Renoprotective Effects of Dapagliflozin in Type 2 Diabetes

Study design A phase 4, monocenter, randomized, double-blind, comparator-controlled, parallel-group, mechanistic intervention trial to assess the effect of 12-week treatment with the sodium-glucose linked transporters (SGLT)-2 inhibitor dapagliflozin versus the sulfonylurea (SU) derivative gliclazide on renal physiology and biomarkers in metformin-treated patients with type 2 diabetes mellitus (T2DM)

Recruitment Subjects will be recruited by researcher physicians involved in this trial using methods that are established practice for all human studies in the Diabetes Center, Vrije Universiteit (VU) University Medical Center (VUMC), via advertisements, use of a Diabetes Center database, through the Diabetes Center website.

Screening and eligibility After giving extensive oral and written information, a written informed consent form will be obtained from the subjects before screening. The screening procedure will consist of obtaining an extensive medical history, complete physical examination, blood analysis, urine screening, and a 12-lead electrocardiogram (ECG). Postvoid residual urine volume will be determined by ultrasonic bladder scan. After inclusion, participants will enroll in a 4-week run-in period to allow for study effects.

Baseline assessments Seven days prior to this visit, subjects will adhere to a 'normal-salt' (9 - 12 grams or 150 - 200 mmol per day) and protein (1.5 - 2.0 g/kg per day) diets, in order to minimize variation in renal physiology due to salt and protein intake. Participants will abstain from alcohol (24 hours), caffeine (12 hours) and nicotine (12 hours) and heavy exercise prior to and during visits 2. One day prior to the testing day, subjects will collect 24-hour urine.

Following an overnight fast, participants will be instructed to drink 500 mL of water, but to delay all morning-time medication (apart from metformin) until conclusion of the examination day. After taking subjects history, current weight and blood pressure, intravenous catheters will be placed in both forearms after which blood and urine will be collected. Thereupon the combined renal and clamp protocol will commence.

Safety and tolerability: Telephone consultation and follow-up Participants will be asked about the occurrence of adverse events and the intake of study drugs will be checked. Furthermore, recent history will be taken, physical examination/anthropometric measurements performed and blood and urine samples collected.

Study Procedures Renal protocol During this protocol a combined inulin/para-aminohippurate (PAH)-clearance technique will be performed to examine the glomerular filtration rate (GFR, ml/min) and effective renal plasma flow (ERPF, ml/min) respectively. In order to examine tubular function, sodium, potassium, chloride, calcium, magnesium, phosphate, urea and glucose will be measured in urine (mmol). In addition, urine osmolality will be determined. Moreover, biomarkers of renal damage (urinary albumin excretion (UAE), Neutrophil gelatinase-associated lipocalin (NGAL) and Kidney Injury Molecule-1 (KIM-1) will be measured in urine

Clamp protocol A combined hyperinsulinemic-euglycemic and hyperglycemic clamp will be performed. During the euglycemic clamp insulin will be infused at 40 microunits/square meter.minute (mU/m2.min) and glucose will be kept at 5 mmol/l by variable glucose 20% infusion. This technique allows for renal measurements during normoglycemia in all participants. After 3 hours of insulin infusion and an one hour rest period for exogenous insulin to be cleared, glucose will be infused in order to reach a plasma concentration of 15 mmol/l. In this matter, renal tests can be performed under stable hyperglycemia. From the euglycemic clamp, insulin sensitivity will be determined from the amount of glucose infused (M-value). Blood will be stored during hyperglycemia in order to be able to obtain measures of beta-cell function, including 1st phase insulin secretion, 2nd phase insulin secretion and arginine-induced insulin secretion.

(cardio)vascular measures After an acclimatization period of >5 minutes, blood pressure will be measured three times at the non-dominant arm, with an automatic oscillometric device (Dinamap®, General Electric (GE) Healthcare). Systemic hemodynamics will be measured by an automated, beat-to-beat blood pressure and ECG recording monitor (Nexfin®, BMEYE, Amsterdam, The Netherlands). Body composition will be assessed by bio impedance analysis using tetrapolar Soft Tissue Analyzer® (STA, Akern, Florence, Italy). Microvascular function will be measured by nail fold skin capillary density on the middle finger, and skin blood flow will be measured using a Laser Doppler probe. Applanation tonometry using the SphygmoCor® system (Atcor Medical, West Ryde, Australia) will be performed to measure variables of arterial stiffness at the radial artery of the non-dominant arm.

Fecal examinations Each participant will collect a stool sample before the baseline tests and once before the long-term follow-up tests. At home, one feces sample must be stored at room temperature and one in the freezer and when arrived at the CRU, the feces will be stored at -20⁰C and -80⁰C, respectively.

