Assessment of dapagliflozin effect on diabetic endothelial dysfunction of brachial artery (ADDENDA-BHS2 trial): rationale, design, and baseline characteristics of a randomized controlled trial

The Assessment of Dapagliflozin effect on Diabetic Endothelial Dysfunction of brachial Artery-Brazilian Heart Study 2 (ADDENDA-BHS2) trial (NCT 02919345) is a single-center, randomized, open, active-controlled, phase-4 trial. All volunteers signed an informed consent form before entering. Patients were recruited through advertisements in newspapers, radio and television. Clinical and laboratory measurements were performed at the Clinical Research Center (CRC) and Atherosclerosis and Vascular Biology Laboratory (AtheroLab) at UNICAMP. The description of the study follows the recommendations of the CONSORT statement.

Inclusion criteria were: (i) high cardiovascular risk, defined by stable coronary artery disease (CAD) or subclinical carotid atherosclerotic disease; (ii) T2DM taking up to two oral hypoglycemic agents; (iii) HbA1c between 7 and 9% at the time of randomization; and (iv) age between 40 and 70 years old. CAD was defined as previous myocardial infarction (MI) at least 6 months before inclusion or angiography showing > 70% stenosis in at least one coronary artery. Subclinical carotid atherosclerotic disease was defined by standard guidelines and diagnosed by the presence of [1] carotid plaque; [2] carotid Intima-Media Thickness (cIMT) ≥ 1 mm; or [3] or cIMT values > p75th for age, gender and race according to the distribution in the Brazilian population [11].

Exclusion criteria were: (i) contraindications to metformin use (estimated glomerular filtration rate (eGFR) calculated by CDK-EPI < 45 ml/min, AST or ALT > 3× upper reference limit); (ii) systolic BP (SBP) ≥ 140 or diastolic BP (DBP) ≥ 90 mmHg after 16 weeks of anti-hypertensive medication adjustment during run-in period; (iii) acute coronary syndrome, stroke or coronary artery revascularization within 6 months prior to enrolment; (iv) plasma triglycerides > 500 mg/dl; (v) polyuria, polydipsia, weight loss, or others clinical signs of volume depletion; (vi) insulin use; (vii) pregnancy or women during reproductive age; and (viii) refusal to participate or sign the Informed Consent Form. The informed consent was obtained by the principal investigator.

After assessing eligibility criteria by preliminary telephone interview, the volunteers underwent detailed clinical evaluation, anthropometry, carotid ultrasound, echocardiography, ophthalmologic evaluation and laboratory evaluation. Patients who fulfilled all the selection criteria above were submitted to a run-in phase to adjust the medications seeking at a HbA1c between 7 and 9% by the addition of extended-release metformin ≥ 1.5 g/day (Glifage XR Merck S.A., Brazil), and BP ≤ 130 × 80 mmHg, with losartan 25 to 100 mg/day (Aradois, Biolab-Sanus, Brazil) and the addition of hydrochlorothiazide 12.5 mg/day (Clorana, Sanofi, Brazil) when necessary. If after 16 weeks HbA1c values are < 7% or > 9% or BP is > 130 × 80 mmHg, patients will be discharged from the study. The Fig. 1 depicts the study protocol.

At each visit, the study participants received a 30-day supply of their medications. On delivery of the medicines, patients were instructed to store the used blisters and bring them on the next visit. The number and lot of the pills were recorded on the Clinical Report Form (CRF), and the adherence was calculated based on the percentage of the medication that was taken. Drug adherence was stimulated by telephone calls conducted by a team member every fortnight. In this same telephone contact, patients were questioned about the manifestation of adverse effects and, if they had, advised to come to a medical visit for examination. These phone calls ensured the complete follow-up of enrolled patients. Characteristics and measures taken for adverse events were recorded in the CRF. Serious adverse events, whether or not considered causally related to the investigational therapy, were reported to the Institutional Ethics in Research Committee and AstraZeneca at the same time. As part of the protocol, patients with adverse events should contact the principal investigator and come to the hospital for evaluation shortly after reporting the event.

