2.1 Trial Design and Oversight
This is a phase 4 (post-marketing), two arm, single site, parallel group, double blind, placebo controlled randomized clinical trial comparing low dose Canagliflozin 100 mg tablets, taken orally, once daily, with matching placebo. The study was conducted in accordance with Good Clinical Practice guidelines set forth by the International Conference of harmonization and any local regulatory guidelines with the approval and oversight of the George Washington University Institutional Review Board. The trial was funded by Janssen Scientific Affairs, LLC and conducted by the Investigator-Sponsor Dr Sabyasachi Sen at the George Washington University.
Subjects were initially pre-screened to assess eligibility. Once determined preliminary eligibility, they were brought in for a screening visit to confirm eligibility via interview, medical record check and laboratory workup once the subject signed the informed consent. The subjects were then enrolled into one of two arms of the study: 100 mg Canagliflozin or matching placebo. 15 subjects were enrolled into the active group and 14 subjects were enrolled into the placebo group.
There were 3 study visits total, first at week 0, second at week 8 (mid-point) and third and last visit at week 16. All three of the visits had same assessments. The assessments that were done were: vital measurements, adverse event (AE) check and a peripheral blood draw. Approximately 80 ml of blood was drawn for CD34 + endothelial progenitor cell harvesting and routine blood work.
Other parameters tested were resting metabolic rate (RMR, energy expenditure), measurement of waist to hip ratio, urine sample collection, Tanita body composition scale, pulse wave analysis and pulse wave velocity to determine arterial stiffness. Subjects were advised to adhere to 150 minutes of weekly aerobic exercise and their activity levels were monitored using ACTi graph activity monitor.
A follow up phone call visit was done 30 days from the last in-person visit to assess for any residual adverse events (AE).
2.2 Participants
Subjects were included if they were between 30 to 70 years old inclusive, with a diagnosis of Type 2 Diabetes for 15 years or less. Glycated hemoglobin level (HbA1c) inclusions were between 7.0 to 10.0% Inclusive. Their baseline medications were stable dose of Insulin (either short acting or long acting) and/or Metformin (1–2 grams per day). A stable dose was considered to be at least the maximum labeled dose or dose not associated with unacceptable side effects. Patients with BMI between 25 and 39.9 kg/m2 were included, thereby excluding severe obesity. Only, patients with impaired renal function were included, with Chronic Kidney Disease stage 1 to 3, with lowest eGFR cut-off of 30 ml/min/1.73 (GFR, as calculated by MDRD formula). During the duration of the study alterations in baseline medications were done to keep HbA1C between 7–8% across all subjects.
Any patients with Type I diabetes, history of Diabetic ketoacidosis, low hematocrit (less than 28 units), history of recent pancreatitis or cancer, recent coronary or cerebrovascular event within 6 months, use of consistent steroid medications, untreated thyroid disease were excluded.
Additional Inclusion and Exclusion criteria can be found in Appendix 1.
2.3 Outcome Objectives
The primary objective is to ascertain if addition of Canagliflozin improves CD34 + cell number, (CD34 + number, %CD34 + of total Mononuclear Cell population) function (cell migration function in response to SDF1α) and gene expression, in Type 2 Diabetes Mellitus, which will be correlated to improvement in 24 hour urinary protein estimation and serum Creatinine Clearance.
The secondary objective is to correlate the cellular outcome measures with other measures of endothelial function such as Arterial Stiffness (measured by Pulse Wave Analysis [Augmentation Index] and Pulse Wave Velocity [m/s]), Serum Biochemistry (CMP, IL6, hsCRP, Leptin, Serum insulin, TNFα), Adiposity ( as % body fat), Resting Energy Expenditure (in kcal) and Glycemic control (through HbA1c).
Body Composition Measurement
Body composition was measuring using Tanita™ BF-350 Body Composition Scale and manually. Manual measurement included height, waist circumference, hip circumference. Tanita scale uses a bio-impedance electrical impulse to measure body fat percent, fat mass (kg), fat free mass (kg), percent body water, water mass (kg) alongside weight. It then calculates the BMI and estimated basal metabolic rate.
Basal Metabolic Rate Measurement
Resting Emergency Expenditure (REE) was measured using KORR REEVUE. Test was conducted with the subject sitting and well rested. Subject was instructed to keep a tight seal around the mouthpiece and use the nose clip to avoid breathing in from the nose. The test ran for about 10 mins. It calculated estimated REE, predicted REE, estimated TEE (Total Energy Expenditure), VO2 Max and estimated calorie intake per day.
