can be one of the following: 
Related: There is a reasonable causal relationship between study drug 
administration and the AE. 
Not related: There is not a reasonable causal relationship between study drug 
administration and the AE.  
The term "reasonable causal relationship" means there is evidence to suggest a causal 
relationship.  
Adverse events can be spontaneously reported or elicited during open-ended questioning, 
examination, or evaluation of a subject. (In order to prevent reporting bias, subjects should 
not be questioned regarding the specific occurrence of one or more AEs.) 
6.1.1 
Serious Adverse Events 
A Serious Adverse Event (SAE) is any untoward medical occurrence that at any dose:  
 results in death 
 is life-threatening (defined as 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 were more severe) 
 requires inpatient hospitalization or causes prolongation of existing hospitalization 
(see note below for exceptions) 
 results in persistent or significant disability/incapacity 
 is a congenital anomaly/birth defect 
 is an important medical event, defined as a medical event that may not be 
immediately life-threatening or result in death or hospitalization but, based on 
appropriate medical and scientific judgment, may jeopardize the subject or may 
require intervention (e.g., medical, surgical) to prevent one of the other serious 
outcomes listed above. Examples of such events include but are not limited to 
intensive treatment in an emergency department or at home for allergic 
bronchospasm; blood dyscrasias or convulsions that do not result in hospitalization. 
Potential drug induced liver injury (DILI) us also considered an important medical 
event (see Section 7.6 for the definition of potential DILI)  
Suspected transmission of an infectious agent (e.g., any organism, virus or infectious 
particle, pathogenic or non-pathogenic) via the study drug is an SAE. 


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Although pregnancy, overdose and cancer are not always serious by regulatory definition, 
these events must be handled as SAEs (See Section 7.5 for reporting pregnancies). 
NOTE: The following hospitalizations are not considered SAEs: 
 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 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). 
 
6.1.2 
Non-serious Adverse Events 
Non-serious adverse events are all adverse events that are not classified as SAEs. 
 
6.1.3 
Adverse Events of Special Interest 
In this study, the following adverse events are to be reported to AstraZeneca, regardless of 
whether these reports are classified as serious or unexpected: 
1. 
liver test abnormalities accompanied by jaundice or hyperbilirubinemia 
2. 
opportunistic infections associated with the use of saxagliptin 
3. 
pancreatitis 
4.  
anaphylaxis 
5. 
angioedema 
6.  
Steven-Johnson’s Syndrome 


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6.2 
Collection and Reporting 
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. 
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 must supply AstraZeneca and the IRB/IEC with any 
additional information requested, notably for reported deaths of subjects. 
6.2.1 
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. 
If applicable, SAEs will be collected that relate to any later protocol-specified procedure 
(e.g., a follow-up skin biopsy).  
The investigator will report any SAE occurring after these time periods that is believed to 
be related to study drug or protocol-specified procedure at 90 days and 180 days period 
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. 
All SAEs, whether related or unrelated to saxagliptin, and all pregnancies will be reported 
to BMS (by the investigator or designee) within 24 hours.  
All SAEs should be reported via confirmed facsimile (fax) transmission, or scanned and 
reported via electronic mail to:  
SAE Email Address: Worldwide.Safety@BMS.com 


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SAE Fax Number: 609-818-3804 
SAEs will be reported on MedWatch Form 3500A, which can be accessed at: 
http://www.accessdata.fda.gov/scripts/medwatch/. 
 
 
 
MedWatch SAE forms should be sent to the FDA at: 
MEDWATCH 
5600 Fishers Lane 
Rockville, MD 20852-9787 
Fax: 1-800-FDA-0178 (1-800-332-0178) 
http://www.accessdata.fda.gov/scripts/medwatch/ 
All SAEs should simultaneously be faxed or e-mailed to BMS at: 
Global Pharmacovigilance & Epidemiology 
Bristol-Myers Squibb Company 
Fax Number: 609-818-3804 
Email: Worldwide.safety@bms.com 
If an ongoing SAE changes in its intensity or relationship to study drug or if new 
information becomes available, a follow-up SAE report should be sent within 24 hours to 
the BMS using the same procedure used for transmitting the initial SAE report. All SAEs 
should be followed to resolution or stabilization. 
6.2.2 
Non-Serious Adverse Events (NSAEs) Collecting and Reporting 
The collection of non-serious adverse event (NSAE) information will begin at initiation of 
study drug. NSAE information will be collected from the start of a placebo lead-in period 
or other observational period intended to establish a baseline status for the subjects. NSAEs 
will be followed to resolution or stabilization, or reported as SAEs if they become serious. 
Follow-up as required for NSAEs that cause interruption or discontinuation of study drug, 
or those that are present at the end of study treatment as appropriate will be undertaken. 
All identified NSAEs will be documented appropriately. 