Data were collected and managed during each visit at CRC using Research Electronic Data Capture (REDCap) tools hosted at Unicamp [12]. Clinical, blood pressure, electrocardiographic, ophthalmologic, anthropometric evaluations were obtained within a week before randomization and after experimental intervention. Echocardiography, laboratory and brachial artery evaluations were obtained on the day of randomization and the day of the 12-week visit. All research data collected is owned by the BHS group. The Principal Investigator of this project, Professor Sposito, takes responsibility for the collection and management of the research data. Study coordinator who is overseen by the Principal Investigator has provided quality assessment in a day-to-day basis. Over the course of the project, data have been collected and stored into the database center of the University following the best practices and standards and in accordance with REDCap rules. Digital video data files generated in the FMD experiments have been processed and stored in AVI format and the same database center. The principal investigators on the project and the BHS group will hold the intellectual property rights for the research data.

After 12-h fasting period blood samples were obtained, centrifuged at 3500 rpm and thereupon measured for urea, creatinine, sodium, potassium, calcium, high-sensitive C-reactive protein (CRP), AST, ALT, glycemia, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) (Cobas c702 Roche, Germany), insulin, thyroid stimulating hormone (TSH) (Cobas e602 Roche, Germany), blood count (Sysmex XN-L-Series, US). Low-density lipoprotein cholesterol (LDL-C) was calculated by a formula [13]. Urinalysis and urinary albumin/creatinine ratio (Dimension RXL MAX, Siemens, Brazil) were performed on the same day. Glycated hemoglobin (HbA1c) was measured by high-performance liquid chromatography (HPLC) (D-100, Bio-rad, Brazil). The Homeostasis Model Assessment version 2 (HOMA2) was used to estimate insulin sensitivity (HOMA2S) using fasting plasma insulin and glucose and calculator version 2.2.

At the randomization and the 12-week visit, blood samples were also obtained, centrifuged and frozen at liquid nitrogen in order to measure: plasma leptin, adiponectin (Invitrogen™ eBioscience™ ProcartaPlex Human leptin and adiponectin simplex kit, USA), Interleukin 6 (IL-6), Interleukin 2 (IL-2), tumour necrosis fact alpha (TNF-alpha), Vascular Cell Adhesion Molecule 1 (VCAM-1), Intercellular Adhesion Molecule 1 (ICAM-1) (Invitrogen™ eBioscience™ ProcartaPlex Human VCAM-1, ICAM-1, IL-6, IL-2, TNF-alpha Simplex kit, USA), Endothelin 1 (ET-1) (ET-1 Human ELISA Kit Thermo Scientific, USA), isoprostane (8-Isoprostane ELISA Kit, Cayman Chemical Company, USA).

Body composition was assessed by measures of weight, height, waist circumference, BMI and dual-energy X-ray absorptiometry (DXA) equipment (Lunar iDXA, GE Healthcare, USA). Briefly, a full-body scanner was performed with information on the body composition and bone mineral density of the femoral neck, Ward triangle, trochanter, and femoral body. The android fat mass was measured between the ribs and the pelvis, and the mass of gynoid fat was measured between the hips and the upper thighs.

Two clinic BP readings were taken at 60-s intervals after 3 min of rest using Omron HEM-705CP (Omron Healthcare, Japan) device, and their mean was defined as the office BP. The participant was then asked to stand up and an orthostatic reading was obtained after 3 min to assess for postural hypotension. Ambulatory blood pressure monitoring (ABPM) was measured as described elsewhere within a week before and after the 12-week experimental treatments (SpaceLabs, model 90207-8Q, USA). Electrocardiogram was recorded at baseline and after follow-up (Micromed Surface Digital Electrocardiogram, Brazil).