Arterial Stiffness
This parameter was measured using AtCor SphygmoCor CP system. We obtained two outcomes such as: Pulse Wave Velocity and Pulse Wave Analysis. The patient was supine on the examination table, 3 leads were attached on right forearm, left forearm and left shin.
Pulse Wave Analysis (PWA) was measured on the left Radial Artery with the subject supine. At least three readings were taken with Operator Index ≥ 80. Measurement includes Augmentation Index (AI), Augmentation Index adjusted for Heart Rate of 75 (AI-75), Augmentation Pressure (AP), Aortic and Radial reading of systolic, diastolic, pulse pressure and mean pressure.
Pulse Wave Velocity (PWV) was measured with the subject supine. This measurement requires a distal and proximal artery. Distal was used as right femoral artery with proximal being the left carotid. Index and ring fingers were used to manually localize the pulse, sometimes an arterial Doppler was used to localize the femoral pulse on patient with challenging body habitus. Once a stable pulse waveform was observed, the probe position was kept stable for 20 more pulses before the reading was finalized. Three readings were taken with standard deviation of less than 10%. The result reported a velocity in m/s, alongside the standard deviation with error.
Biological Sample and Vital Collection
A venous blood sample was collected from the Antecubital fossa. About 80 ml of blood was collected. 60 ml for EPC analysis and 20 ml for standard of care blood works which included Basic Metabolic Panel, Lipid Panel, HbA1c, hsCRP, IL6, Adiponectin and Insulin. ELISA was performed to analyze serum GLP1 and SDF1α using ELISA Immunoassay kit (Raybiotech, Norcross, GA) for GLP1 and Sandwich ELISA (EHCXCL12A, Thermo Scientific) for SDF1α. Urine sample was collected for urine Microalbumin and Creatinine ratio. Vitals were gathered on the left arm, Systolic Pressure, Diastolic Pressure and heart Rate, along with sublingual temperature. Serum NAD/NADH were measured by using NAD/NADH assay kit from Abcam, MA, USA (Catalog No. ab65348) and Ketone bodies were from serum were measured by using Ketone Body Assay Kit from Millipore Sigma, MO, USA (Catalog No. MAK134).
ACTi graph Activity Monitor
Subjects level of activity was measured using Actigraph wGT3x-BT activity monitors. Subjects were advised on diet and exercise instructed to wear the meter during all waking hours and was advised to adhere to 150 min of moderate intensity aerobic exercise per week. Actigraph served as a measure of this exercise compliance, and to verify for exercise as a confounding variable.
Polyethylene Glycol (PEG) enrichment of Extracellular vesicles
The cells debris and large apoptotic bodies were removed from the urine samples by centrifugation at 500 g for 5 minutes followed by 3000 g for 30 minutes at 4 0 C. Transfer supernatant into ultracentrifugation tubes and centrifuged at 100,000 g at 4 0 C for 75 min (Optimal XPN-100 centrifuge, Beckmann Coulter Inc, US). After ultracentrifugation the pellet was dissolved in RIPA buffer with protease inhibitor cocktail and stored the sample at -800 C for further analysis.
Western blotting: Extracellular vesicle extracts were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min. The membrane was washed once with TBST and incubated with antibodies against CD9 (1:1000), CD81 (1:1000), CD63 (1:1000), HSP70 (1:1000), anti-podocalyxin (PODXL, 1:1000), anti- wilms tumor protein (1:1000) and anti-Nephrin antibody (1:1000) at 4 °C for 12 h. Membranes were washed three times for 10 min and incubated with a 1:20000 dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody for 90 min at room temperature. Blots were washed with TBST three times and developed with Pierce ECL kit (ThemoFisher Scientific, USA).
Cellular and Clinical Assessments
CD34+ Endothelial Progenitor Cell Analysis
Peripheral blood samples (approximately 60 ml) were drawn from patients and phosphate buffered saline (1:1) was added. Identification and quantification of circulating cell phenotypes was performed on fresh blood samples, within 3 hours after collection, using flow cytometry. Briefly, mononuclear cells (MNCs) were then isolated from whole blood using a Ficoll density centrifuge method. MNCs were counted and aliquot was used for CFU-Hill colony formation assay following the manufacturer’s instruction (Stem Cell Technologies, Vancouver, BC, Canada). Colony forming unit was counted at day14. A fraction of the MNC were stained with fluorescein isothiocyanate (FITC)-conjugated antihuman CD34, Allophycocyanin (APC) conjugated antihuman CD184 (CXCR4) and FITC conjugated antihumanCD31 antibodies (Miltenyi Biotec GmbH, Bergisch-Gladback, Germany) in order to analyze specific progenitor cell surface marker (CD34) and mature endothelial cell surface markers (CD31) or receptor for SDF1a ligand, CXCR4) by flow cytometry. After gating mononuclear cells in the side scatter (SSC)-A vs forward scatter (FSC)-A plot, CD34/CD184 single- and double-positive cells were identified. Cells were acquired on a fluorescence-activated cell sorter (FACS) Canto instrument (Becton Dickinson) and scored with the Flo-Jo software.
