SIALIDASE INHIBITION AS ANTIATHEROSCLEROTIC THERAPY

• Rationale

Dietary supplement "Tertinat" is based on green tea extract with a standardized content of epigallocatechin-3-gallate. According to in-silico and in vitro data, epigallocatechin-3-gallate is an inhibitor of neuraminidases, thus capable of preventing desialylation of low-density lipoprotein (LDL). In pilot study it has been demonstrated that peroral intake of Tertinat leads to a sustained increase in the level of LDL sialic acid content in the blood of study participants in vivo, as well as a decrease in the serum-induced intracellular cholesterol accumulation by macrophages in vitro.

The available published data demonstrate the anti-inflammatory effects of epigallocatechin-3-gallate and its ability to regulate lipid metabolism, which potentially indicate to the multiplicity of mechanisms of its anti-atherogenic action.

• Study Objectives

The objective of the study is to evaluate the effect of long-term intake of dietary supplement Tertinat on the rate of major adverse cardiovascular events (MACE).

• Study Hypothesis

Oral administration of Tertinat in addition to the main therapy reduces the rate of cardiovascular events in patients with coronary atherosclerosis via restoring LDL sialic acid content and lowering serum atherogenicity.

• Primary Endpoints:

The 6-point MACE (Major Adverse Cardiovascular Events) is established as the composite endpoint to represent the combined occurrence of severe heart- and blood vessel-related medical complications:

• Cardiovascular death (fatal cardiovascular events)
• Non-fatal myocardium infarction
• Stroke / Transient ischemic attack
• Unstable angina / Acute coronary syndrome
• Heart failure / Hospitalization due to critical ischemia
• Revascularization

The rate of MACE is estimated at 12 and 24 months after inclusion in the study.

The deaths from other non-coronary cardiovascular diseases and from definitively non-atherosclerotic causes of death are registered but not considered as primary endpoints.

• Secondary Endpoints:

• Change in the extent of stenosis of the affected arteries
• Change in LDL sialic acid content
• Change in blood serum-induced intracellular cholesterol accumulation
• Change in total cholesterol, HDL cholesterol, triglycerides, LDL cholesterol
• Change in the cholesterol level of circulating immune complexes

Secondary endpoints are estimated at 12 and 24 months after inclusion in the study.

• Study Design

A prospective double-blinded randomized placebo-controlled multicenter study evaluating the effect of oral administration of the dietary supplement "Tertinat" (green tea extract with a standardized content of epigallocatechin-3-gallate) at a dosage of 330 mg/day in atherosclerotic patients after coronary revascularization on the background of standard therapy.

Study duration: 24 months after inclusion in the study, or until a statistically significant rejection of the null hypothesis.

A simple randomization method is used (random assignment of patients to groups of verum- and placebo-recipients) followed by adaptive randomization for the purpose of statistically based balancing the number of group participants by gender and age.

The calculated minimum sample size for detecting the difference in MACE rate between verum- and placebo-recipients is 588 patients per group, with anticipated 15% difference in MACE rate by the end of the study at alpha 0.05 and 80% statistical power.

• Withdrawal from the Study and Replacement of Participants

Participants may withdraw from the study at any time without giving reasons and without any consequences for the patient's further management at the clinic. Replacement of withdrawn participants is not anticipated; the planned sample size considers the possible withdrawal of some participants from the study.

Potential reasons for participant withdrawal include:

• withdrawal at the participant's own decision;
• a decision made by the participant due to adverse events while taking the investigated dietary supplement;
• withdrawal due to a decision by the principal investigator or an expert body regarding the unsafety of continuing the study;
• loss of contact with the participant;
• participant compliance of less than 80%.
• Informed Consent, Patient Information, CRF

All patients who are potential candidates for screening must sign written informed consent before participation.

Participants must sign the current version of the informed consent form before any interventions related to the specific trial. The personnel conducting the study must explain to participants that even if he/she or they agree to participate in the study and sign the informed consent form, the assessment of the inclusion/non-inclusion criteria may show that the patient is not a suitable candidate for the study and, therefore, will not be admitted to participation.

• Procedures Performed:

9.1. The following information is collected about study participants: gender; age; diagnosis; the history of cardiovascular events (according to clause 4), the dates of previous cardiovascular events; smoking history; weight; height; concomitant diseases; type of surgical revascularization procedure performed; the date of surgery; medication therapy prescribed at discharge (antiplatelet therapy, lipid-lowering therapy, antihypertensive therapy, glucose-lowering medications).

9.2. Collection of the history of cardiovascular events from the patient or his/her representative, including analysis of medical documentation (if cardiovascular events were accompanied by hospitalization at one of the study centers). During history collection, the following is noted: type of cardiovascular event (acute myocardial infarction and acute coronary syndrome, acute cerebrovascular accident, progressive heart failure, progressive atherosclerotic lesions of the limb arteries, repeated surgical intervention on vessels), date of manifestation. The cases of MACE are recorded during a call to schedule the next visit, during the interview of the contact person, or from medical center documentation data. During information collection, the following is recorded: the type of cardiovascular event that led to death (acute myocardial infarction and acute coronary syndrome, acute cerebrovascular accident, progressive heart failure, other types of death of cardiovascular origin), date of death.