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Date: 12/21/2015 
6.3 
Laboratory Test Abnormalities 
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: 
 any laboratory test result that is clinically significant or meets the definition of an 
SAE 
 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. 
6.4 
Overdose 
An overdose is defined as the accidental or intentional administration of any dose of a 
product that is considered both excessive and medically important. All occurrences of 
overdose will be reported as an SAE. 
6.5 
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 during at least 6 half lifes after 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. 
The investigator or designee will immediately notify the BMS (or designee) Medical 
Monitor of this event and complete and forward a Pregnancy Surveillance Form to BMS 
(or designee) within 24 hours and in accordance with SAE reporting procedures described 
in Section 7.2.1.  


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Follow-up information regarding the course of the pregnancy, including perinatal and 
neonatal outcome and, where applicable, offspring information will be reported on the 
Pregnancy Surveillance Form. 
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. 
6.6 
Potential Drug-Induced Liver Injury (DILI) 
Wherever possible, timely confirmation of initial liver-related laboratory abnormalities 
should occur prior to the reporting of a potential DILI event. All occurrences of potential 
DILIs, meeting the defined criteria, must be reported as SAEs (see Section 7.2.1 for 
reporting details). 
Potential drug induced liver injury is defined as  
1. AT (ALT or AST) elevation > 3 times upper limit of normal (ULN) 
AND 
2. Total bilirubin > 2 times ULN, without initial findings of cholestasis (elevated 
serum alkaline phosphatase), 
AND 
3. No other immediately apparent possible causes of AT elevation and hyper-
bilirubinemia, including, but not limited to, viral hepatitis, pre-existing chronic or 
acute liver disease, or the administration of other drug(s) known to be hepatotoxic. 
 
6.7 
Other Safety Considerations 
Any significant worsening noted during interim or final physical examinations, 
electrocardiograms, x-rays, and any other potential safety assessments, whether or not 
these procedures are required by the protocol, should also be recorded as a nonserious or 
serious AE, as appropriate, and reported accordingly. 
7 
DATA MONITORING COMMITTEE 
Not Applicable. 


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8 
STATISTICAL CONSIDERATIONS 
Statistical Analysis:  
Sample Size Estimation:  
The total sample size requested, accounting for attrition over the 12-week period, is 21 
subjects per group or 42 subjects total. 
 
Sample size estimates were based on the effects of exercise on CD34+/KDR+ cells and 
VEGF, as described in the literature 47-50.  The effect of a single session, as well as 
extended training, on healthy subjects or those with existing cardiovascular conditions 
appears to increase the CD34+/KDR+ cells and VEGF.   
 
To compute sample size we used the approach suggested by Diggle, Liang, & Zeger 
(1994)51 which compares the rates of change in the two study groups over time.  This 
approach incorporates the number and interval of time points and the correlation among 
repeated measures.  For this study, we will employ one baseline and two follow-up 
measures at 6 and 12 weeks.  Further, we will assume a correlation 0.60 among repeated 
measures of the outcome. [We consider this a conservative estimate since Frison and 
Pocock (1992) 52 suggest a correlation of 0.65 as reasonable in the absence of an existing 
estimate. We also note that as this correlation increases, statistical power also increases.  
 
The results in the table below show the expected mean difference in study groups at the 
end of follow-up, as well as the average rate of change in the two groups at 80% power 
and 90% power.  To estimate the effect of saxagliptin on the CD34+/KDR+ cells, we 
expect that the effect would be at least 25% greater than the effect seen for exercise 
alone.  Using the results from Sandri et al. (2005)49 for the rate of change and the 
variability, the CD34+/KDR+ cells increased an average rate of about 4/wk with a 
standard deviation of about 15.  Thus, for a 25% increase in the rate of change for the 
CD34+/KDR+ cells due to saxagliptin, a sample size of 18 subjects per group would 
provide about 84% power, assuming measures taken at baseline and 2 equally-spaced 
time points over 12 weeks. At the conclusion of follow-up, we would expect study groups 
to differ by an average of 12 cells.  If the effect of saxagliptin is only 20%, a sample of 18 
would provide about 70% power, whereas a sample of 20 would provide about 73% 
power. 
 
