The SAMAS study is designed as a single, blinded, randomized trial of 12 months duration, with the aim to investigate the anti-atherosclerotic mechanisms of oral semaglutide versus placebo in patients with T2D. The participants will be randomized in a 1:1 ratio to the oral semaglutide or placebo treatment groups. They will be recruited from the diabetes outpatient clinic of the University Medical Centre Ljubljana, Ljubljana, Slovenia. The study was approved on 23 August 2021 by the Slovenian National Medical Ethics Committee (Approval No. 0120–245/2021/3), and it will be conducted in accordance with the Declaration of Helsinki and Good Clinical practice guidelines. The study is supported by a grant of the University Medical Centre Ljubljana, Ljubljana, Slovenia (Grant No. 20210043). Participants will have to sign informed consent to participate.
The aim is to recruit 100 consecutive patients with T2D of both genders, aged between 30 and 60 years. They will have to fulfil all of the inclusion criteria while not meeting any of the exclusion criteria (listed in Table 1). As basic antidiabetic therapy, participants will have to be treated with at least the optimal dose of metformin (i.e. 2000 mg daily) with or without sulphonylurea. The required glycaemic control at the entry to the study, based on HbA1c, will be ≤ 8.5%. The rationale underlying this choice is based on these patients being naïve for antidiabetic therapy with additional protective effects on the cardiovascular system (e.g. for treatment with SGLT-2 inhibitors). The presence of additional cardiovascular risk factors (smoking, arterial hypertension, dyslipidaemia and chronic kidney disease stage 1–3), but not overt cardiovascular disease, allow the patients to be eligible for acceptance into the trial. In this regard, other cardioprotective therapy, i.e., angiotensin-converting enzyme inhibitors/sartans, statins or beta blockers, will be maintained during the study, with the aim to strict and optimal regulation of cardiovascular risk factors.
Patients will be recruited from the diabetes outpatient clinic, where we already have a large pool of patients who are registered as having T2D. Additionally, patients are continuously referred to our outpatient clinic by general practitioners, and these patients also represent additional pool of possible recruitment candidates. Therefore, in accordance with the inclusion and exclusion criteria, we expect the pool of patients to be sufficient. The patients will receive written and oral information about the study at the outpatient clinic, they will have sufficient time to consider enrolling up to the first visit, when they will sign written informed consent. At the first visit, all the inclusion and exclusion criteria (Table 1) will be rechecked. Patients will be randomized to the study, after signing a written informed consent form, in 1:1 fashion to receive either oral semaglutide or placebo. They will be followed for 12 months, i.e. the treatment period. The inclusion frequency is expected to be stable and continuous and will be regularly evaluated.
Withdrawal From the Study
All participants will be free to withdraw from the study at any time. They would not be requested to provide any reason for their withdrawal, and their withdrawal from the study will not influence their further treatment at the diabetes outpatient clinic of the University Medical Centre Ljubljana. Anticipated reasons for withdrawal may include: consent withdrawal, adverse events, pregnancy discovered during the trial or failure to comply with the trial medication or protocol. Missing ≤ 5 drug doses will not be the reason for exclusion, as once the steady state is achieved, the therapeutic concentration of the drug in plasma remains stable for a certain amount of time and missing some pills does not influence it. Persons with newly diagnosed carotid plaques will be withdrawn from the study and, if necessary, referred for further diagnostics. Also, all the participants with major pathologic findings on clinical examination will be withdrawn from the trial at the discretion of the investigator. In the case of findings that merit further attention, the participants will be referred to the appropriate specialist(s). If additional cardioprotective therapy needs to be introduced in the treatment regimen, the participants will also be withdrawn from the study.
The sample size calculation was performed for the primary endpoint of our study, i.e. to explore whether treatment with oral semaglutide can reduce the cIMT; the cIMT was calculated based on data from a previous study , and the standard deviation was estimated to be 0.29 mm. Setting the targeted statistical power of analysis at 90% with two-sided test alpha (α) value of 0.05 and aiming to show a decrease of 15% after treatment with oral semaglutide, the required number of patients was calculated to be 92. Based on our previous experience, the drop-out rate is estimated to be around 8–10%; therefore, our aim is to randomize a total of 100 participants to either the oral semaglutide or placebo arm.