SAFETY (REPORTING) In accordance with the guidelines of ICH-GCP, the European Clinical Trials Directive (2001/20/EC) and the local regulations, the investigator will inform the subjects and the reviewing Ethical Review Board (ERB) if anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly greater than was foreseen in the research proposal.

Reporting The investigator will be responsible for reporting the SAEs and SUSARs to the accredited ERB. This will be done within 15 days after first knowledge, using the specified web portal ('ToetsingOnline'), which automatically leads to informing the respective national health authorities. For fatal or life threatening SAEs and SUSARs this term will be 7 days for a preliminary report ('expedited') with another 8 days for completion of the report.

The involved pharmaceutical company (AstraZeneca) will be notified of all SAEs, pregnancy, clinical relevant overdosing and adverse events of special interest (AESIs), if any.

Monitoring As required by GCP, a monitoring procedure will be performed in order to oversee the progress of the clinical trial and ensure that it is conducted, recorded and reported in accordance with the protocol, standard operating procedures (SOPs), GCP and the applicable regulatory requirements.

Statistical Analysis Plan All tests of differences between treatments will be conducted at a two-sided significance level of <0.05. In case of missing data, a multiple imputation technique will be applied and compared with complete case analysis. The efficacy analysis population will be based on the per protocol (PP) principle - i.e. all subjects who completed the entire treatment period using the treatment as originally allocated. Safety analysis will be performed in all patients who received one or more doses of the study medication.

In order to compare the effects between the two study arms the investigators will perform t-tests and/or multivariable linear regression models. The latter technique allows us to use continuous outcome variables and both continuous and dichotomous independent variables. The efficacy endpoint of interest will be added to the model as dependent variable.

Our primary outcomes will be the most important outcomes for testing the hypothesis that SGLT-2 inhibitor dapagliflozin versus the SU gliclazide causes an improvement in renal hemodynamics. The large amount of secondary/exploratory endpoints potentially justifies the use of corrections for multiple tests. However, there is an increasing debate on whether this should be done, because of the increased risk of type 2 errors which could lead to prematurely discarding of potential useful observations. As recently pointed out, in a hypothesis-generating study - such as the current study - correction for multiple outcomes can be detrimental. A final decision will be made in the detailed Statistical Analysis Plan (SAP) prior to the conduction of the analysis.

1. ETHICS The trial will be conducted in accordance with the Declaration of Helsinki (64th World Medical Association (WMA) General Assembly, Fortaleza, Brazil, October 2013) for biomedical research involving human subjects and in accordance with ICH-GCP. The investigator will ensure that all aspects of the institutional ERB review are conducted in accordance with current institutional, local and national regulations.

Study Medication Participants will be randomized for a 12 week treatment with two different active study agents. Consequently, independent of treatment allocation, beneficial effects can be expected, as both SGLT-2 inhibitors and SU derivatives improve glycemic control. Both study medications have been approved for blood-glucose lowering treatment in T2DM patients and, based on currently available data, are considered to be safe. Furthermore, SGLT-2 inhibitors in general may decrease blood pressure and body weight compared to gliclazide.

The most common side effects for dapagliflozin are hypoglycemia (especially when used in combination with a SU derivative or insulin) and genital mycotic- and urinary tract infections. For gliclazide, hypoglycemia and blurred vision in the initiation phase of treatment (due to changes in blood glucose levels) are the most common side effects. As in all drug intervention trials, in this study, the investigators will closely monitor patients for adverse drug and study events during the follow-up visit and by telephone consultation according to GCP. Participants can contact the research staff 24 hours a day.

Safety issues The study examinations/tests are considered to be safe. No invasive procedures (besides intravenous peripheral catheters) are involved. During the study tests, two 'diagnostic agents' (i.e. inulin and PAH) need to be administered; both agents are inert and have no side effects. The total amount of drawn blood will be 500 mL during a total period of 16 weeks. Side effects are not expected because the blood volume taken per visit is relatively small, especially in comparison with regular blood donation, with 500 mL per donation (and is allowed 5 times a year for men and 3 times for women).

Publication policy AstraZeneca will be provided will all intended publication(s) and abstract(s), preceding submission at least 30 days and 7 days, respectively, AstraZeneca can make suggestions to change the wording of any planned publication or abstract in relation to the study and/or the results. The sponsor-investigator is under no obligation to implement such changes, unless local regulatory requirements specify otherwise.

Data will be presented at relevant national and international scientific meetings, and published in peer reviewed journals.