Role of Canagliflozin on CD34+ Cells in Patients With Type 2 Diabetes

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

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

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

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

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

Inclusion Criteria:

- Age 30-70 years

- Currently treated with any combination of the following anti-diabetic therapies:
metformin (1-2 grams), insulin, GLP-1 agonists, a DPP-IV inhibitor, or sulfonylureas

- Hemoglobin A1C (HbA1C) between 7.0% and 10.0%

- Body Mass Index (BMI) between 25 and 39.9 kg/m^2 (both inclusive)

Exclusion Criteria:

- Type 1 diabetes

- History of hyperosmolar nonketotic coma

- History of diabetic ketoacidosis in the last 3 months

- Abnormal CBC that is judged by physician to be unsafe to enroll or low hematocrit (<28
UNITS).

- History of pancreatitis

- History of diabetic ketoacidosis in the last 3 months

- History of cancer (except basal cell carcinoma and cancer that is cured or not active
or being treated in the past 5 years)

- Heart attack or stroke within 6 months of screening

- Clinically significant coronary and/or peripheral vascular disease that would be
unsafe to enroll in the study.

- Statin use started or dose change in the last 3 months

- CKD Stages 3,4 and 5

- Use of oral or injectable anti-diabetic medication other than any combination of the
following anti-diabetic therapies: metformin (1-2 grams), insulin, GLP-1 agonists, a
DPP-IV inhibitor, or sulfonylureas currently, or in the past 1 month.

- Use of consistent long-term steroid medication (oral, inhaled, injected) within the
last 3 months

- Uncontrolled inflammatory disease, or current chronic use of anti-inflammatory drugs
within the last 3 months. **This will be judged on a case by case basis by the PI**

- Implanted devices (e.g., pacemakers) that may interact with Body Composition scale

- Untreated Systolic Blood Pressure > 150 mmHg and diastolic Blood Pressure > 90 mmHg

- Active wounds or recent surgery within 3 months

- Untreated hyper/hypothyroidism

Physical and Laboratory Test Findings:

- Pre-existing liver disease and/or ALT and AST >2.5X's UNL

- Serum creatinine levels ≥2.0

- Estimated CrCl < 60 mL/min (measured by eGFR value)

- Triglycerides >450 mg/dL

Allergies and Adverse Drug Reactions:

- Subjects with a history of any serious hypersensitivity reaction to Cana or another
SGLT2 inhibitor.

Sex and Reproductive Status:

- Women in reproductive age group will be included in the study but encouraged to use
contraceptive method to avoid pregnancy within 16 weeks of study duration.

- Women who are pregnant or breast-feeding will be excluded.

Other Exclusion Criteria:

- Prisoners or subjects who are involuntarily incarcerated.

- Subjects who are compulsorily detained for treatment of either a psychiatric or
physical (e.g., infectious disease) illness.

- Patients who are active smokers

- Patients who are pregnant

- Nursing women

- Post-menopausal women who are on estrogen hormone replacement therapy will be
excluded.

- Patients on low dose oral contraceptives will be allowed to participate as these
formulations contain very low amounts of estrogens.

- 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.