The participants would be randomized into two groups by personnel performing the randomization process; these persons will not be blinded. In contrast, the investigators, those performing the ultrasonography and other metrics, those assessing outcomes, those performing the data analysis and patients will remain blinded through the whole course of the study. Unblinding of the participants will be permissible in case of medical emergencies that require knowledge of treatment allocation and in the cases of serious adverse events or suspected unexpected serious adverse reaction(s).
Patients will be assigned to two groups, with one receiving the active agent and the other receiving placebo. The active substance will be the oral GLP-1 receptor agonist semaglutide (Rybelsus 3 mg, 7 mg, 14 mg tablets; Novo Nordisk A/S Bagsvaerd, Denmark). The drug will have to be taken on an empty stomach in the morning after at least an 8 h fasting period. After the oral semaglutide tablet is ingested, the subjects will have to wait at least 30 min before eating, drinking or taking other oral medications. The oral semaglutide tablet must be taken with no more than a half glass of water (equivalent to 120 ml), must be swallowed whole and can not be split, crushed or chewed. The starting dose of oral semaglutide will be 3 mg; after 30 days, a dose escalation to 7 mg is anticipated; and after another 30 days, dose escalation to 14 mg, i.e., the maintenance dose, is expected. A dose escalation regimen is needed to avoid the development of the potential side effects (listed in Table 2). Dose escalation will be encouraged by notifying the patient by phone, with the call made 1 day prior to the day on which the new dose starts. If treatment with oral semaglutide is associated with unacceptable gastrointestinal side effects (moderate to severe), dose reduction or extension of dose escalation intervals will be allowed as judged by the unblinded nurse involved in the randomization process.
The placebo and semaglutide tablets will be provided in identical cases, will be identically shaped and will be packed in identically shaped bottles that will be labelled with numbers. The patients will be advised to contact the study team in case of any problems or adverse events during the study.
After the screening, the participants will be randomized to one of the two study groups in 1:1 ratio. A non-blinded study nurse (otherwise not associated with the trial) will allocate study participants into the groups evenly. Patients will be given a sufficient number of tablets (placebo or semaglutide) in bottles labelled by numbers, for the first 6 months of therapy. An additional drug package will be provided to each patient at the 6-month interval.
Trial Visits and Examinations
The trial will consist of three visits (V1, V2, V3) and three phone calls (P1, P2, P3) (Fig. 1). After initial screening for suitable candidates at the diabetes outpatient clinic, those eligible will be invited for the V1 (day 0) at which time a complete clinical examination (including blood pressure measurement and anthropometric measurements) will be performed, followed by ultrasound and endothelial function measurements as well as determination of arterial stiffness. A package of drugs (comprising of 6 bottles labelled 1–6) for the first 6 months will be handed over. Detailed instructions will be provided on the use of the oral semaglutide tablets (as explained above) and on the sequence at which the numbered bottles should be opened and the tablets taken. After the imaging and functional studies, blood samples will be collected. Similar procedures will be performed at V2 (6 months after the V1) and at the V3 (12 months after V1, i.e. the end of the study). At V2, glycaemic control will be re-evaluated, and in the case of insufficient control, rescue therapy with basal insulin will be initiated. Patients will also be educated on the use of insulin and dose titration.
The glycaemic control at V2 is defined as insufficient if the HbA1c value decreases by < 15% compared to the value at V1, or if the HbA1c value persists at or rises > 8.0%. At P1 (30 days after V1), an assessment of patient adherence and potential side effects will be made, and encouragement for dose titration (opening of the next numbered bottle) from day 31 will be given. The same steps will be taken on day 60, with encouragement given for the higher dose (opening of the next numbered bottle) to be initiated on day 61. At 30 days after the full oral dose of semaglutide is reached, the patient will receive P3, with inquiries about adherence and potential side effects. Also, from V1 onwards, in case of hypoglycaemia events or significant blood glucose reduction, patients will be encouraged to reduce the sulphonylurea dose or discontinue using it completely, while still adhering to treatment with oral semagultide, as this agent alone or in combination with metformin does not cause hypoglycaemia.
All measurements will be performed in a standardized, comfortable, warm environment, under the same conditions at every visit. All patients will be asked to overnight fast for at least 8 h prior to each examination. The measurements will be performed in the early morning before the patient’s first meal. Patients will also be asked to refrain from smoking and drinking alcoholic beverages or coffee for the same amount of time.