The best corrected visual acuity (BCVA) was tested, following the Early Treatment Diabetic test Retinopathy Study (ETDRS) in the distance evaluation, standardized to 4 m patient, and Jaeger’s table at close range, at a distance of 30 cm [14]. Biomicroscopy was performed on the anterior segment evaluation and, after maximum pupillary dilation (Mydryacil1% 2× e 1× Phelinefrine eye drops), patients underwent evaluation of the lens and retina. Lens opacification was classified based on Locs III [15]. Retina was appraised through posterior pole retinography by VISUCAM^® device (NM/FA Carl Zeiss, USA) and indirect ophthalmoscopy, following the International Classification of Diabetic Retinopathy and diabetic macular edema classification, proposed by Wilkison et al. [16].

Finally, patients underwent Optical Coherence Tomography-Spectral Domain (SD-OCT) macular examination, with the SPECTRALIS^® SD-OCT (Heidelberg Engineering, Inc., USA), according to International Council of Ophthalmology guideline classification [17]. The retinal layers were analyzed in the macular region, the thickness retinal region in the foveal region and the maximum thickness of the choroid in the subfoveal area was calculated, through the Enhanced Deep Imaging (EDI) method.

Patients were screened for clinical or subclinical atherosclerosis by carotid arteries Doppler ultrasound with a 2.4–10 MHz linear array transducer (Vivid-S60, GE Healthcare, Milwaukee, Wisconsin). The patients were evaluated in a supine position with the head slightly extended and turned away from the side of the scanned carotid. After a transversal scan of the arteries, bilateral common, internal and external carotid arteries, as well as vertebral arteries, were imaged longitudinally, and ideally perpendicular to the transducer, by either anterior, lateral or posterior windows, whichever provided the highest image definition of the intima, media and adventitia layers. Images were recorded at frame rates of at least 20 per second and stored in a dedicated workstation for off-line assessments. Intima-media complex thickness (IMT) was assessed at end diastole 20 mm from the carotid bulb and at least 10 mm from the bifurcation. Focus was set to provide the best near and far wall resolution and at a depth of no more than 3–4 cm.

Atherosclerosis occurrence was defined as an IMT above the 75th percentile, IMT ≥ 1 mm, or the presence of plaque in the common carotid artery [11] as preconized by local guidelines. A plaque was defined as a localized protrusion of the arterial wall of more than 1.5 mm into the lumen or thickening of 50% of the arterial compared with an adjacent portion of the vessel wall. Apart from IMT evaluation, manual measurements of the intima, media and adventitia layers, separately, were performed after finding an optimized and zoomed still-frame image in order to individualize and estimate the thickness of each of these layers. These measures were made at the same spot defined for IMTs assessments. Doppler flow velocities of each artery and the respective resistivity index were calculated using the formula [(Peak systolic velocity-end diastolic velocity)/Peak systolic velocity].

Brachial arteries were measured using a high-resolution ultrasound (Vivid S6, GE Medical System, Milwaukee, WI, USA) according to previously published guidelines [18]. Briefly, the procedure takes place after over-night fasting and withdrawal of any vasoactive medications for the previous 24 h [19]. After 10 min of quiet resting in a supine position, and in a room with a controlled temperature around 22–25 °C, the brachial artery was located above the antecubital fossa, and a longitudinal image of 6 to 8 cm of the artery was considered as the baseline scan. The probe was then fastened to a probe holder (Quipu, Pizza, Italy) and sustained in an upright position. Continuous recording of real-time 2D image and simultaneous Doppler flow of the brachial artery was started by using a video capture device (Epiphan’s DVI2USB 3.0™, Epiphan Video, Ottawa, Canada) connecting the ultrasound to a dedicated computer. An appropriately sized blood pressure cuff was placed around the forearm and inflated up to 50 mmHg above the systolic blood pressure for 5 min and then deflated. The scan recording was obtained for 11 min (baseline: 1 min, ischemia: 5 min, and hyperemia and dilation: 5 min). Automatic edge-detecting software (LabVIEW 6.02, National Instruments) was used for online and off-line assessment of artery dilation and flow changes. The percentage change in diameter and flow for FMD was calculated in comparison with baseline scans. Other secondary parameters were also assessed offline by means of the same software, namely: the velocity–time integral of the antegrade and retrograde brachial flow, as determined by Doppler flow evaluation; the area under the curve (AUC) of the diameter versus time scatterplot; time to maximum vasodilation; and the derived parameters of blood flow (the product of cross-sectional area and Doppler velocity) and shear rate (4 times velocity divided by arterial diameter). These are validated methods used for covariate adjustment and microvascular endothelial function analysis. Two FMD assessments were performed in two different time-points—at the time of randomization and after 12 weeks of intervention (or control)—totalizing 4 FMDs for each patient. Fifteen minutes after the first FMD, reperfusion injury of the brachial artery was performed by cuff-induced ischemia (50 mmHg above the systolic blood pressure) for 15 min, followed by 15 min of reperfusion. At that moment, the second FMD analysis was repeated and the same parameters described above assessed.

