Effect of Saxagliptin and Dapagliflozin on Endothelial Progenitor Cell in Patients With Type 2 Diabetes Mellitus

The Investigator hypothesize that Dapagliflozin will improve EPC number and function AND Saxagliptin in addition to Dapagliflozin (additive effect) may improve EPC number and function even more than Dapa alone, compared to placebo.

The Investigator propose a 3-arm randomized, parallel group, longitudinal study of 16-week intervention duration. Participants will be randomized to 3 groups:

Group A: Dapa (10 mg) + Saxa Placebo, Enroll n=15, retain n=12 Group B: Dapa (10 mg) + Saxa (5 mg), Enroll n=15, retain n=12 Group C: Dapa Placebo + Saxa Placebo, Enroll n=15, retain n=12

The Investigator hypothesize that Dapagliflozin will improve EPC number and function AND Saxagliptin in addition to Dapagliflozin may have an additive effect to improve EPC number and function even more than Dapa alone, compared to placebo.

In this proposal the investigator plan to conduct a placebo matched study with type 2 diabetes subjects on any doses of metformin or Insulin or a combination of both and has no history of DPP4 ( Dipeptidyl Peptidase-4) DPP4 inhibitor, incretin mimetic or SGLT2 inhibitor intake history. Participants will have known macrovascular complications (such as Cardiovascular Disease (CVD), Cerebrovascular Accident (CVA), and Peripheral Vascular Disease (PVD).

3 STUDY OBJECTIVES

PRIMARY OBJECTIVE:

CELLULAR BIOMARKER OF ENDOTHELIUM

The primary objective is to ascertain if 16 weeks of Dapa or Dapa+Saxa Combo therapy will improve :

CD34+ cell number, CD34+ migratory function and CD34+ gene expression in type 2 diabetes with CVD.

SECONDARY OBJECTIVE:

ARTERIAL STIFFNESS AND RENAL FUNCTION, NON-CELLULAR MARKERS OF ENDOTHELIUM To determine whether use of Dapa or Dapa+Saxa Combo alters markers of endothelial function such as: arterial stiffness measures (via tonometry), biochemical measures derived from plasma, pertaining to endothelial function (hs-CRP, IL-6, TNF-alpha), renal function such as proteinuria (microalbumin/creatinine ratio) and urine exosome study to determine podocyte health. The secondary measures are indirect measures of endothelial inflammation in early type 2 diabetes patients.

Effect on Arterial Stiffness:

I. Pulse Wave Analysis and Vascular Flow will be assessed using SphygmoCor CP system from ATCOR as a measure of central arterial pressure and arterial stiffness.

II. Vessel health will be assessed by degree of arterial stiffness, using arterial tonometry.

III. The central and the aortic pressure is assessed by pulse wave analysis (PWA) and pulse wave velocity (PWV).

Effect on Blood Biochemistry:

The Investigator believes cell based biomarkers are superior to traditional serum and plasma biomarkers and the outcome report will be stronger if one can show positive correlation between the two outcome measures. The Investigator therefore will be looking at:

I. Inflammation, apoptosis and anti-oxidant protein levels: Highly selective C-reactive protein (hs-CRP), IL-6, TNF-alpha.

II. Plasma SDF1 alpha (ELISA) and GLP-1 and Ghrelin (ELISA) will be estimated to assess endothelial health and factors that may influence CD34+ cell chemotaxis III. Podocyte health via urine exosome analysis. IV. The glomerular filtration rate (GFR) will be estimated by MDRD equation.

a. GFR = 141 X min (Scr/κ,1)α X max(Scr/κ,1)-1.209 X 0.993Age X 1.018 \[if female\] X 1.159 \[if African American\]; where Scr is serum creatinine (mg/dL), κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min indicates the minimum of Scr/κ or 1, and max indicates the maximum of Scr/κ or 1.32

TERTIARY OBJECTIVE:

METABOLISM MARKERS The tertiary objective is to determine whether use of Dapa or Dapa+Saxa Combo alters body composition, fasting lipid profile, and levels of insulin, glucose, and appetite controlling hormones.

Effect on Blood Biochemistry:

The Investigator believes cell based biomarkers are superior to traditional serum and plasma biomarkers and the outcome report will be stronger if one can show positive co-relation between the two outcome measures.

I. Fasting glucose, and insulin. a. Glycemic control will be evaluated by measuring fasting blood glucose, insulin levels and HbA1c. Fasting blood glucose, insulin and lipid profile will be used to assess insulin resistance.28,31 II. Lipid profile III. Appetite controlling hormones via LabCorp: Leptin, Adiponectin IV. Appetite controlling hormones, via ELISA: GLP1, Ghrelin

Effect of Dapa and Dapa+Saxa Combo on Body Habitus (Determination of body composition and visceral fat) The Investigator plans to study cardio-metabolic effect of Dapa and Dapa+Saxa Combo.