History and Clinical Examination
Detailed history and standard clinical examination of all organ systems will be performed. In the case that certain major pathologic features associated with the exclusion criteria are found, the participant will be withdrawn from the study and referred to the appropriate specialist(s).
Anthropometric measurements will include height, weight, waist circumference. Height will be measured using the standard wall-mounted stadiometer and expressed in centimetres. Weight will be measured using the standard body scale and expressed in kilograms. Body mass index will then be calculated and expressed in kilograms per square meter. Waist circumference will be measured using non-rigid measurement tape and expressed in centimetres.
Blood Pressure Measurement
An automated sphygmomanometer will be used for blood pressure measurements and expressed in millimetres of mercury (mmHg).
Ultrasound Measurements of the Carotid Intima Media Thickness
Ultrasound cIMT measurements will be performed by the single investigator who is blinded to the patient’s therapy; the investigator will also not have access to the previous ultrasound scans when follow-up measurements are made. The ultrasound examination will be performed using the General Electric Logiq F6 (GE Healthcare, Chicago, IL, USA) ultrasound machine. The L6-12-RS linear probe with the frequency of 3.7–13.0 MHz will be used for vascular examination, as per manufacturer's instructions. The cIMT measurements will be performed in the standard manner, i.e. supine position of the participant and standard angles of insonation of carotid arteries.
Complete wall segments of the carotid artery branches will be carefully scanned bilaterally, starting with the common carotid artery just above the clavicle, sliding upwards towards the bifurcation and along the internal carotid artery. Near and far planes of each of the arterial segments will be scanned, while measurements will be made starting 1.0 cm proximal to the bifurcation, followed by the bifurcation itself and then 1.0 cm of the proximal internal carotid artery segment . The auto-IMT modality of the ultrasound machine software will be used, which will automatically—and independently of the observer—detect the cIMT, considering that the distance between the echogenic line represents the intimal blood interface and the outer echogenic line represents the adventitial junction. The maximum cIMT value, expressed in millimetres, will be used for analysis, and determined as the mean of all six measurements performed bilaterally [23, 24].
Measurement of Endothelial Function
The endothelial function measurement will be performed on the peripheral arterial tone using the EndoPAT 2000 device (Itamar Medical Ltd., Caesarea, Israel); this device has been approved by the US Food and Drug Administration (FDA) for determination of endothelial dysfunction. It will produce standardized measurements that are mostly independent of the investigator. Patients will be lying in a semi-supine position, with their forearms extended on the foam arm rest at a 40º angle as per manufacturer’s instructions. Pneumatic probes will be placed on the index fingers bilaterally and inflated to 70 mmHg to squeeze the veins and prevent the veno-arteriolar vasoconstrictor reflex. At this point of time, a baseline signal will be recorded. After 5 min of baseline recording, a blood pressure cuff on one forearm will be inflated to at least 60 mmHg above the patient's systolic blood pressure for another 5 min. The blood pressure cuff will then be rapidly deflated, causing reactive hyperaemia, and the signal will then be followed for the last 5 min. The reactive hyperaemia index (RHI) will be calculated using the manufacturer’s algorithm based on the ratios between pulse wave amplitudes during the reactive hyperaemia and baseline phases [25,26,27].
Measurement of Arterial Stiffness
Arterial stiffness will be measured using the SphygmoCor device with CvMS software (version 9) (AtCor Medical Pty Ltd., West Ryde, Australia). For this measurement, the investigator will again be blinded regarding the therapy that a patient is taking as well as the results of the previous measurements. The measurements will be performed according to the Consensus document on arterial stiffness for measurement of carotid-femoral pulse wave velocity (cfPWV) . The pulse waveforms will be collected at two sides on the right-hand side of the body, with the patient lying in the supine position. Pulse waveforms will first be obtained at the common carotid artery, then on the femoral artery. The distances from the sternal notch to the common carotid artery and from the sternal notch to the femoral artery will be measured. The CvMS software will then calculate the cfPWV automatically as the ratio of the pulse wave distance travelled and the transit time needed [28, 29].
Venous blood samples will be taken after an overnight fast of at least 8 h. If required, selected blood samples will be centrifuged within 30 min after collection. Aliquots of both serum and plasma will be collected, and some will be used for immediate analyses and will be immediately frozen at − 80 °C.