To isolate EPCs (CD34+), MNCs were magnetically sorted through a column after cells were stained with CD34+microbeads antibody (Miltenyi Biotec GmbH, Bergisch Gladback, Germany). An aliquot of CD34+ cells were then stained with trypan blue and counted using an Auto Cellometer Mini (Nexcelom Bioscience, Lawrence, MA) to assess viability.
CD34+ gene expression analysis was performed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) as previously described (33). CD34 +ve cell total mRNA was extracted and purified using the RNeasy Minikit (Qiagen, Germany). mRNA was then converted into cDNA by using the high capacity cDNA reverse transcriptase kit (Thermo Fisher Scientific, MA) Possible gene expression changes promoted by Canagliflozin was assessed by a CFX96 real-time PCR systems (Bio-Rad, CA.) using Taqman Universal masters Mix II (Thermo Fisher Scientific, MA) and inventoried probes. The gene expression analysis included antioxidants, apoptosis, endothelial functions, chemotaxis, inflammation and endothelial lineage cell surface markers. The expression of each individual gene was normalized to either housekeeping 18S or GAPDH and calculated using C-ddct method considering the difference in cycle threshold between visit 2 and 3 and baseline (Visit 1). mRNA gene expression of CD34- cell population (from MNC population) was also analyzed along with CD34+ cells.
The migratory capacity of CD34+ was evaluated using the CytoSelect 24-well Cell Migration Assay kit (Cell Biolads, Inc., San Diego, CA). Cells were suspended in Serum free media and seeded at 100,000 cells per insert. Migration of the cells through a 3 um polycarbonate membrane to the wells containing a serum-free media (control) and chemoattractant SDF-1α (10 or 100 ng/mL) was assessed after cells were kept overnight in incubator. Migratory cells were dissociated from the membrane and subsequently lysed and quantified by fluorescence (480 nm/530 nm) using CyQuant GR dye (Cells Biolabs, Inc, San Diego, CA). The fluorescence ratios between cells exposed to the chemotactic factor and cells exposed to chemoattractant-free media (control) along the visits were used to analyze the migratory capacity of the cells.
As is the case with diabetic patients, number of isolated CD34+ cells is usually not as high as anticipated due to established endothelial damage and because the progenitor cells are very susceptible to apoptotic death in hyperglycemia. Hence in order to understand some of the effect of protein upregulation we have done the analysis on all remaining CD34- cells as they are the remaining 99% of Polymorphonuclear cells. Idea is that any gene expression upregulation on CD34+ cells should also be prevalent in CD34- cells and should by and large reflect the mRNA analysis of unsorted MNC population.
2.5 Statistical Analysis
Statistical Analysis
Continuous variable distributions were examined for skewness or outliers using histograms. When these were present, we log-transformed the variables. Outliers > 5sd from the mean were capped at 5sd from the mean. We used 2-tailed between-groups t-tests to examine differences between treatment groups at baseline on continuous variables, and either chi-square or the Fishers Exact test for categorical variables. To examine differences between treatment groups across all time points, as well as time effects, and whether the slope of change over time differed between treatments, we used random effects mixed model regression, examining the main effects of treatment (Canagliflozin vs placebo), and time (v1, v2, v3), and the treatment by time interaction. This method allows us to use all non-missing subject data and adjusts for within-subject auto-correlation. For variables with significant effects in the mixed models, we examined the means graphically. SAS (version 9.4, Cary, NC) was used for data analysis with p<.05 considered significant.
Since subjects were randomized to treatment, chance of baseline subject characterizations acting as confounders are minimized. Therefore, randomized control trials do not usually adjust for baseline differences. However, in small studies, imbalances may exist as a result of random group assignment, and may thus function as mediators of an association between treatment and outcome. In this situation, one would need to adjust for the mediators in order to obtain the direct effect of treatment on outcome, which is the effect of interest.