For VEGF, the results from Sandri et al. (2005)49 were mixed with one study group 
showing a .6 pg/mL per week increase and another showing a 0.5 pg/mL per week 
decrease after 4 weeks of training.  If we assume that saxagliptin will increase VEGF 
consistently at 0.3 pg/mL per week difference in slopes compared with exercise alone, 
then assuming a standard deviation of 5 and correlation or 0.60 among repeated 
measures, a sample size of 18 per group would provide about 77% power to detect a 
difference in slopes of 0.3 pg/mL per week or a mean difference in the groups at the end 


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of the study of 3.6 pg/mL.  A sample size of 27 would provide about 90% power to detect 
the same effects. 
 
Thus, we feel that a sample size of at least 18 subjects per group with complete data 
would provide sufficient power for the study outcomes.  In order to insure that we will 
have 18 per group who complete the study, we will enroll 20 subjects per group in order 
to account for attrition over the 12-week period. 
 
 
 
 
Biochemical 
Measure 
Mean difference  
at end of 12 weeks 
Sample 
Size per 
group 
Power 
CD34+/KDR+ 
cells 
12 cells 
(25% increase;  
4/wk vs. 5/wk) 
18 
0.84 
22 
0.90 
VEGF, pg/mL 
3.6 pg/mL 
(50% increase;  
(0.6/wk vs. 0.9/wk) 
18 
0.77 
27 
0.90 
 
Data Analysis.   
 
The distributional assumptions of all measures will be examined. Means and standard 
deviations will be computed for continuous measures and proportions for categorical 
variables. Graphical representations of the mean group slopes and individual slopes will 
be generated and inspected.  Study groups will be compared to determine whether any 
imbalance between the groups on patient characteristics remain after randomization.  If 
imbalance is evident, by inspection, all models will be adjusted for the unbalanced 
covariates.  Change in outcome over time will be examined using a multilevel approach 
with linear mixed models (LMM) 53,54.  The longitudinal multilevel modeling approach 
will enable us to examine characteristics of within-person change, as well as between-
group differences that may influence change.  LMMs handle missing data more 
efficiently than traditional ANOVA designs. In the linear mixed models, the patient is 
considered a random effect and the outcome measured at specific time points is nested 
within patient.   In an initial model containing only the variable TIME, we will use a 
likelihood ratio test to determine whether a random effect for time should be modeled.  
The model below represents a random intercept for patient and a random coefficient for 
TIME. 
 
 
ij
ij
i
i
ij
ij
Time
u
u
Time
Y








1
0
1
0


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We will examine whether TIME may be modeled linearly by testing the added 
contribution of TIME (represented with two indicator variables) to a model with TIME as 
a single continuous variable.  Assuming time may be modeled as a continuous variable, 
subsequent analyses will add study group and other patient characteristics as covariates. 
We are particularly interested in whether there is a significant interaction between study 
group and time, adjusting for other patient level covariates.  A significant interaction 
would indicate that the slopes in outcome over time differ by study group.  If the global 
test of interaction is significant, subsequent significance testing of the pair wise 
comparisons of group slopes group will be adjusted for multiple comparisons.  The model 
also includes coefficients for the group-by-time interactions and random effects for 
patient and time. 
 
 
In the event that TIME is modeled using indicator variables, two indicator variables will 
be used to represent the three time points, with baseline serving as the reference category.  
The interaction between GROUP and TIME will be modeled in a similar fashion using 
two additional indicator variables.   The above multivariable model enables us to adjust 
for time-varying covariates, such as glucose.  Since glucose will be measured 
contemporaneously with the outcomes at each visit, however, causal interpretations of 
glucose and any outcome are suspect.  As such, we will consider glucose as a nuisance 
covariate, and consider any relationship between glucose and an outcome as merely 
associative.   
9 
STUDY MANAGEMENT 
9.1 
Compliance with the Protocol 
9.1.1 
Compliance with the Protocol and Protocol Revisions 
The study shall be conducted as described in this approved protocol. All revisions to the 
protocol must be discussed with, and be prepared by, BMS. The investigator should not 
implement any deviation or change to the protocol without prior review and documented 
approval/favorable opinion from the IRB/IEC of an amendment, except where necessary 
to eliminate an immediate hazard(s) to study subjects. 
If a deviation or change to a protocol is implemented to eliminate an immediate hazard(s) 
prior to obtaining IRB/IEC approval/favorable opinion, as soon as possible the deviation 
or change will be submitted to: 
 IRB/IEC for review and approval/favorable opinion 
 AstraZeneca Pharmaceuticals LP 
 Regulatory Authority(ies), if required by local regulations 
ij
ij
i
i
i
ij
i
ij
ij
Time
u
u
Group
Time
Group
Time
Y