HbA1c, fasting blood glucose, creatinine, electrolytes, hepatic enzymes and lipids (total cholesterol, triglycerides and high-density lipoproteins) will be measured by standard laboratory methods using the VITRO 5.1FS Chemistry System (Ortho Clinical Diagnostics, Raritan, NJ, USA). LDL will be calculated using the Friedewald formula. HsCRP will be measured in serum using the VITROS Chemistry Products hsCRP Reagent (Ortho- Clinical Diagnostics, Inc., Rochester, NY, USA). Uric acid will be determined by enzymatic reaction with uricase and Trinder-like endpoint product detection (ADVIA Chemistry Systems, Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA). The CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) formula will be used to calculate estimated glomerular filtration rate. Parameters of inflammation will also be analysed, and additional parameters of coagulation will be determined.
The insulin resistance index (HOMA-IR) will be calculated from fasting insulin (FI) values in venous blood samples and fasting blood glucose (FBG) according to the following equation: FBG × FI/22.5.
For analyses of the lipoprotein subclasses, the samples will be stored at − 80 °C immediately after collection and then analysed at a later time point to determine at least 11 lipoprotein subclasses, including very-low-density lipoproteins, intermediate-density lipoprotein subclasses (IDL-A, IDL-B and IDL-C) and seven LDL subclasses (LDL1–7); the US FDA-approved non-denaturing, linear polyacrylamide gel electrophoresis system (Lipoprint; Quantimetrix Corp., Redondo Beach, CA, USA) will be used for determiniation of the LDL subclasses . Electrophoresis will be performed for 60 min at 3 mA in a gel tube, with each chamber having two quality controls; after electrophoresis, the gel tubes will be scanned using the ArtixScan F1 densitometric flatbed scanner (Microtek Int. Corp., Hsinchu, Taiwan) combined with a Mac personal computer (Apple Inc, Cupertino, CA, USA) with the ScanWizard Pro software installed (Microtek Int. Corp.) . The relative area of each lipoprotein band will be determined and multiplied by the total cholesterol concentration of the sample, and LDL subclasses will be defined as seven bands (LDL1-7, respectively) [30, 31].
The main objectives of the present study, as explained above, are to examine the mechanisms responsible for anti-atherosclerotic and cardioprotective actions of the oral semaglutide formulation in patients with T2D without manifested cardiovascular disease.
The anticipated primary outcome is the reduction in cIMT, improvement of endothelial function and reduction of arterial stiffness, after 12 months of treatment with oral semaglutide.
The anticipated secondary outcomes comprise reduction in atherogenic small dense LDL, improvement of HbA1c and reduction of hsCRP, after 12 months treatment with oral semaglutide. Additionally, possible correlations between the primary and secondary endpoints will be sought. Also, the revaluation visit at 6 months will enable the pace of the oral semaglutide’s anti-atherosclerotic effect to be evaluated.
Participants will be identified by a personal study identification (ID). All venous samples will also be labelled by a study ID. Study data (history and clinical examination data and anthropometric measurements) will be collected as paper case report forms that will be stored securely. The data will also be entered into the electronic database on an on-going basis. The results of measurements of cIMT, endothelial function examinations and arterial stiffness examinations will be supported by data from the measurement devices. Laboratory data will be printed out from the laboratory analyses and stored in paper form; the data will also be transferred electronically to the electronic database system of the study. The electronic database will be stored in secured hard drives with backups. The trial adheres to the Data Protection Act that requires the data anonymisation as soon as this is possible in the study.
All data and information relating to the study subjects will be saved and recorded on password-protected computers and will only be accessible to the research team. A database will also enable the research team to track each participant’s progress throughout the trial, including the scheduling of visits and appointments.
Data Analysis and Statistical Analysis
Statistical analysis of the stored data, using the two-way analysis of variance test, will be performed at the end of the study. Where significant differences will be found, subsequent tests will be performed. Spearman’s test will be used to assess the associations between different parameters. P values < 0.05 will be considered to be statistically significant. Microsoft Office Excel (Microsoft Corp., Redmond, WA, USA), GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA) and SPSS (SPSS, IBM Corp., Armonk, NY, USA) software will be used for the statistical analysis assessment.