9.3. Laboratory examination data at the clinical center (information is taken from the clinical chart): blood glucose level before surgery; serum C-reactive protein level before surgery.

9.4. Procedures for instrumental assessment of the degree of arterial damage:

9.4.1. Assessment of coronary blood flow (in case of suspected silent myocardial ischemia, performed as part of the preoperative examination): coronary angiography or CT (in case of coronary heart disease as the underlying disease) with assessment of the severity of stenosis of the left main coronary artery, left anterior descending artery, right coronary artery, circumflex artery, obtuse marginal branch, posterior descending artery, posterolateral artery, diagonal artery, and ramus intermedius artery.

9.4.2. Ultrasonography (US): Determination of the severity of stenosis of the brachiocephalic vessels: right and left common carotid, internal carotid, and external carotid arteries; rught and left femoral arteries. The severity (%) of stenosis is calculated using the NASCET method, using the formula (1-(N/D))*100%, where N is the diameter of the residual lumen, D is the diameter of the healthy internal carotid (or femoral) artery in the area above the stenosis site, where the vessel walls are parallel.

9.4.3. The intima-media thickness (IMT) of right and left common carotid arteries is assessed by ultrasound examination. The IMT of the common carotid arteries is defined as the distance between the vessel lumen boundary and the media-adventitial boundary. The IMT of the far wall of common carotid arteries is measured at 1 cm adjacent to the carotid bulbus at anterior, lateral, and posterior angles of interrogation.

9.5. Procedure for blood collection for special studies.

Blood from study participants is collected for biochemical analysis of lipid profile (serum total cholesterol level; serum LDL cholesterol level; serum HDL cholesterol level; serum triglyceride level); LDL sialic acid content; the cholesterol content of circulating immune complexes, and to measure blood serum atherogenicity (serum-induced cholesterol accumulation in cultured macrophages in vitro).

In all patients, venous blood is collected into three Vacutainer-type tubes without anticoagulant, with a volume of 10 ml (a total of 30 ml of blood). After complete blood clotting (after 30 minutes, but no later than 4 hours), the blood is centrifuged in a laboratory centrifuge with a swinging rotor at 1500g for 10 minutes to separate the serum.

The resulting serum is transferred into prepared tubes with a weighed portion of sucrose (60 mg) strictly up to the mark (corresponding to 10 ml), closed with a cap, and mixed by inversion until the sucrose is completely dissolved. The tube is labeled according to the rule:

Tube labeling: center number/study participant number/blood collection date/SERUM

In the tubes into which blood was collected (without anticoagulant, three tubes of 10 ml each), after centrifugation and serum collection, a blood clot remains. These tubes are closed with a cap and labeled according to the rule:

Tube labeling: center number/study participant number/blood collection date/CLOT

All labeled tubes are placed in a freezer (minus 18-20 degrees), accumulated, and stored until shipment to the Central Laboratory (Moscow) in a box with cold packs. The central laboratory performs the measurement of lipid profile parameters (serum total cholesterol level; serum LDL cholesterol level; serum HDL cholesterol level; serum triglyceride level); LDL isolation from serum samples by ultracentrifugation in KBr density gradient; measurement of LDL sialic acid content; measurement of serum-induced cholesterol accumulation (serum atherogenicity) in cultured macrophages in vitro; measurement of cholesterol content in circulating immune complexes.

• Data Collection and Processing

Participant data collected during the performance of procedures (clause 10) will be stored in a database in the form of an Excel spreadsheet in an anonymized form.

Statistical data processing will be carried out: at the stage of recruiting study participants in order to ensure the homogeneity of the groups according to the characteristics: gender, age, diagnosis, type of treatment performed (e.g., emergency surgeries, elective surgeries, minimally invasive vascular surgeries, open vascular surgeries), and degree of vascular occlusion according to ultrasound data. At the data processing stage, the collected data are used to assess the difference in endpoint parameters between the study groups.

The following statistical methods are used: The Shapiro-Wilk test to assess the normality of distribution; descriptive statistics; T-test, or Mann-Whitney U test to assess differences in quantitative indicators between groups; Chi-Square or Fisher's exact test - to assess the difference in the frequency of cardiovascular events in the groups; Cox regression, or proportional hazards model to assess the time of occurrence of cardiovascular events; Repeated Measures Analysis or Mixed-Effects Models: to assess the correlation between repeated measurements of secondary endpoints within a single study participant; analysis of covariance (ANCOVA): to compare results between groups with adjustment for baseline differences. Statistical analysis is performed in Central laboratory by the staff blinded for prescription of the dietary supplement under study to trial participants.

• Regimen for Dispensing Medications. Monitoring of Participant Adherence (Compliance).

The dietary supplement under study is dispensed to the study participant with a calculation for the period until the next scheduled visit (12 months). Each protocol participant takes a medication, which batch number begins with the same digit (1 or 2), since the batch number encodes whether the capsules contain verum or placebo.