Nitrite and nitrate (NOx) levels in plasma were measured just before FMD and 1 and 5 min after brachial artery cuff deflation. This collection was repeated during the second FMD after ischemia and reperfusion. Samples were collected in a tube containing heparin, centrifuged at 3500 rpm in a refrigerated centrifuge and stored in liquid nitrogen in up to 3 min for further analysis of NOx by a NO chemiluminescence analyzer (Model NOA, Sievers Instruments, Boulder, CO, USA).

Patients underwent cardiac ultrasound analysis with a 1.5–4.5 MHz phased array transducer (Vivid-S60, GE Healthcare, Milwaukee, Wisconsin) on the day of the randomization and again at the end of the intervention (or control) periods. Still images and cine-loops were acquired, as necessary, for an off-line evaluation by an experienced physician blinded to patients’ characteristics and allocation to intervention or control arms. Cardiac chamber quantification was performed according to the current American Society of Echocardiography (ASE) Guidelines [20]. Left ventricular diastolic function status was determined by evaluation of tissue Doppler myocardial velocities (acquired in early diastole at the anterior, inferior, lateral and septal mitral annulus portions), mitral wave inflow velocities, body surface area-indexed left atrial volumes and tricuspid regurgitation peak velocities, as recommended by ASE guidelines [21]. Left atrial strain complemented left ventricular diastolic function evaluation as proposed recently [22]. Finally, the global longitudinal strain and post-systolic strain (PSS) were assessed by speckle tracking. All the exams were recorded and reviewed by three experienced echocardiographers and in the absence of consensus the evaluations were reprocessed.

Randomization was made in 1:1 ratio on strata according to gender, baseline levels of HbA1c (7–7.9% or 8–9%) and BMI (below or above 30 kg/m²) for dapagliflozin (10 mg/day) or glibenclamide (5 mg/day) on top of metformin. The REDCap system was used for randomization at the study site by the principal investigator. Glibenclamide was selected as a comparator so that it could be based on the known effect and tested on the RCT with hard events in this population and thus estimate a potential gain with the new therapy [23‐25]. The doses of dapagliflozin and glibenclamide were chosen in order to obtain similar magnitudes of reduction of glycemia. After randomization, patients were evaluated every 30 days in order to check clinical status, treatment adherence, adverse events, use of concomitant medications.

The primary objective was the comparison of the FMD change at rest and after IR between a 12-week period of treatment with dapagliflozin or glibenclamide on top of metformin. Secondary outcomes were the difference between treatments regarding: (i) plasma NOx change after FMD; (ii) plasma isoprostane; (iii) plasma levels of the following vascular inflammatory markers ICAM-1, VCAM-1 (iv) plasma ET-1; (v) plasma levels of adipokines: adiponectin and leptin; (vi) systemic inflammatory markers: hs-CRP, TNF-alpha, IL-6 and IL-2; (vii) anthropometric measures: waist circumference, weight, body-mass index, body composition (% of fat mass and % free fat mass) and android/gynoid percent fat ratio, (viii) glucose control parameters: fasting glycemia, insulin, HOMA2S, HbA1c, (ix) office and ambulatory BP control, (x) systolic and diastolic function, and cardiac structure assessed by echocardiography, and (xi) retinopathy change during treatment.