I. Using body composition scale:

1. Height and weight will be measured and the body mass index (BMI=kgm2) used as an indicator of relative weight.
2. The body composition scale calculates body fat%, total body water%, fat free mass, etc., in addition to BMI.

   The secondary outcome markers (arterial stiffness and renal outcome measures) and tertiary outcome markers (serum biochemistry) are crucial in order to corroborate the cellular findings with currently accepted clinical efficacy outcome measures such as arterial stiffness and serum biochemistry. This design is similar to our recently published manuscript on Saxagliptin and cellular outcome measures.

   4 INVESTIGATIONAL PLAN

   STUDY DESIGN AND DURATION

   +/- 6 day window for visits

   \*Assessed at week 0, 8 and 16: Primary, Secondary \& Tertiary Outcomes.

   Week 20: A telephone call to subjects will be made 4 weeks after last dose of study medication to determine if there have been any adverse events.

Eligibility Criteria:
Inclusion Criteria:

1. Able to understand the study, and provide a signed \& dated informed consent.
2. Diagnosis of Type 2 diabetes mellitus using criteria of the American Diabetes Association.
3. 30-70 years old.
4. HbA1C 7 to 10%, both inclusive
5. BMI of 25 - 39.9 kg/m2 both inclusive.
6. Taking a stable dose (for 12 weeks) of Metformin (any dosage) and/or Insulin (any dosage) for the treatment of T2DM
7. Patients with current Cardiovascular Disease (CVD) in tye 2 diabetes patients, defined by ≥ 1 of the following:

   1. MI \>2 months prior
   2. Multivessel CAD
   3. Angina (intermittent or chronic)
   4. Single vessel CAD with positive stress test or UA hospitalization in prior year
   5. UA \>2 months prior and evidence of CAD
   6. Stroke \>2 months prior
   7. Occlusive PAD
   8. Proteinuria of more than 30mg/dl

Exclusion Criteria:

1. Planned CV surgery or angioplasty in 1 month
2. Prior surgery with chronic malabsorption (eg, bariatric) in prior 1 year
3. Diagnosis of Type 1 diabetes mellitus
4. History of GAD antibody positive status
5. Uncontrolled Inflammatory Disease/Inflammatory drug use. \*\*Evaluated by PI on case-by-case basis\*\*
6. Recent history of diabetic keto-acidosis in the past 3months, or recurrent history of diabetic ketoacidosis (≥ 3 times)
7. Active bladder cancer
8. Active wounds (e.g. Diabetic ulcers) or recent surgery within 1 month
9. Untreated hyper/hypothyroidism
10. Women of child bearing potential who are not willing to use a contraceptive method to avoid pregnancy for the 16 weeks of study duration
11. Women who are pregnant or breastfeeding
12. Implanted devices (eg. Pacemaker) that may interact with Tanita scale
13. Any other clinical condition that would jeopardize patients safety while participating in this clinical trial Concomitant Medications
14. Taking any other oral anti-diabetic agent other than Metformin and/or Insulin for their treatment of T2DM
15. Beginning statin medications in the past 1 month or change in statin dose in the past 1 month
16. Use of consistent long-term steroid medication (oral, inhaled, injected) within the last 1 month
17. Treatment with a strong cytochrome P450 3A4 (CYP34A) or P-gp inducer (ie. Rifampin)
18. Subjects with a history of any serious hypersensitivity reaction to Dapagliflozin / Saxagliptin or another SGLT-2 inhibitor/ DPP4 inhibitor Laboratory Findings
19. Uncontrolled hyperglycemia, defined as a fasting glucose \>240 mg/dL (\>13.3 mmol/L) at screening.
20. Liver disease with ALT, AST or ALP x3 ULN
21. eGFR \< 60 mL/min/1.73 m2 by MDRD equation in the past 3 months
22. Clinically significant RBC disorders such as hemoglobinopathies
23. Serum creatinine levels ≥1.8 mg/dL with estimated eGFP \< 60 mL/min
24. Triglycerides \> 450 mg/dL
25. Baseline Hematuria (judged by a urinalysis dipstick at screening) Social History
26. Active smokers
27. Chronic or persistent alcohol or drug abuse
28. Prisoners or subjects who are involuntarily incarcerated
29. Subjects who are compulsorily detained for treatment of either a psychiatric or physical (eg. infectious disease) illness
30. Participation in another trial with an investigational drug within 30 days prior to informed consent