Two weeks before the scheduled visit, study participants are contacted by phone to schedule the visit date, and they are asked to bring the remaining medication capsules for the issuance of a new package. Based on the number of remaining capsules, compliance is calculated using the formula: (number of capsules used / number of capsules assuming daily intake) *100%. For example, a participant was given 365 capsules to take 1 capsule per day. The participant's next visit occurred after 351 days, and he brought 50 unused capsules. The patient's compliance is - ((365-50)/351)*100% = 90%.

An indirect method of assessing compliance in the form of a participant survey will also be applied.

• Schedule of Visits

Visit 0 (inclusion in the study)

Conducting an interview with the patient and obtaining informed consent for participation in the study Making a decision on compliance with the inclusion criteria (clause 7) Collection of clinical data (clause 9.1.) Collection of the history of cardiovascular events (clause 9.2.) Collection and entry into the database of coronary angiography parameters (performed only if there are clinical indications for coronary angiography, in case of CHD) (clause 9.4.1.) Collection and entry into the database of laboratory parameters (clause 9.3.) Determination of the severity of carotid and femoral artery stenosis (clause 9.4.2) and entry into the database Determination of the intima-media thickness (IMT) parameters of the common carotid arteries (clause 9.4.3) Collection of 30 ml of blood from a vein for special studies (clause 9.5.) Randomization (clause 6), recording of the batch number of the dispensed medication (clause 6) in the database Dispensing the medication to the participant for a period of 365 days, explaining the regimen for taking the medication (clause 12), scheduling the date of the next visit Completing the Case Report Form Exchange of contact details (telephone, messengers, email) between the study participant and study coordinators - for answering questions, consultations, and calling for subsequent visits

Visit 1 (12 months ±14 days after Visit 0):

Preliminary agreement with the study participant on the possibility and date of his/her arrival to the center; reminder about the need to bring the capsules of the medication remaining after the previous issuance.

Collection of the history of cardiovascular events (clause 9.2.) Determination of the severity of carotid and femoral artery stenosis (clause 9.4.2) and entry into the database Determination of the intima-media thickness (IMT) parameters of the common carotid arteries (clause 9.4.3) Collection of 30 ml of blood from a vein for special studies (clause 9.5.) Monitoring of medication intake (clause 12) Assessment of the possibility or impossibility of the participant's further inclusion in the study (clause 7 and clause 8) Dispensing the medication, the batch number of which begins with the same digit as the batch previously dispensed to the participant, for a period of 365 days, explaining the regimen for taking the medication (clause 12), scheduling the date of the next visit.

Visit 2 (24 months ±14 days after Visit 0, either 12 months ±14 days after Visit 1):

Preliminary agreement with the study participant on the possibility and date of his/her arrival to the center; reminder about the need to bring the capsules of the medication remaining after the previous issuance.

Collection of the history of cardiovascular events (clause 9.2.) Determination of the severity of carotid and femoral artery stenosis (clause 9.4.2) and entry into the database Determination of the intima-media thickness (IMT) parameters of the common carotid arteries (clause 9.4.3) Collection of 30 ml of blood from a vein for special studies (clause 9.5.) Monitoring of medication intake (clause 12)

Inclusion criteria

• Men and women aged 45-75
• Patients with atherosclerotic cardiovascular diseases who have passed revascularization procedure within last 3 months prior to the inclusion in the study
• Atherosclerotic cardiovascular diseases may include coronary heart disease, and/or coronary atherosclerosis, and/or atherosclerosis of brachiocephalic / limb / renal arteries.
• Patients who have undergone instrumental and laboratory examinations (ECG, ultrasound / CT / angiography, biochemical tests - total cholesterol, triglycerides, LDL cholesterol, HDL cholesterol, blood glucose, C-reactive protein), and the data from above examinations from medical records are available.
• The principal patient's abitity for follow-up questioning and examination at 12 and 24 months apart from inclusion in the study.
• The signed informed consent.

Exclusion criteria

• Critical and urgent cardiovascular conditions: tissue ischemia stage III-IV, stroke, acute coronary syndrome, acute myocardial infarction, chronic heart failure III and IV class NYHA (New York Heart Association).
• Other critical and urgent conditions not associated with cardiovascular diseases, including the need for urgent interventions, chronic renal failure stages IV-V (creatinine clearance < 30 ml / min according to the Cockcroft-Gault Equation)
• Systemic autoimmune diseases in medical history, including: rheumatoid arthritis, systemic lupus erythematosus, autoimmune thyroiditis, autoimmune vasculitis, ulcerative colitis.
• Significant weight loss (> 10% of body weight in the previous year) of unknown etiology.
• Conditions that limit adherence to participation in the study (dementia, neuropsychiatric diseases, drug addiction, alcoholism, etc.).
• Participation in other clinical studies (or use of investigational substances) within 3 months prior to study entry.
• Carriers of HIV or viral hepatitis
• Pregnancy or breast feeding
• Refusal to participate in the study / to sigh informed concent.