Based on previous observations, we assumed a 3% change in FMD between arms (reduction of 1% in glibenclamide and increase of 2% in dapagliflozin), with a mean pre-treatment value of 5.5% and a standard deviation of 3.9% [26]. As we had a double primary outcome, i.e. rest and post-IRI FMD, we performed the Bonferroni’s method of adjustment and, hence, considered an alpha value of 0.025 and beta level of 90%. Under these conditions, it would take 44 patients per arm to achieve the necessary statistical power. However, as the researchers took into account that this study has a large number of secondary endpoints, the total sample was defined in 98 individuals, 49 in each arm. The sample size was calculated with G*Power 3.1 Mac version (Heinrich-Heine-Universität, Germany).

Estimates of change from baseline will be compared between treatments for each primary endpoint. Results will be analyzed on an intention to treat principle. This study will be considered a success if one or both of the primary endpoints differ between treatment groups with statistical significance. Similar comparisons of secondary endpoints will be made with adjustment for multiplicity, and statistical significance will be inferred at a two-sided Type I error level of 0.025. Adjustment for potential confounders such as statin use will be employed for the results analyses. The data are shown as the mean ± standard deviation (SD) for normal distribution or median and interquartile range (IQR) for non-normal distribution data. Baseline continuous and categorical data were compared by Wilcoxon–Mann–Whitney test and a two-tailed t test or Fisher exact test. Analyses were performed using the SPSS 22, Mac version (IBM, USA).

Recruitment of participants for the Addenda-BHS2 trial began in September 2016 and was completed in December 2018. A total of 674 subjects were evaluated and 134 met the selection criteria for the run-in phase when they underwent adjustment of medications and clinical control (Fig. 2). Of these, 98 individuals were enrolled into the trial. The main reasons for screen failure were HbA1c < 7% or > 9% (28.6%), low cardiovascular risk (12.8%), insulin use or age < 40 or > 70 years (6%). At the first visit, enrolled patients were using antidiabetic, antihypertensive and lipid-lowering medications in similar proportions (Table 1). At this time, lipid-lowering therapies were maintained at the same doses and were not introduced for those patients who were not yet in use to avoid interfering upon the primary endpoint of the study. Antidiabetic and antihypertensive medications were replaced with study drugs for a 16-week run-in period.

The baseline characteristics of the participants are shown in Table 2. It is a population with a mean age of 57 years, most males (61%) and about 10 years of T2DM (median 8 and interquartile range of 10 years). Prior coronary artery disease was found in 8 patients (8%) and cIMT above the 75th percentile for Brazilian population was the criteria for 90 patients (92%). Consistent with a tight metabolic control, the majority (98%) had eGFR > 60 ml/min/1.73 m² (57 to 142 ml/min/1.73 m²). All patients were normoalbuminuric (urinary albumin/creatinine ratio range from 0.01 to 2 mg/g). Systolic and diastolic blood pressure measured in the office were on average 137 and 82 mmHg, respectively, and 79% of the participants (77) were taking antihypertensive medication. The mean systolic and diastolic blood pressure obtained during 24-h ambulatory monitoring was 126 and 77 mmHg, respectively. The mean levels of LDL-C, HDL-C and triglycerides were 95 mg/dl, 41 mg/dl and 181 mg/dl, respectively. A total of 42 (43%) and 1 (1%) participants were taking statins and ezetimibe, respectively. Although no patient enrolled into the trial were active smoker, about 42% smoked in the past and stopped 19 ± 9 years ago.