1
0
3
2
1
0
)
(


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Documentation of approval signed by the chairperson or designee of the IRB(s)/IEC(s) will 
be sent to BMS. 
If an amendment substantially alters the study design or increases the potential risk to the 
subject: (1) the consent form will be revised and submitted to the IRB(s)/IEC(s) for review 
and approval/favorable opinion; (2) the revised form will be used to obtain consent from 
subjects currently enrolled in the study if they are affected by the amendment; and (3) the 
new form must be used to obtain consent from new subjects prior to enrollment. 
If the revision is an administrative letter, investigators must inform their IRB(s)/IEC(s). 
9.2 
Records Retention  
9.2.1 
Records Retention 
The investigator will retain all study records and source documents for the maximum 
period required by applicable regulations and guidelines, or institution procedures, or for 
the period specified by BMS, whichever is longer. 
If the investigator withdraws from the study (e.g., relocation, retirement), the records shall 
be transferred to a mutually agreed upon designee (e.g., another investigator, IRB). Notice 
of such transfer will be given in writing to BMS.  
9.2.2 
Study Drug Records  
Investigator will ensure that a current disposition record of investigational product (those 
supplied by the BMS) is maintained at each study site where study drug and non 
investigational product(s) is/are inventoried and dispensed. Records or logs will comply 
with applicable regulations and guidelines and will  include: 
 amount received and placed in storage area 
 amount currently in storage area 
 label ID number or batch number  
 amount dispensed to and returned by each subject, including unique subject 
identifiers 
 amount transferred to another area/site for dispensing or storage 
 non-study disposition (e.g., lost, wasted)  
 amount destroyed at study site, if applicable 
 amount returned to the AstraZeneca Pharmaceuticals LP 
 retain samples for bioavailability/bioequivalence, if applicable  


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 dates and initials of person responsible for Investigational Product (IP) 
dispensing/accountability, as per the Delegation of Authority Form. 


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10 
GLOSSARY OF TERMS  
Term 
Definition 
Adverse Reaction 
An adverse event that is considered by either the 
investigator or the sponsor to be related to the 
investigational product 
Expedited Safety Report 
Rapid notification to investigators of all SAEs that are 
suspected (related to the investigational product) and 
unexpected (i.e., not previously described in the 
Investigator Brochure), or that could be associated with 
the study procedures.  
SUSAR 
Suspected, Unexpected, Serious Adverse Reaction as 
termed by the European Clinical Trial Directive 
(2001/20/EC).  
Unexpected Adverse 
Reaction 
An adverse reaction, the nature or severity of which is 
not consistent with the applicable product information 
(e.g., Investigator Brochure for an unapproved 
investigational product) 
 


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11 
LIST OF ABBREVIATIONS  
A1C 
Glycosylated Hemoglobin 
AE 
Adverse Event 
BMS 
Bristol-Myers Squibb 
CYP3A4 
Cytochrome P450 3A4 
DPP-4 
Dipeptidyl peptidase 4 
ESR 
Expedited Safety Report 
FDA 
Food and Drug Administration 
FSH 
Follicle-Stimulating Hormone 
GCP 
Good Clinical Practice 
GIP 
Glucose dependent insulinotropic peptide 
HCG 
Human Chorionic Gonadotropin 
HRT 
Hormone Replacement Therapy 
IB 
Investigator Brochure 
ICH 
International Conference on Harmonisation 
IEC 
Independent Ethics Committee 
IND 
Investigational New Drug (Application) 
IRB 
Institutional Review Board 
IST 
Investigator-Sponsored Trial 
MWG 
Mean Weighted Glucose 
NSAE 
Non-Serious Adverse Event 
PPG 
Post-Prandial Glucose 
SAE 
Serious Adverse Event 
SU 
Sulfonylurea 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TDZ 
Thiazolidinedione 
WOCBP 
Women of Child-Bearing Potential 
 


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Saxagliptin-Sen ISR CV181-305 
Protocol Version: 5.3 
Date: 12/21/2015 
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